On Naivety of T Cells in Inflammatory Bowel Disease: a Review

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carmen horjus

Mucosal immunology and imaging in early Inﬂammatory Bowel Disease

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radboud universiteit nijmegen

Mucosal immunology and imaging in early Inﬂammatory Bowel Disease

proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magniﬁcus prof. dr. J.H.J.M. van Krieken, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 1 februari 2017 om 14.30 uur precies

door

Carmen Simona Horjus Talabur Horje

geboren op 13 mei 1977 te Oradea (Roemenië)

Promotor: prof. dr. j.p.h. drenth

Copromotoren: dr. e.g. van lochem (rijnstate, arnhem) dr. p.j. wahab (rijnstate, arnhem) dr. m.j. groenen (rijnstate, arnhem)

Manuscriptcommissie: prof. dr. m.g. netea (voorzitter) prof. dr. w.m. prokop prof. dr. g.r. van den brink (amc, amsterdam)

“ A thing of beauty is a joy for ever: Its loveliness increases; it will never Pass into nothingness;”

john keats

table of contents

chapter 1 Introduction and outline of the thesis 9

part i Clinical aspects of the primary diagnosis in Inﬂammatory Bowel Disease 21

chapter 2 Time trends in forty years of diagnostic procedures in the primary diagnosis of Inflammatory Bowel Disease 23 chapter 3 Prevalence of upper gastrointestinal lesions at primary diagnosis in adults with Inflammatory Bowel Disease 39 chapter 4 MR Enterography next to ileocolonoscopy in newly diagnosed adults with Crohn’s disease 53 chapter 5 Contrast Enhanced abdominal Ultrasound in the assessment of ileal inflammation in Crohn’s disease in comparison to MR Enterography 67

part ii Immunological characteristics in early Inﬂammatory Bowel Disease 87

chapter 6 Naive T cells in the gut mucosa of newly diagnosed, untreated Inflammatory Bowel Disease patients 89 chapter 7 On naivety of T cells in Inflammatory Bowel Disease: A review 105 chapter 8 High endothelial venules associated with naïve and central memory T cells in the inflamed gut of newly diagnosed Inflammatory Bowel Disease patients 119

7 table of contents

part iii Discussion 141

chapter 9 Summary and general discussion 143

chapter 10 Dutch summary 153

part iv Appendices 163

appendix i List of abbreviations 165 appendix ii List of publications 167 appendix iii Acknowledgement/Dankwoord 179 appendix iv About the author 173

8 chapter 1

Introduction and outline of the thesis

Inflammatory bowel disease (IBD) comprises several chronic inflammatory disorders of the gastrointestinal tract with a heterogeneous presentation and potentially severe disease course. The initial diagnosis of IBD as well as diferentiation between diferent phenotypes ofen represents a challenging clinical issue, as no single diagnostic criterion is available. Like many other immune mediated diseases the complex etiopathogenesis of IBD is known to be multifactorial based on interactions between genetic and environmental factors, gut microbiota and the immune system, leading to ongoing inflamma- tion1,2. However neither the single role of these factors nor their supposed interactions has yet been completely elucidated. Knowledge of the initial alterations of the immune responses in the early phase of IBD, without the influence of immunosuppressive medication, might, in part, elucidate the first stages of disease development and may therefore help to better understand pathogenesis and subsequent clinical heterogeneity and clinical disease course.

Clinical aspects in IBD

Historical remarks In all probability we shall never know who first diagnosed IBD and how the first diagnosis was made. Hippocrates (460-377 BC) already wrote about perianal fistula and was aware of the fact that diarrhoea was not a single entity, although he could not distinguish between an infectious and non-infectious cause3,4. In the first century AD, various forms of non-contagious diarrhoea were described freely in the literature by physicians such as Aretaeus of Cappadocia (A.D 300). During the nineteenth century, non-contagious diarrhoea was commonly reported under diferent names, such as “the flux of Sydenham”5. The first to refer to the name ulcerative colitis was Sir Samuel Wilks in 1859, when he described inflammation in the distal ileum and colon at autopsy of a young woman who died afer a period of illness with bloody diarrhoea and abdominal pain, in a report entitled “The morbid appearance of the intestine of Miss Banks”6. A few years later, in 1875, Wilks together with Moxon, described the histopathological findings of UC in the second edition of the Lectures on Pathological Anatomy7. With today’s knowledge it could be argued that Miss Banks was actually sufering from Crohn’s disease instead of UC, given the extended inflammation of the ileum. The first physician to describe Crohn’s disease was, notwithstanding its currently coined name, not Burrill Bernard Crohn. The first narrative describing ileal ulcerations and enlarged mesenteric lymph nodes in a deceased young man was made by Morgagni (1682-1771) in 17618. However Morgagni’s findings may also have fitted the description of infectious enteritis, such as tuberculosis or Yersinia infection. Another early report of non-infectious enteritis came from the Scottish surgeon Thomas Kennedy Dalziel in his

11 chapter 1

publication of 1913, entitled “chronic interstitial enteritis”9. He described in that paper a series of patients sufering from chronic stricturing inﬂammation of the small and large bowel with no evidence of tuberculosis or other active infection. The entity of chronic regional enteritis, however, has been attributed to the American gastroenterologist Burrill Bernard Crohn who authored, together with his colleagues Leon Ginzburg and Gordon D Oppenheimer, the paper “Regional Ileitis: A pathological and clinical entity” in 193210. Since then, UC and CD have steadily increased in scientiﬁc popularity. During the last decades numerous studies on the incidence, course and prognosis of the disease have been carried out, reporting an increasing incidence of IBD ranging from 0.7 to 14.6/100,000 for CD, and from 1.5 to 24.5/100,000 for UC, varying greatly between regions11-19. In Eu- rope, the reported incidence for CD is 3-8/100,000 and 3-14/100,000 for UC20-21.

Clinical diagnosis and disease course

Classification of IBD A single criterion for diagnosing IBD is not available and therefore a combination of clinical, endoscopic, histologic and radiologic criteria is used as a gold standard22. IBD comprises diferent entities currently classified as Crohn’s disease (CD), ulcerative colitis (UC), IBD unclassified (IBDU) and indeterminate colitis (IC)23. IBDU should be used in cases where diferentiation between CD and UC is not possible afer endoscopy with biopsies and radiologic examinations. The term IC should be preserved for pathologists describing a colectomy specimen when definite discrimination between CD and UC cannot be made24.

Ulcerative colitis Ulcerative colitis is generally limited to the colon and is classified by the extent of inflammation and severity of symptoms23. The symptoms of UC include rectal bleeding, diarrhoea and abdominal tenderness. The severity and combination of symptoms de- pend on the extent of the inflammation. In case of proctitis, rectal bleeding is the main symptom, accompanied by rectal urgency, tenesmus and loss of mucopurulent exu- date. In extensive and lef-sided colitis the main symptoms are bloody diarrhoea and abdominal pain. Recent guidelines recommend ileocolonoscopy with biopsies as the most appropriate examination in the primary diagnosis of UC, with the exception of severe colitis with large ulcerations when complete colonoscopy may increase the risk of complica- tions25-27. The endoscopic findings in UC typically start in the rectum and may extend more proximally. In mild disease activity, erythema, loss of vascular pattern and granu- larity of the mucosa are seen, whereas in severe disease ulcerations and spontane- ous bleeding occur. Correlation between symptoms and disease activity in UC is quite

12 introduction and outline of the thesis

reliable, which can be very helpful in assessing clinical remission and the efectiveness of therapy. Radiological techniques have a limited role in the diagnosis of UC. Plain X rays of the abdomen or CT scanning may suggest colitis, but endoscopy and histology remain the gold standard.

Crohn’s disease Crohn’s disease presents more heterogeneously than UC, with regard to disease location in that it can develop anywhere in the gastrointestinal tract, as well as with regard to disease behaviour and perianal manifestations. Symptoms associated with CD vary greatly between heavy abdominal pain, bloody diarrhoea and weight loss, to mild abdominal tenderness, while some patients may even be symptom-free. Diferent clinical CD phenotypes are currently classified according to the Montreal classification23. A clear explanation for the variation in clinical presentation and disease phenotype of CD is lacking. Unfortunately, up till now, little attention has been given to the potential immunological pathways underlying diferent CD phenotypes. Interest- ingly, it has recently been demonstrated that colonic CD is associated with a diferent genetic profile compared with ileal CD and therefore it has been suggested to be a genetic intermediate between ileal CD and UC28. At present, complete ileocolonoscopy with biopsies is the recommended examination for diagnosing CD26,29, in combination with upper GI endoscopy and radiological imaging of the small bowel in all newly diagnosed patients. Endoscopically, CD is characterized by discontinuous lesions, such as erythema, and ulcerations that can vary in size between small aphthous ulcers and large longitudinal ulcerations forming the characteristic “cobble stone” appearance, separated by areas of normal tissue30. The histopathological changes of IBD can be subdivided in two sections: architectural changes and inflammatory features. Architectural abnormalities include: crypt distortion crypt branching, decreased crypt density and irregular mucosal surface. Inflammatory features consist of: increased lamina propria cellularity, large numbers of neutrophils especially organized in crypt abscesses and epithelioid granulomas31. Most of these features occur in both CD and UC, but have quite diferent distribution and localization: difuse and superficial in UC versus discontinuous and transmural in CD32. Furthermore, granulomas, when non-IBD aetiology has been excluded, are thought to be an exclusive CD characteristic, being present in 21-37% of the endoscopic biopsies31,33.

Imaging in Crohn’s disease Assessment of the small bowel is possible with diferent techniques, such as balloon- assisted enteroscopy, wireless capsule endoscopy (video capsule endoscopy) and radiological imaging. Single or double balloon enteroscopy combines the endoscopic visualisation of the entire small bowel with the possibility of taking biopsies, however, the invasive character and length of the procedure limits its clinical use in the primary diag-

13 chapter 1

nosis of CD. Video capsule endoscopy allows visualisation of the entire small bowel with minimal discomfort but without the ability for tissue sampling. Notably, because of the risk of capsule retention in a stenotic small bowel segment, capsule endoscopy should be preceded by cross-sectional imaging techniques in CD patients. Cross sectional imaging techniques, such as abdominal ultrasonography (US), Magnetic Resonance Imag- ing (MRI) and Computed Tomography (CT) are being recommended in order to stage the disease and monitor follow-up. Several guidelines have stated that MR Enterography (MRE) might be the most useful imaging modality to assess bowel damage given its high contrast resolution, lack of ionizing radiation and ability to cover the whole abdo- men26,29,34. However MRI is a costly technique requiring elaborated protocols that make quantification of active inflammation, by scoring the contrast uptake, rather complex and time-consuming. Newer, less expansive options have emerged, such as contrast- enhanced ultrasound (CEUS), that allow visualization and quantification of bowel wall perfusion have emerged35. The diagnostic accuracy of CEUS compared to MRE, and its place in the diagnostic work-up of CD patients, still needs to be studied. Systematic research on the frequency and the clinical impact of findings during upper gastrointestinal (GI) endoscopy and small bowel imaging in newly diagnosed adult CD patients is lacking. Nevertheless recent European guidelines recommend routine upper GI endoscopy and small bowel imaging, such as Magnetic Resonance Enterogra- phy (MRE) in all newly diagnosed CD patients irrespective of the findings by ileocolonos- copy29. These recommendations are merely based on expert opinion as no studies have addressed the aspects of the diagnostic algorithm in adult IBD patients.

Immunological aspects

Whether diferent IBD phenotypes are fundamentally diferent nosological entities or part of a mechanistic continuum is still an unanswered question, with both conceptual and therapeutic implications. The most important factors involved in the aetiology of IBD are the genetic makeup, the environment, gut microbiota and immune respons- es36-38. Interaction between these components ultimately leads to chronic inflammation. The study of immune abnormalities in IBD has initially concentrated on the adaptive immune system and lately on innate immunity39. Based on available research, it is nowadays thought that the recognition of commensal-derived antigens by the adaptive immune system and its continuous stimulation by the innate immune system play a key role in the immunopathogenesis of IBD36,39. The present knowledge of immunopathogenesis originates mostly from research on genetically engineered animal models and patients with established, longstanding disease under anti-inflammatory treatment. In particular, inducible and adoptive transfer mouse models of intestinal inflammation have shown the key role of the efector T lymphocytes for the inflammatory process

14 introduction and outline of the thesis

in the gut38,39. Both T helper 1 (Th1) and Th2 T-cell subsets have been found to cause chronic gut inflammation, with CD having a predominantly Th1 cytokine profile (IFN- gamma, IL12, IL23, TNF-alpha) and UC a predominantly Th2 profile (IL-4, IL-13)40. More recently data has also emerged on the efects of other T-cell subsets (regulatory T cells, Th17, cytotoxic T cells) as well as endothelial, epithelial and mesenchymal intestinal cells in the pathogenesis of IBD41,42. In order to understand the complex initial interaction between microbiota, the immune system and genetic background, one might start by studying the diferent immune cell subsets in the gut of newly diagnosed, untreated patients. Information on the early immunologic dysregulation and potentially diferent immunologic pathways between patients may deliver new predictive factors for the course of the disease and response to therapy.

Early IBD as research model

The complex clinical and pathological nature of IBD, as well as its chronic unpredictable course, makes it an extremely interesting research model. The most important questions arise at the beginning of the disease course and have implications for the whole course of the disease. Which diagnostic modalities are needed in an individual patient in order to establish a certain diagnosis as well as the severity and extent of the disease? Is there an immuno-pathological explanation for diferences in clinical phenotypes at diagnosis? Could we identify integrated clinical and immuno-pathological phenotypes at initial presentation that might predict disease course and response to therapy? In order to study these questions one must consider early IBD as a research model, including the study of early immunological changes without the disadvantage of immunosuppression. In this thesis we wanted to focus on diagnostic aspects in the early phase of IBD, integrating both clinical and immuno-pathological characteristics. The heterogeneity of the clinical phenotype by presentation requires a diferent diagnostic work-up for diferent phenotypes and might rely on diferent immunological mechanisms. Our hypothesis was that initial diagnostic strategies could be individualized according to clinical phenotype at presentation and that integration of clinical and immuno-pathological features might identify diferent immune-based phenotypes with potential value in prognosis and guidance for treatment options.

Research questions In order to test the hypothesis we formulated the following research questions: 1 Which diagnostic modalities have been used over time in the primary diagnosis of IBD patients in daily practice and what is the current diagnostic work-up? (Chapter 2)

15 chapter 1

2 What is the prevalence of upper GI lesions in adult IBD patients at first presentation and are there discriminating features between phenotypes? (Chapter 3) 3 What is the additional value of small bowel imaging in diferent CD phenotypes at first presentation and which modality is most suitable at presentation and during follow-up? (Chapter 4,5) 4 Can we identify diferent immunological phenotypes in the inflamed gut mucosa of early IBD? (Chapter 6) 5 How do naive T cells enter the inflamed mucosa of IBD patients? (Chapter 7,8)

Diferent study models To answer the first question, we have performed a retrospective cohort analysis of the diagnostic modalities used in the IBD population of a secondary care hospital over the last four decades. To answer the second question, a prospective cohort study was performed in a consecutive group of newly diagnosed adult IBD patients before initiating immunosuppressive medication. We chose for this approach in order to study the prevalence of upper GI involvement without the influence of immunosuppression and to identify possible discriminating features between CD and UC at the time of primary diagnosis. We addressed the third question by performing two studies. First, we examined the additional value of small bowel imaging by MRE at the time of primary diagnosis next to ileocolonoscopy in a retrospective observational cohort study. Recent European guidelines recommend small bowel imaging in all newly diagnosed CD patients independent of ileocolonoscopy. By identifying clinical phenotypes at presentation where small bowel imaging might add new findings or have no additional value next to ileocolonoscopy, we might individualize the diagnostic work-up and increase the adherence to guidelines in daily practice. Secondly, we studied the diagnostic accuracy of CEUS compared to MRE in the assessment of terminal ileitis by performing a prospective cohort study in CD patients, using ileocolonoscopy as a reference standard for active inflammation. In order to answer the fourth question we performed a prospective patient-control study of diferent immunological subsets in the gut mucosa of newly diagnosed IBD patients before start of medication and healthy controls. We focused on diferent lymphocyte subsets and T-cell maturation status. The fifh question was arrived at afer observation of high numbers of naïve and central memory T cells in a subgroup of the patients. We first performed a review of the current literature on the presence and homing process of naïve and memory T cells into the gut mucosa. Tertiary lymphoid organs (TLOs) have been suggested to play a role in the homing process of naïve T cells in other systemic diseases and malignancies, however this has not yet been studied in IBD patients.

16 introduction and outline of the thesis

We therefore chose to study the presence of TLOs in the biopsy specimens of the newly diagnosed IBD patients and their correlation with the presence of naïve and central memory T cells.

Outline of the thesis

The heterogeneity and unpredictability of IBD makes clinical decision-making difcult. Combining the clinical and immunological characteristics of diferent phenotypes early in the disease course might help target clinical decision-making and facilitate faster and more efective medical care. The aim of this thesis is to provide evidence for an individualized diagnostic work-up at first presentation and help develop a classification of specific disease phenotypes that enable clinicians to apply more focused, personalized medicine in this heterogeneous population of IBD patients. The first part of the thesis focuses on clinical aspects of the primary diagnosis in IBD. Chapter 2 provides a historical overview of the changes in endoscopic and radiologic examinations for diagnosing IBD. The prospective study on the endoscopic and histologic alterations during upper GI endoscopy in newly diagnosed IBD patients is presented in Chapter 3. In Chapter 4, the findings of MRE alongside those of ileocolonoscopy in the primary diagnosis of dif- ferent subgroups of CD patients, are provided. In Chapter 5, a prospective comparison is presented between contrast enhanced abdominal ultrasound (CEUS) and MRE for the detection of disease activity in the terminal ileum of CD patients. The second part of the thesis focuses on immunological characteristics in the gut mucosa of newly diagnosed, untreated IBD patients. In Chapter 6 the distribution of diferent T-cell subsets in the mucosa of newly diagnosed IBD patients, including an increased number of naïve and central memory T cells in a subgroup of patients, is reported. Chapter 7 is a review of the migration pathways of naive T cells and their potential role in the inflammatory process in IBD. Finally, Chapter 8 focuses on the presence of ectopic lymphoid organs, so called tertiary lymphoid organs, that may play a role in the accumulation of naive and central memory T cells in inflamed gut mucosa.

17 chapter 1

References

1 Shanahan F, Bernstein CN. The evolving epidemiology of inflammatory bowel disease. Gastroenter- ology. 2009; 25:301–305. 2 Loddo I, Romano C. Inflammatory bowel disease: genetics, Epigenetics, and pathogenesis. Front Immunol. 2015; 6:551. 3 Sainio P. Epidemiology. In: Robin KS, Lunniss PJ. Anal fistula. London: Chapman & Hall, 1996:1–11. 4 Kirsner JB. Historical origins of current IBD concepts. WJG 2001 5 Sydenham T. Complete works ”History and cures of acute diseases and the shortest and safest way of curing most chronical disease” 3rd ed. Corrected from the original Latin by John Pechey. London: R. Wellington; 1701. 6 Wilks Sir S: The morbid appearance of the intestine of Miss Banks. Medical times & Gazette, 2: 264, 1859, quoted by Crohn 7 Wilks Sir S, Moxon W: Lectures on Pathological Anatomy, 2nd ed, Churchill. London. p. 408, 1875, quoted by Goligher 8 Morgagni G. De sedibus et causis morborum per anatomen indagatis libri quinque Venice: typo- graphia Remondiniana; 1761. 9 Dalziel TK. Chronic interstitial enteritis. BMJ 1913;ii: 1068 -70. 10 Crohn BB, Ginzburg L, Oppenheimer GD. Regional ileitis: a pathologic and clinical entity. JAMA 1932:99. 11 Hawkins HP. An address on the natural history of ulcerative colitis and its bearing on treatment. The British Medical Journal 1909, March 27. 12 Ekbom A, Helmick CG, Zack M, Holmberg L, Adami HO. Survival and causes of death in patients with inflammatory bowel disease: a population-based study. Gastroenterology. 1992;103:954 –960. 13 Jess T, Winther KV, Munkholm P, Langholz E, Binder V.. Mortality and causes of death in Crohn’s disease: follow-up of a population-based cohort in Copenhagen County, Denmark. Gastroenterol- ogy. 2002;122:1808 –1814. 14 Winther KV, Jess T, Langholz E, Munkholm P, Binder V. Survival and cause-specific mortality in ulcerative colitis: follow-up of a population-based cohort in Copenhagen County. Gastroenterology. 2003;125:1576 –1582. 15 Lofus EV Jr, Silverstein MD, Sandborn WJ, Tremaine WJ, Harmsen WS, Zinsmeister AR. Ulcer- ative colitis in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and survival. Gut. 2000;46:336 –343. 16 Lofus EV Jr, Silverstein MD, Sandborn WJ, Tremaine WJ, Harmsen WS, Zinsmeister AR. Crohn’s disease in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and survival. Gastroen- terology. 1998;114:1161–1168. 17 Wolters FL, Russel MG, Stockbrugger RW. Systematic review: has disease outcome in Crohn’s disease changed during the last four decades? Aliment Pharmacol Ther. 2004; 20:483– 496. 18 Langholz E. Ulcerative colitis. An epidemiological study based on a regional inception cohort, with special reference to disease course and prognosis. Dan Med Bull. 1999;46:400–415. 19 Lofus EV. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology. 2004; 126:1504-1517. 20 Vind I, Riis L, Jess T, et al. Increasing incidences of inflammatory bowel disease and decreasing surgery rates in Copenhagen City and County, 2003-2005: a population-based study from the Danish Crohn colitis database. Am J Gastroenterol 2006;101:1274– 1282. 21 Cosnes J, Gower-Rousseau C, et al. Epidemiology and Natural History of Inflammatory Bowel Dis- eases. Gastroenterology 2011; 140:1785–1794.

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22 Lennard-Jones JE. Classification of inflammatory bowel disease. Scand J Gastroenterol 1989;/24 Suppl 170:/2-6. 23 Satsangi J, Silverberg MS, Vermeire S and Colombel J-F. The Montreal classification of inflammatory bowel disease: controversies, consensus and implications. Gut. 2006; 55:749-753. 24 Guindi M, Riddell RH. Indeterminate colitis. J. Clin. Pathol. 2004; 57:1233-1244. 25 Stange EF, Travis SPL, Vermeire S, et al. for the ECCO. European evidence based consensus on the diagnosis and management of ulcerative colitis: definition and diagnosis. Journal of Crohn’s and Colitis; 2008, 2:1-23. 26 Richtlijn Diagnostiek en behandeling van inflammatoire darmziekten bij volwassenen 2009. ISBN 978-90-8523-196-7. 27 Kornbluth A., Sachar DB and The Practice Parameters Committee of the American College of Gas- troenterology. Ulcerative Colitis Practice Guidelines in Adults: American College of Gastroenterol- ogy, Practice Parameters Committee. Am J Gastroenterol. 2010; 105: 501-523. 28 Cleynen I, Boucher G, Jostins L, et al. Inherited determinants of Crohn’s disease and ulcerative colitis phenotypes: a genetic association study. Lancet. 2016 Jan 9;387 (10014):156-67 29 Van Assche G, Dignass A, Panes J et al. The second European evidence-based Consensus on the diagnosis and management of Crohn’s disease: Definitions and diagnosis. J Crohn’s Colitis. 2010 Feb;4(1):7-27. 30 Annese V, Daperno M, Rutter MD et al. European evidence based consensus for endoscopy in inflammatory bowel disease. J Crohn’s Colitis 2013; 7: 982–1018. 31 Jenkins D, Balsitis M, Gallivan S et al. Guidelines for the initial biopsy diagnosis of suspected chronic idiopathic inflammatory bowel disease. The British Society of Gastroenterology Initiative. J Clin Pathol. 1997;50:93-105. 32 Geboes K1, Riddell R, Ost A, Jensfelt B et al. A reproducible grading scale for histological assessment of inflammation in ulcerative colitis. Gut. 2000 Sep; 47(3): 404-9. 33 Seldenrijk CA1, Morson BC, Meuwissen SG et al. Histopathological evaluation of colonic mucosal biopsy specimens in chronic inflammatory bowel disease: diagnostic implications. Gut. 1991 Dec; 32 (12):1514-20. 34 Rimola J, Rodriguez S, Garcia-Bosch O, Ordas I, Ayala E, Aceituno M, et al. Magnetic resonance for assessment of disease activity and severity in ileocolonic Crohn’s disease. Gut. 2009; 58:1113–1120. 35 Migaleddu V, Scanu AM, Quaia E et al. Contrast-enhanced ultrasonographic evaluation of inflammatory activity in Crohn’s disease. Gastroenterology 2009; 137:43–52. 36 Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshif mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001; 411 (6837): 603-6. 37 Geremia A, Biancheri P, Allan P et al. Innate and adaptive immunity in inflammatory bowel disease. Autoimmunity Reviews 2014; 13; 3-10. 38 Powrie F, Leach MW, Mauze S, et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity. 1994;1:553-562. 39 Kanai T, Kawamura T, Dohi T, et al. TH1/TH2-mediated colitis induced by adoptive transfer of CD4+CD45RBhigh T lymphocytes into nude mice. Inflamm Bowel Dis. 2006;12:89-99. 40 Neurath MF. Cytokines in inflammatory bowel disease. Nature Reviews 2014;14: 329-342. 41 Liu Y, Lan Q, Lu L et al. Phenotypic and functional characteristics of a newly identified CD8+ Foxp3- CD103+ refgulatory T cells. Journal of Molecular Cell Biology (2014), 6, 81 – 92. 42 Mei Lan Chen BS, Sundrud MS. Cytokine networks and T-cell subsets in Inflammatory Bowel Dis- eases. Inflamm Bowel Dis 2016; 22:1157-1167.

19 chapter 1

20 part I Clinical aspects of the primary diagnosis in Inﬂammatory Bowel Disease

21 chapter 2

Time trends in forty years of diagnostic procedures in Inﬂammatory Bowel Disease

(submitted)

CS Horjus Talabur Horje1

MGH van Oijen2

R. Bruijnen3

FBM Joosten3

MJM Groenen 1

PJ Wahab 1

DJ de Jong2

1 Department of Gastroenterology, Rijnstate Hospital, Arnhem, the Netherlands 2 Department of Gastroenterology and Hepatology, Nijmegen Medical Center, Radboud University Nijmegen, the Netherlands 3 Department of Radiology, Rijnstate Hospital, Arnhem, the Netherlands

Abstract background and aim The diagnostic work-up of Inﬂammatory Bowel Disease (IBD) patients has changed in the last decades. We aimed to study historical trends in the usage of diagnostic procedures over the last forty years and evaluate the present clinical standards for the primary diagnosis of Crohn’s disease and ulcerative colitis, in a Dutch secondary care teaching hospital. methods Patients from an administrative hospital database, with conﬁrmed Crohn’s disease (N=421) or ulcerative colitis (N=391), diagnosed between 1970 and 2007 were included. The number of endoscopic and radiological procedures at the time of the primary diagnosis were collected retrospectively and analysed for time trends. results In diagnosing CD, between 1970-1980, small bowel enteroclysis (SBE) and colonography accounted together for 71% of all diagnostic procedures. In the last period of time (2005-2007), the use of SBE and colonography was replaced by MRI enterography, and the use of endoscopy increased to 85% of all procedures. In diagnosing UC, between 1970-1980, colonography accounted for 33% of all diagnostic procedures. In the course of time the use of colonography was totally replaced by endoscopy. conclusion We describe a considerable change in the use of procedures for the primary diagnosis of both CD and UC patients, in the last forty years. At present, ileocolonoscopy and MRI enterography account for the majority of diagnostic modalities, which is in line with recommendations from recent IBD guidelines.

25 chapter 2

Introduction

Chronic inflammatory bowel disease (IBD) consists of two major clinical entities known as Crohn’s disease (CD) and ulcerative colitis (UC), with a label of IBD “unclassified” (IBDU) or indeterminate colitis (IC) when there is difculty diferentiating between CD and UC, before or afer bowel resection, respectively. Since the first description of the disease as “chronic interstitial enteritis” in 1913 by Dalziel et al., and later as “regional enteritis” in 1932 by Crohn et al., there has been ongoing research regarding the etiology of these chronic entities as well as the appropriate approach to their diagnostic investigation and management1,2. During the last decades, a relatively large number of population-based studies on the incidence, course and prognosis of the disease have been carried out, showing an increasing incidence of IBD reaching incidence rates ranging from 0.7 to 14.6/100,000 for CD, and from 1.5 to 24.5/100,000 for UC, varying greatly between regions3-12. Only few studies have addressed the aspects of the diagnostic algorithm in IBD. An exact diagnosis and subclassification is of great importance but remains difcult in up to 10-15% of patients, recommending a systematic follow-up with a re-evaluation of the diagnosis in this group of patients13,14. A single gold standard for diagnosing IBD is not available and therefore the diagnosis is based on a combination of clinical, endoscopic, histologic and radiologic criteria15. A systematic description of the diagnostic procedures used in the primary diagnosis of IBD is not available and most of the national and international recommendations and evidence based guidelines, on diagnosing IBD are very recent16-20. We believe that the choice of the endoscopic and radiologic procedures used for diagnosing IBD, have changed in time, depending on the technical innovations made in these fields and also on the feasibility and availability of the diagnostic methods in a general secondary care hospital. A view of the time trends in the use of the procedures in the primary diagnosis of IBD may give us a better understanding of the history of the diagnostic process of this disease and help us implementing future diagnostic guidelines with new modalities. The aim of this study is to examine the changes in the usage of diagnostic procedures over the last nearly forty years, the relation between the diagnostic procedures used and reclassification of the diagnosis and to evaluate the present clinical standards in the primary diagnosis of Crohn’s disease (CD) and ulcerative colitis (UC), in a Dutch secondary care teaching hospital.

Materials and methods

Study setting and Patients Inpatients and outpatients who were diagnosed with Crohn’s disease (CD) or ulcerative colitis (UC) in the Rijnstate Hospital (Arnhem, the Netherlands) in the period between

26 time trends in the primary diagnosis of ibd

1970 and 2007 were identified using the diagnostic codes in the administrative hospital database. Chart review of all these patients was undertaken retrospectively. Individual information on sex, age, date of diagnosis, smoking habits, initial symptoms and disease related characteristics at diagnosis according to the Montreal classification, as well as the endoscopic and radiologic procedures used in the primary diagnosis of IBD were recorded onto a standard proforma by detailed case-note review. Patients diagnosed with incomplete data over the diagnostic procedures at the time of the primary diagnosis were excluded Figure 1. The Montreal classification includes a classification for Crohn’s disease by disease location in L1 (ileal), L2 (colonic), L3 (ileocolonic) and +L4 (upper intestinal disease) which can be added to any of the other three locations, and by disease behaviour in B1 (non- stricturing, non-penetrating), B2 (stricturing), B3 (penetrating), with +P as perianal disease modifier, that can be added to any of the other phenotypes. Based on the distribution of the disease (Montreal classification) ulcerative colitis has been classified in proctitis (E1, limited to the rectum), lef-sided colitis (E2, limited to the colon distal the splenic flexure) and extensive colitis (E3, involvement extending proximal to the splenic flexure, including pancolitis)16. The time of primary diagnosis was defined as the date of the procedure, which revealed objective lesions positive for IBD, for the first time. Furthermore, all examinations within one month from the time of primary diagnosis were regarded as procedures at diagnosis.

Figure 1 Inclusion diagram

Administratieve database N=995

Excluded Incomplete data N=147 IBD unclassiﬁed N=36

Initial diagnosis Initial diagnosis Crohn’s disease Ulcerative colitis N=380 N=432

N=41

Deﬁnitive diagnosis Deﬁnitive diagnosis Crohn’s disease Ulcerative colitis N=421 N=391

27 chapter 2

The diagnosis of CD was based on the presence of two or more of the following criteria: (I) history of abdominal pain, diarrhoea and weight loss; (II) macroscopic appearance at endoscopy (or operation) showing segmental, discontinuous lesions with or without rectal involvement, aphtous ulceration, fissuring and penetrating ulceration, cobble- stones or strictures; (III) radiological evidence of stenosis or mucosal inflammation in the small bowel, segmental colitis, or fistulae; (IV) microscopic evidence compatible with CD including epithelial granulomas and transmural inflammation. The diagnosis of UC was based on the presence of three or more of the following criteria: a history of diarrhoea and/or bloody stools; macroscopic appearance on endoscopy of continuous mucosal inflammation afecting the rectum extending to some of all of the colon; microscopic features on biopsy compatible with UC; and no suspicion of CD on biopsy, ileocolonoscopy and small bowel imaging.

Diagnostic procedures The endoscopic procedures were done using standard flexible video endoscopy. An ileocolonoscopy is defined as the endoscopic procedure with intubation of the colon and terminal ileum, while during a colonoscopy and sigmoidoscopy the colon intubation reaches the coecum or the descending colon, respectively. The small bowel radiologic examination with barium sulfate suspension was done using the enteroclysis technique (nasal intubation with a small-bore enteric tube, di- rected by fluoroscopic guidance into distal duodenum or jejunum). The colonography was performed using the double-contrast method obtained by combined use of barium suspension and air. The magnetic resonance of the small bowel was performed using the per-oral method, which was done afer a period of 5h fasting and ingestion of 1 liter of a 6% mannitol solution in the last hour before the MRI. The MRI protocol consisted of multiple pre- and post-contrast sequences, with fat saturation afer intravenous administration of gadolinium DTPA (Dotarem; Guerbet Nederland B.V.). In our hospital, computed tomography enterography (CTE) is only used in patients with absolute contraindications for MR enterography.

Statistical analysis All data were entered in a structured database, and were extracted for analysis with SPSS statistical sofware, version 15.0. Frequency tables were provided for demographics and clinical characterization (including Montreal classiﬁcation) at the time of diagnosis, and presented for either Crohn’s disease or ulcerative colitis over ﬁve diferent time cohorts: 1970-1979, 1980-1989, 1990-1999, 2000-2004 and 2005 -2007. The period between 2000-2007 was divided in two because of the introduction of the MR enterography in 2005. The median number and type of diagnostic procedures were assessed over the same time cohorts and plotted over time for both Crohn’s disease and ulcerative colitis.

28 time trends in the primary diagnosis of ibd

Finally, we analyzed all patients that were reclassified from ulcerative colitis to Crohn’s disease, or vice versa, during the course of disease. For characterization of all reclassified patients we described demographics, clinical characterization and diagnostic procedure used at time of diagnosis, followed by the procedures used for reclassification.

Results

We identified a total of 995 patients diagnosed with IBD during the study period. A number of 147 patients were excluded because of incomplete data and 36 (3%) patients received the diagnosis IBD unclassified (IBDU) Figure 1. The definitive diagnosis of CD was made in 421 patients (52%), and UC in 391 (48%) patients. In the course of time we observed an increase in the number of newly diagnosed patients, as well as a higher age at the time of diagnosis, in both Crohn’s disease and ulcerative colitis Table 1.

Findings in patients diagnosed with Crohn’s disease The mean age at diagnosis of the 421 patients with CD was 34 years (SD 14) of whom 63% were female. Regarding disease location, we found an increase in the number of patients with disease location in the upper digestive tract Table 1. In the seventies, the radiological procedures such as small bowel enteroclysis and colonography were mostly used in diagnosing CD Figure 2a. Sigmoidoscopy was used in 53% of the patients, being the only available endoscopic procedure at that time Table 2. In the eighties, the complete colonoscopy was performed in 13% of the patients, followed by a decrease in the use of small bowel enteroclysis and colonography, in favour of the endoscopic methods, in the nineties Table 2. In the last period, small bowel

Figure 2a Time trends in the use of radiological procedures in diagnosing Crohn’s disease

100 MRI enterography 90 80 colonography 70 SBE 60 50 40 30 20 10 0 1970-1979 1980-1989 1990-1999 2000-2004 2005-2007

29 chapter 2

Table 1 Demographic and clinical characteristics of patients diagnosed with IBD during the study period

1970-1979 1980-1989 1990-1999 2000-2004 2005-2007

CD UC CD UC CD UC CD UC CD UC

n=15 n=12 n=40 n=44 n=123 n=119 n=133 n=129 n=110 n=87

F/M 11/4 10/2 27/13 23/21 84/39 55/64 77/56 69/60 66/44 40/47

Age at diagnosis (%)

A1 (<16) 1 (7) 1 (8) 3 (8) 1 (3) 6 (5) 3 (2) 2 (2) 8 (6) 1 (1) 0

A2 (17-40) 14 (93) 9 (75) 29 (73) 27 (61) 81 (66) 58 (49) 88 (66) 59 (46) 64 (58) 39 (45)

A3 (>40) 0 2 (17) 8 (20) 16 (36) 36 (29) 58 (49) 43 (32) 62 (48) 45 (41) 48 (55)

Extend of CD at diagnosis (%)

L1 4 (25) 24 (60) 49 (40) 35 (26) 31 (28)

L2 8 (53) 8 (20) 58 (47) 58 (44) 49 (45)

L3 4 (25) 7 (18) 16 (13) 38 (29) 30 (27)

+L4 0 1 (3) 5 (4) 18 (14) 10 (9)

+P 2 (13) 5 (13) 22 (18) 13 (10) 11 (10)

Disease behavior of CD at diagnosis (%)

B1 8 (53) 19(48) 75 (61) 79 (59) 79 (72)

B2 6 (40) 14 (35) 24 (19) 40 (30) 18 (16)

B3 0 3 (8) 18 (15) 9 (7) 11 (10)

B4 1 (7) 3 (8) 7 (6) 3 (2) 3 (3)

Location of UC at diagnosis (%)

E1 9 (75) 17 (39) 37 (31) 41 (32) 32 (37)

E2 1 (8) 23 (52) 59 (50) 62 (48) 36 (41)

E3 1 (8) 4 (9) 23 (19) 26 (20) 19 (22)

Smoker (%) 10(67) 5 (42) 17(43) 7 (16) 51 (41) 9 (8) 45 (34) 12 (9) 40 (36) 8 (9)

enteroclysis was replaced by MR Enterography Figure 2a. Regarding the endoscopic procedures, we observed an obvious growth in the number of patients undergoing an (ileo)colonoscopy (83% between 2005-2007), as well as an increase in the use of upper endoscopy (20% between 2005-2007) Table 2, Figure 2b.

30 time trends in the primary diagnosis of ibd

Figure 2b Time trends in the use of endoscopic procedures in diagnosing Crohn’s disease

100 upper endoscopy 90 80 sigmoidoscopy 70 colonoscopy 60 50 ileocolonoscopy 40 30 20 10 0 1970-1979 1980-1989 1990-1999 2000-2004 2005-2007

Table 2 Diagnostic procedures per number of patients, during the study period

1970-1979 1980-1989 1990-1999 2000-2004 2005-2007

CD UC CD UC CD UC CD UC CD UC Total number N=15 N=12 N=40 N =44 N=123 N 119 N=133 N=129 N=110 N =87 of patients

Sigmoidoscopy 8 (53) 10 (83) 16 (40) 38 (86) 49 (40) 89 (75) 37 (28) 95 (74) 18 (16) 59 (68) (%)

Colonoscopy (%) 0 0 5 (13) 7 (16) 32 (26) 18 (16) 42 (32) 18 (14) 20 (18) 15 (17)

Ileocolonoscopy 0 0 0 0 16 (13) 3 (3) 43 (32) 13 (11) 72 (65) 13 (15) (%)

Upper endoscopy 0 0 1 (2) 0 13 (11) 0 23 (17) 0 22 (20) 0 (%)

SBE (%) 9 (60) 0 30 (75) 0 55 (45) 0 67 (50) 0 7 (6) 0

Colonography (%) 11 (73) 5 (42) 15 (37) 18 (41) 30 (24) 20 (17) 14 (10) 14 (11) 0 2 (2)

MR enterography 0 0 0 0 0 0 1 (1%) 0 18 (16) 0 (%)

Median number 2 (2) 1,5 (2) 2 (2) 1 (2) 2 (3) 1 (2) 2 (3) 1(1) 2 (2) 1 (1) of procedures/ patient (Range)

31 chapter 2

Figure 3 Time trends in the use of radiologic and endoscopic procedures in diagnosing ulcerous colitis

100 sigmoidoscopy 90 80 colonoscopy 70 ileocolonoscopy 60 50 colonography 40 30 20 10 0 1970-1979 1980-1989 1990-1999 2000-2004 2005-2007

Findings in patients diagnosed with ulcerative colitis The mean age at diagnosis of the 391 patients with UC was 41 years (SD 16), of whom 50% were female. In the course of time, we noticed a growing number of patients with an extensive UC at the time of diagnosis Table 1. The number of patients diagnosed by colonography decreased from 42% in the seventies, to 2% in the most recent period of time. Between 2005-2007, 32% of the patients were diagnosed by a (ileo)colonoscopy and 68% by a sigmoidoscopy Table 2, Figure 3.

Findings in patients diagnosed with IBD unclassiﬁed The mean age at diagnosis of the patients diagnosed with IBDU was 39 (SD 11) of whom 12 were female. IBDU was diagnosed between 2005-2007. A number of 20 patients were diagnosed by ileocolonoscopy, 4 patients by colonoscopy and 2 patients by sigmoidoscopy. In 2 patients a MRE was performed, and other 2 patients underwent a gastro- scopy. None of the patients diagnosed with IBDU has been reclassiﬁed during follow-up.

Reclassification of the diagnosis during follow-up A total of 41 patients were reclassified from the diagnosis ulcerative colitis into Crohn’s disease. Most of these patients were diagnosed in the nineties (51%) and the median value of the period between the primary diagnosis and reclassification was 5 years with a range 1-32 years. A total of 33 patients (80%) were diagnosed at first by sigmoidoscopy alone, or in combination with colonography or small bowel enteroclysis. Most patients (n=26, 63%) were reclassified by use of ileocolonoscopy Table 3.

32 time trends in the primary diagnosis of ibd

Table 3 Reclassiﬁcation of the diagnosis from UC into CD

Number (%)

Female/male ratio 25/16

Age at diagnosis (mean) 35 years (SD 15)

Date of primary diagnosis

1970-1979 2 (5) 1980-1989 5 (12) 1990-1999 21 (51) 2000-2004 8 (20) 2005-2007 5 (12)

Period of time till reclassiﬁcation (median) 5 years (Range 1-32)

Diagnostic procedures at primary diagnosis

Sigmoidoscopy 21 (51) Sigmoidoscopy + colonography 9 (22) Sigmoidoscopy+ SBE 3 (7) Colonoscopy 3 (7) Colonoscopy +colonography 2 (5) Colonography 3 (7)

Diagnostic procedures at reclassiﬁcation

Ileocolonoscopy 26 (63) Upper endoscopy 2 (5) Sigmoidoscopy 4 (10) SBE 3 (7) MRE 2 (5) Surgery 4 (10)

Discussion

In this retrospective study, over a period of almost forty years we reported a clinical significant change in the use of diagnostic modalities for the primary diagnosis of IBD. According to our results, forty years ago CD was mostly diagnosed using radiologic procedures, such as small bowel enteroclysis and colonography, while combining colonography with sigmoidoscopy made the diagnosis of UC. In the last twenty-five years technical innovations have led to an expansion in the use of flexible video-endoscopy. Starting in the nineties there is an unmistakable increase in the use of (ileo)colonoscopy, although in a minority of patients the primary diagnosis of colonic CD is still made by sigmoidoscopy. Furthermore we observed a growing use of upper endoscopy

33 chapter 2

at the time of primary diagnosis of CD, which could form an explanation for the higher number of patients diagnosed with an additional location in the upper digestive tract. Regarding UC, in the last period the use of (ileo)colonoscopy has completely replaced the colonography, while a relative high number of patients (68%) were still being diagnosed by sigmoidoscopy alone. Recent American guidelines recommend sigmoidoscopy as well as colonoscopy in the primary diagnosis of UC17. In our study 10% of the UC patients were later reclassified as having Crohn’s disease. 80% of these patients were initially diagnosed by using sigmoidoscopy alone or in combination with SBE or colonography. In the majority of these patients the findings by ileocolonoscopy decided the definitive classification. These data support the recommendation in the most recent European guidelines that ileocolonoscopy is needed in the work-up of all patients suspected of IBD18-22. Sigmoidoscopy alone or in combination with radiological procedures, represents an incomplete diagnostic work-up in the primary diagnosis of IBD. The group of patients with endoscopic and histopathological findings that cannot diferentiate between CD and UC should be diagnosed as IBD unclassified (IBDU), according to the Montreal classification. In our population the group of IBDU patients turned out to be small, as the diagnosis was only made afer 2004. The clinical relevance of an accurate classification lies also in the fact that a number of patients with IBDU could later turn out to have a diferent type of colitis due to infections of drugs23-24. At present, endoscopic examination has become the gold standard in the diagnosis of IBD as it provides direct macroscopic and histologic information for evaluating the colon, the terminal ileum and upper digestive tract. According to most recent Dutch recommendations, initial diagnostic colonoscopy should intubate the terminal ileum and include systematic biopsies from each anatomic segment of the bowel (rectum, sigmoid, lef, transverse and right colon, and ileum) while upper endoscopy is still not recommended in all patients but is reserved for symptomatic patients. However some authors, including recent European guidelines regard upper-gastrointestinal Crohn’s disease as a challenging and underestimated diagnostic problem, and claim that all newly diagnosed patients with Crohn’s disease should undergo upper endoscopy25. Furthermore, recent European guidelines recommend small bowel imaging in all newly diagnosed CD patients irrespective of ileocolonoscopy for assessment of disease extent and eventual complications. Such general, extensive diagnostic work-up is based on merely expert opinion and has not yet been proven to be clinical relevant or cost-efective in the daily practice26,27. The role of newer techniques for examination of the small bowel, such as video capsule examination and balloon-assisted enteroscopy in the primary diagnosis of CD, is quite limited at the moment. The true benefit of the video capsule examination is not yet clear, as there are presently no standardized criteria for the diagnosis of CD by using this investigation28. Balloon-assisted enteroscopy allows visualization of the small bowel with the possibility of obtaining biopsy samples and eventual endoscopic

34 time trends in the primary diagnosis of ibd

therapy (e.g. dilatation of stricture, cauterization of a bleeding site). In a recent paper complete enteroscopy rates of 66% and 22 % for double-balloon and single-balloon enteroscopy were reported, respectively29. Therefore double-balloon enteroscopy has been recommended as the non-surgical gold standard procedure for deep small-bowel endoscopy29,30. However balloon-assisted enteroscopy is still not generally available and regarded as an invasive endoscopic procedure. The diagnosis of small intestinal CD complications relies mostly on radiologic imaging modalities. Small bowel enteroclysis is widely available and historically the most used technique for detection of small bowel lesions when CD is suspected30. However small bowel enteroclysis has major disadvantages, as it is invasive, burdensome and accompanied by a relatively high dose of radiation exposure. Furthermore the detection of the small bowel wall alterations is indirect, without providing any information about the extra-intestinal abdomen (visceral fat, the lymph nodes and the vascular structures) or possible complications. In our cohort, MR enterography has replaced small bowel enteroclysis, in the last 3 years. The high tissue contrast obtained using the MRI, combined with the absence of ionizing radiation, makes it suitable for repetitive imaging in patients with Crohn’s disease. At present the work-up in the primary diagnosis of an IBD patient includes an ileocolonoscopy and in patients suspected of small bowel CD an additional cross-sectional imaging modality such as CT enterography or MR enterography. Both radiological methods are very accurate for the detection of IBD of the small bowel, but the use of the CT is limited by the ionizing radiation needed, especially in the follow-up of young patients31. Recently CT enterography was demonstrated to be a cost-efective alternative for small bowel follow-through in diagnosing small bowel Crohn’s disease. Although they did not study MR enterography, a fundamental ﬁnding was that the accuracy and not the direct costs of a procedure, is the primary driver of the cost-efectiveness of a diagnostic test for Crohn’s disease32. Careful evaluation of disease characteristics at baseline is essential for diferential diagnosis and assessment of disease severity and behaviour, requiring a combination of endoscopic and cross-sectional modalities33. However the heterogeneity of the clinical presentation of IBD patients suggests that diagnostic work-up might be individualized according to clinical phenotype34. In conclusion, this study demonstrated strong time trends in the primary diagnostic process of IBD, in the period before publication of the most national and international guidelines. Recent guidelines generally recommend a complete assessment at baseline, endoscopic and radiologic, however without robust evidence of clinical beneﬁt or cost- efectiveness. Unanswered questions over the role of upper GI endoscopy or radiologic imaging in the early phase of IBD still need to be studied, in order to improve the diagnostic process and facilitate guidelines implementation in the daily practice.

35 chapter 2

References

1 Dalziel TK. Chronic interstitial enteritis. BMJ 1913;ii:1068-70. 2 Crohn BB, Ginzburg L, Oppenheimer GD. Regional ileitis, a pathologic and clinical entity. JAMA 1932:99. 3 Ekbom A, Helmick CG, Zack M, et al. Survival and causes of death in patients with inflammatory bowel disease: a population-based study. Gastroenterology. 1992; 103:954 –960. 4 Jess T, Winther KV, Munkholm P, et al. Mortality and causes of death in Crohn’s disease: follow-up of a population-based cohort in Copenhagen County, Denmark. Gastroenterology. 2002; 122:1808 –1814. 5 Winther KV, Jess T, Langholz E, et al. Survival and cause-specific mortality in ulcerative colitis: follow-up of a population-based cohort in Copenhagen County. Gastroenterology. 2003; 125:1576 –1582. 6 Lofus EV Jr, Silverstein MD, Sandborn WJ, et al. Ulcerative colitis in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and Survival. Gut. 2000; 46:336–343. 7 Lofus EV Jr, Silverstein MD, Sandborn WJ, et al. Crohn’s disease in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and survival. Gastroenterology. 1998; 114:1161–1168. 8 Ekbom A, Helmick C, Zack M, et al. Increased risk of large-bowel cancer in Crohn’s disease with colonic involvement. Lancet. 1990; 336: 357–359. 9 Jess T, Winther KV, Munkholm P, et al. Intestinal and extra-intestinal cancer in Crohn’s disease: follow-up of a population-based cohort in Copenhagen County, Denmark. Aliment Pharmacol Ther. 2004; 19:287–293. 10 Wolters FL, Russel MG, Stockbrugger RW. Systematic review: has disease outcome in Crohn’s disease changed during the last four decades? Aliment Pharmacol Ther. 2004; 20:483– 496. 11 Langholz E. Ulcerative colitis. An epidemiological study based on a regional inception cohort, with special reference to disease course and prognosis. Dan Med Bull. 1999; 46:400–415. 12 Lofus EV. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology 2004; 126:1504-1517. 13 Henriksen M., Jahnsen J. B Moum & The Ibsen Study Group. Change of diagnosis during the first five years afer onset of Inflammatory Bowel Disease: Results of a prospective follow-up study (the IBSEN study). Scand. J. Gastroenterol. 2006; 41:1037-1043. 14 Moum B, Ekbom A, Fausa O et al. Inflammatory bowel disease: re-evaluation of the diagnosis in a prospective population based study in south eastern Norway. Gut 1997; 40:328-332. 15 Lennard-Jones JE. Classification of inflammatory bowel disease. Scand J Gastroenterol 1989;/24 Suppl 170:/2-6. 16 Satsangi J, Silverberg MS, Vermeire S and Colombel J-F. The Montreal classification of inflammatory bowel disease: controversies, consensus and implications. Gut, 2006; 55:749-753. 17 Kornbluth A., Sachar DB and The Practice Parameters Committee of the American College of Gas- troenterology. Ulcerative Colitis Practice Guidelines in Adults: American College of Gastroenterol- ogy, Practice Parameters Committee. Am J Gastroenterol 2010; 105: 501-523. 18 Stange EF, Schreiber S, Fo¨lsch UR, et al. Diagnostik und Therapie des M. Crohn-Ergebnisse einer evidenzbasierten Konsensuskonferenz der Deutschen Gesellschaf fu¨r Verdauungs- und Stof- wechselkrankheiten. Z Gastroenterol 2003; 41:19–68. 19 Stange EF, Travis SPL, Vermeire S, et al. for the ECCO. European evidence based consensus on the diagnosis and management of Crohn’s disease: definition and diagnosis. Gut 2006; 55, i1-i15. 20 Stange EF, Travis SPL, Vermeire S, et al. for the ECCO. European evidence based consensus on the diagnosis and management of ulcerative colitis: definition and diagnosis. Journal of Crohn’s and Colitis; 2008, 2:1-23.

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21 Richtlijn Diagnostiek en behandeling van inflammatoire darmziekten bij volwassenen 2009. ISBN 978-90-8523-196-7. 22 Schussele Filliettaz S, Juillerat P, Burnand B, Vader JP et al. Appropriateness of colonoscopy in Eu- rope (EPAGE II) Chronic diarrhea and known inflammatory bowel disease. Endoscopy 2009; 41:218- 226. 23 Moum B, Vatn MH, Ekbom A, et al. Incidence of ulcerative colitis and indeterminate colitis in four counties of southeastern Norway, 1990-93. A prospective population-based study. Scand J Gastro- enterol. 1996;31:362–366. 24 Notteghem B, Salomez JL, Gower-Rousseau C, et al. What is the progosis in unclassified colitis? Results of a cohort study of 104 patients in Northern-Pas-de-Calais region. Gastroenterol Clin Biol. 1993;17:811–815. 25 Hommes DW, van Deventer SJ. Endoscopy in inflammatory bowel diseases. Gastroenterology 2004;126-1561-73. 26 Naganuma M, Hosoe N, Kanai T et al. recent trends in diagnostic techniques for Inflammatory Bowel Diseases. Korean J Intern Med, 2015; 30:271-278. 27 Haas K, Rubesova E, Bass D. Role of imaging in the evaluation of Inflammatory Bowel Diseases: How much is too much? World J Radiol 2016; 28; 8(2):124-131. 28 Triester SL, Leighton JA, Leontiadis GI et al. A meta-analysis of the yield of capsule endoscopy compared to other diagnostic modalities in patients with non-stricturing small bowel Crohn’s disease. Am J Gastroenterol 2006;101:954-64. 29 May A, Färber M, Aschmoneit I et al. Prospective Multicenter Trial Comparing Push-and-Pull Enter- oscopy with the Single- and Double-Balloon Techniques in Patients with Small-Bowel Disorders. Am J Gastronterol, 2010; 105(3):575-81. 30 Bernstein CN, Fried M. Krabshuis JH et al. World Gastroenterology Organization Practice Guidelines for the Diagnosis and Management of IBD in 2010. Inflamm Bowel Dis 2010; 16:112-124. 31 Horsthuis K, Stokkers PCF, Stoker J. Detection of inflammatory bowel disease: diagnostic performance of cross-sectional imaging modalities. Abdom Imaging 2008; 33:407-416. 32 Levesque BG, Cipriano LE, Chang SL et al. Cost-efectiveness of Alternative Imaging Strategies for the Diagnosis of Small-bowel Crohn’s Disease. Clin Gastroenterol and Hepatol, 2010; 8(3):261-7. 33 Papay P, Ignjatovic A, Karmiris K et al. Optimising monitoring in the management of Crohn’s disease: A physicians perspective. J Crohn’s Colitis 2013; 7:653-669. 34 Zhulina Y, Udumyan R, Tysc C et al. The changing face of Crohn’s disease: a population based study of the natural history of Crohn’s disease in Orebro, Sweden 1963-2005. Scand J Gastroenterol 2016; 51(3): 304-313.

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38 chapter 3

Prevalence of upper gastrointestinal lesions at primary diagnosis in adults with Inﬂammatory Bowel Disease

inflamm bowel dis. 2016 aug; 22(8): 1896-901

Carmen S. Horjus Talabur Horje1

Jos Meijer2

Lian Rovers3

Ellen G van Lochem1

Marcel JM Groenen1

Peter J Wahab1

1 Department of Gastroenterology and Hepatology, Rijnstate Hospital, Arnhem, the Netherlands. 2 Department of Pathology, Rijnstate Hospital, Arnhem, the Netherlands. 3 Department of Epidemiology and Statistics, Rijnstate Hospital, Arnhem, the Netherlands.

Abstract background The prevalence of upper gastrointestinal (GI) involvement in adult inflammatory bowel disease (IBD) has mostly been studied in patients with long-standing disease. The aim of this study was to prospectively evaluate the prevalence of upper GI involvement in a consecutive series of newly diagnosed, treatment-naive adult IBD patients, irrespective of upper GI tract symptoms. methods Consecutive patients with suspected IBD underwent combined ileocolonoscopy and upper endoscopy with biopsies. Patients diagnosed with either Crohn’s disease (CD) of ulcerative colitis (UC), denying use of NSAID were included in the study. Helico- bacter pylori infection was diagnosed histologically and positive patients were excluded from the analysis. Endoscopic and histologic lesions in the stomach and duodenum were recorded. Upper GI location (+L4) was defined as a combination of endoscopic and histological lesions. results A total of 152 patients, (108 CD and 44 UC) were analyzed. Endoscopic lesions were only seen in CD patients (60/108, 55%). Histological lesions were present in both CD and UC patients: focally enhanced gastritis in 58 CD (54%) and 10 UC (23%), granulomas in 30 CD (28%). Upper GI disease location was diagnosed in 44 CD (41%) and no UC patients. Upper GI tract symptoms were reported in 14/44 (32%) patients with upper GI location. conclusion A high prevalence of upper GI involvement was observed in newly diagnosed CD patients, with a majority of the patients being asymptomatic. Focally enhanced gastritis was common in both CD and UC patients, while granulomatous inflammation was restricted to CD.

41 chapter 3

Introduction

The clinical work up of newly diagnosed adult patients with Crohn’s disease (CD) has not yet been studied prospectively. Ileocolonoscopy with biopsies is the golden standard for establishing the diagnosis1-4. Whether upper endoscopy should be added to ileocolonoscopy in all patients, in order to examine the disease extent at primary diagnosis is still unclear. Recent European and American guidelines recommend further examination of the upper gastrointestinal (GI) tract in all newly diagnosed patients, irrespective of the findings at ileocolonoscopy in order to determine the location and extent of any CD le- sions1-3. However, evidence of frequency, type of lesions and clinical impact of these findings in newly diagnosed adult patients is missing. Recently it has been stated that nearly half of the IBD guideline recommendations are solely based on expert opinion5. CD is classically defined as an inflammatory bowel disease with fissural, granulomatous ulceration afecting any part of the digestive tract, while in ulcerative colitis (UC) more superficial inflammation is by definition limited to the colon-rectum4,6-8. The dif- ference in distribution throughout the intestinal tract is one of the major discriminators between CD and UC6,9-12. The paradigm regarding location and extent of these two diferent types of inflammation in Inflammatory Bowel Disease (IBD) is at present being challenged by recent reports of gastroduodenal lesions in adult patients with ulcerative colitis (UC)13. The so-called ulcerative gastroduodenal lesions (UGDL) or gastroduodenitis associated with UC (GDUC) appear incompatible with the original disease concept of UC being confined to the large intestine. On the other hand, these clinical entities seem exclusively to be associated with postcolectomy status and pouchitis13-15. In adult patients with Crohn’s disease (CD), the prevalence of upper gastrointestinal lesions irrespective of gastrointestinal (GI) symptoms, has been reported to be up to 16%16. The Montreal classification facilitates the classification of the upper GI involvement in CD, yet a clear definition and consensus on the macroscopic and microscopic lesions confirming the diagnosis is lacking17. In approximately 10-15% of patients with the primary diagnosis of idiopathic chronic colitis, it is difcult to distinguish between CD and UC, reflected by the diagnostic category IBD-unclassified (IBDU)12,17-18. It has been suggested that a routinely performed upper GI endoscopy at the time of primary diagnosis, may be useful in diferentiating between CD and UC. This requires data on the prevalence, endoscopic and histologic nature of upper GI findings in both CD and UC adult patients, at the time of primary diagnosis. Up till now, gastroduodenal endoscopic and histological lesions in treatment naïve newly diagnosed, adult IBD patients have not yet been sys- tematically studied. Available data in adult patients originate primarily from retrospective studies or case series performed in patients already using IBD- related anti-inflammatory medication such as 5-ASA compounds, corticosteroids and thiopurines19-24.

42 upper gastrointestinal lesions at primary diagnosis of ibd

The aim of the present study was to prospectively evaluate the prevalence of upper GI involvement in a consecutive series of newly diagnosed, treatment-naive adult IBD patients irrespective of upper GI tract symptoms.

Materials and Methods

Patients Consecutive, adult patients with symptoms of chronic diarrhea, rectal blood loss, abdominal pain or weight loss, underwent combined ileocolonoscopy and upper endoscopy. From this group, newly diagnosed CD and UC patients were included in the study. Use of non-steroidal anti-inflammatory drugs (NSAID) or proton pump inhibitors (PPI) during the preceding 4 weeks and diagnosis of IBD unclassified (IBDU) were considered exclusion criteria (Figure 1). The diagnosis of CD and UC was established and certified by clinical, endoscopic and histological standard criteria1,3,6. During ileocolonoscopy the type and distribution of the lesions were recorded and multiple biopsies were taken from ileum and colon in order to establish the diagnosis. Microscopic features such as difuse versus focal chronic inflammation and crypt disruption, superficial versus transmural, absence or presence of granuloma (not related to crypt injury) and presence of paneth cell metaplasia and crypt abscesses were used to diferentiate between UC and CD. Patients with inflammatory disease limited to the colon and no histologic criteria or other evidence to favor either CD or UC were diagnosed as IBDU. Data concerning age, sex, upper GI symptoms and smoking status were recorded.

Figure 1 Inclusion diagram of the patients

Suspected IBD N=235

Excluded

N=39 No informed consent N=16 No IBD diagnosis N=11 NSAIDs or PPI use N=6 IBDU diagnosis

Included IBD (CD or UC) N=163

43 chapter 3

Upper GI endoscopy and histology

The endoscopic examinations were performed under conscious sedation using a fore- head-view instrument (Olympus, type CF 180). During upper GI endoscopy, the presence of mucosal lesions in the esophagus, stomach and duodenum including granular, erythematous mucosa, erosions, ulcers, nodular lesions and strictures was assessed. Mucosal biopsy specimens were sampled before any anti-inflammatory (IBD) treatment. During upper endoscopy, four mucosal biopsies of normal gastric corpus and antrum and two biopsies of normal duodenal mucosa were taken for histopathological examination. Additional biopsies were taken from specific macroscopic lesions if observed. The tissue was fixed in 10% bufered formalin, routinely processed and stained with he- matoxylin and eosin. Presence of Helicobacter pylori (H. pylori) was identified on biopsy samples by Giemsa stain. Histological features analyzed were: acute or chronic inflammatory infiltrate, focal crypt destruction, ulceration, ‘focally enhanced gastritis’ (FEG) defined by the presence of a peri-foveolar or peri-glandular cellular infiltrate containing mononuclear cells and granulocytes, and epithelioid granulomas. An epithelioid cell granuloma was defined as a focal granulomatous inflammation, consisting of aggregation of macrophages that are transformed in epithelial-like cells. These epithelioid cells can occasionally fuse to form multinucleated giant cells and can be surrounded by a collar of lymphocytes. One pathologist who was blinded to the fi- nal diagnosis, highly experienced in histopathological evaluation of the gastrointestinal tract, examined each biopsy specimen.

Definitions

Upper GI disease location of CD, +L4 according to the Montreal classification, was defined based upon both macroscopic and microscopic criteria. Macroscopic criteria were erosions, aphthous lesions, ulcerations and strictures. Aspecific findings such as erythema and oedema were also recorded but were not seen as an independent criterion for the diagnosis of IBD. The diagnosis of upper GI CD was confirmed when the macro scopic findings were accompanied by microscopic features such as focal enhanced gastritis, focal crypt irregularity/crypt distortion, fissural ulceration and/or granuloma’s in the absence of H. pylori infection.

Statistical analysis Descriptive statistics of continuous data are reported as means or medians with standard deviations (SD) or interquartile ranges [IQR], respectively. Categorical data are reported as absolute numbers and percentages. All analyses were performed using SPSS Statistics [version 21 Chicago, IL USA].

44 upper gastrointestinal lesions at primary diagnosis of ibd

Ethical considerations Regional medical ethics committee approval was obtained prior to the study, and all patients provided written informed consent. The study was performed according to the principles of the Declaration of Helsinki.

Results

A total of 115 CD and 48 UC patients were included in the study. H. pylori infection was present in 7 (6%) CD and 4 (8%) UC patients and these patients were excluded from

Table 1 Baseline characteristics and Montreal classiﬁcation of patients at presentation

Patient characteristics Crohn’s disease Ulcerative colitis

Number of patients 108 44

Gender (male/female) 46/62 16/28

Age at diagnosis • A2 (between 17-40 years old) 67 20 • A3 (above 40 years old) 41 24

Disease location (N(%)) • L1 (small bowel only) 34 (32%) • L2 (large bowel only) 23 (21%) • L3 (small and large bowel) 51 (47%) • +L4 (upper GI) 44 (41%) • +P (perianal disease) 13 (12%) • E1 (proctitis) 8 (18%) • E2 (lef sided colitis) 8 (18%) • E3 (pancolitis) 28 (64%)

Disease behaviour (N(%)) • B1 (non-stricturing non-penetrating) 81 (75%) • B2 (stricturing) 16 (15%) • B3 (penetrating) 11 (10%) • S1 (mild UC) 10 (23%) • S2 (moderate UC) 23 (52%) • S3 (severe UC) 11 (25%)

Upper GI symptoms 23 (21%) 3 (7%) L4 with upper GI symptoms 14 L4 without upper GI symptoms 9

Smoking status • Active smoking 49 (45%) 8 (18%) • Ex smoking 9 (8%) 7 (16%) • No smoking 50 (47%) 29 (66%)

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further analysis. Baseline clinical characteristics according to Montreal classiﬁcation at initial presentation and demographics of all included patients are presented in Table 1. In all patients, endoscopic and histologic inﬂammatory disease activity in the ileum, colon or both was observed. A minority of patients, 23/108 CD (21%) and 3/44 (7%) UC patients reported upper GI tract symptoms, such as pain localized in epigastrium accompanied by nausea and/or vomiting. Upper GI disease location (+L4) was present in 14/23 (61%) of the symptomatic CD patients.

Endoscopic ﬁndings Macroscopic ﬁndings were detected at upper GI endoscopy in 60/108 (55%) CD patients. Endoscopic lesions, such as erythema, aphthae, ulcers and nodules were restricted to the stomach in 19 patients and to the duodenum in 15 patients (Figure 2). In 26 patients both gastric and duodenal lesions were observed. The distribution of the endoscopic

Figure 2 Endoscopic ﬁndings of Crohn’s disease- related upper gastrointestinal lesions

A B

C D

A Nodular antrum gastritis. B Ulceration and erythematous thickening of the inﬂamed mucosa in the antrum. C Aphthous ulcerations on top op thickened mucosal folds in the antrum. D Aphthous ulcerations in the second part of the duodenum.

46 upper gastrointestinal lesions at primary diagnosis of ibd

Table 2 Histologic Findings Presented According to the Presence and Location of Endoscopic Lesions

Gastric endoscopic ﬁndings Duodenal endoscopic ﬁndings Erythema Aphthae Ulcers Nodules Strictures NF Erythema Aphthae Ulcers Strictures NF

Crohn’s disease N9 N24 N6 N5 N1 N64 N10 N22 N9 N0 N67

Histological ﬁndings

Chronic inﬂammation N3 N4 N8 N4 N13 N3 N7

Erosions N1 N2 N3 N2

Ulcerations N2 N2

FEG N2 N18 N6 N5 N1 N27

Granuloma N1 N12 N5 N5 N1 N9 N2 N2

Normal ﬁndings N4 N2 N27 N6 N4 N60

Ulcerative Colitis N3 N41 N44

Histological ﬁndings

Chronic inﬂammation N1 N9

Erosions

Ulcerations

FEG N10

Granuloma

Normal ﬁndings N2 N23 N44

N: numbers referring to frequency of observed changes; NF: normal ﬁndings; FEG: focally enhanced gastritis.

ﬁndings in the stomach and duodenum and the corresponding histologic lesion for each location are described in the cross Table 2. Furthermore 4/60 (7%) patients had aphthous ulcerations located in the esophagus. Deﬁned upper GI location of CD (+L4), based on combined macroscopic and microscopic criteria, was established in 44/108 (41%) patients. Upper GI disease (+L4) was accompanied by ileum location (L1) in 13/44 (29%) patients, by colon location (L2) in 6/44 (14%) and by ileo-colonic location (L3) in 25/44 (57%) patients. Upper GI symptoms were present in 14/44 (32%) patients with upper GI disease location.

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Figure 3 Histological ﬁndings of IBD- related upper gastrointestinal lesions

A B

A Focally enhanced gastritis characterized by collections of lymphocytes, neutrophils and macrophages surrounding a small group of pits and/or glands (haematoxylin and eosin stain, original magnification × 100). B Granulomatous inflammation (haematoxylin and eosin stain, original magnification × 200)

In UC patients, no macroscopic lesions were identiﬁed, except atypical gastric erythema in 3/44 (7%) patients. The three symptomatic UC patients had no lesions on upper GI endoscopy.

Histological findings Histological abnormalities were detected in 77/108 (71%) CD patients and are summarized in Table 2. FEG was found in 58/108 (54%) CD patients (Figure 3A). Granulomas in stomach and/or duodenum biopsies were found in 34/108 (31%) CD patients (Figure 3B). Histological inflammation without endoscopic lesions included granuloma formation in 9 patients, FEG in 27 patients, chronic idiopathic gastritis in 8 patients and chronic idiopathic duodenitis in 7 patients. In UC, histological findings included chronic idiopathic inflammation and FEG, which were identified in 19 out of 44 patients (43%). FEG was found in 10 out of 44 UC patients (23%) (Table 2).

Discussion

The prevalence of upper GI endoscopic lesions was 55% in treatment-naive, newly diagnosed adult CD patients, irrespective of upper GI symptoms. The most common macroscopic ﬁndings in H. pylori-negative CD patients were (aphthous) ulcerations in the duodenum or stomach. In 41% of all CD patients, macroscopic lesions were accompanied by histological ﬁndings, thus qualifying for the diagnosis of upper GI CD (+L4).

48 upper gastrointestinal lesions at primary diagnosis of ibd

In newly diagnosed UC patients, no macroscopic aphthous lesions or ulcerations were observed in H. pylori-negative patients, but microscopic inflammation was observed in 43% of UC patients. Granulomatous inflammation in stomach or duodenum biopsies was exclusively observed in CD patients, irrespective of endoscopic findings. Focally enhanced gastritis was present in both CD and UC patients, either in combination with granulomas in CD or as an isolated finding, regardless of the presence of mucosal lesions during upper endoscopy. Most of the histological findings reported in gastroduodenal biopsies of CD and UC are not specific for one of the main two phenotypes of IBD. Initially, FEG was thought to be a diagnostic hallmark of CD inflammation in the gastric mucosa25. However, in several studies, it has been shown that even if FEG is more common in CD, it may also be a feature in some UC patients26-28. The currently presented findings confirmed these data as we identified FEG in 23% of UC patients. For upper GI lesions associated with UC, a new entity called Ulcerative Gastroduo- denal Lesions (UGDL) has been proposed13,14. The authors defined UGDL as difusely inflamed, friable or granular mucosa with multiple erosions, similar to the characteristic colorectal inflammation of UC. The prevalence of UGDL in two diferent studies was 4,7% (15/322) and 7,6% (19/250); remarkably, all patients with upper GI lesions had a pancolitis or postcolectomy status13. Furthermore, UGDL was associated with pouchitis in up to 61,5% of patients14,15. Although we found a high count (43%) of microscopic inflammation in our UC patients, we encountered no such lesions in our population. This may be explained by the cohort size, the early phase of the disease course, or exclusion of IBD-related drug therapy in the current UC patient group. In a recent prospective study the occurrence of upper GI involvement was analyzed in a heterogeneous group of asymptomatic CD patients (45 newly diagnosed and 74 previously diagnosed CD patients). The reported overall prevalence for upper GI CD involvement was 16% (19/119), of which 68% (13/19) were newly diagnosed CD patients16. Others observed upper GI findings in known CD and UC patients, reporting minute endoscopic lesions in 78% (18/23) CD and 53% (10/19) UC patients whilst using IBD-related anti-inflammatory medication21. In a retrospective study on known CD patients with long-standing disease, the reported prevalence of CD related upper GI endoscopic lesions reached 48%, most of the lesions being found in the stomach22. Furthermore, in recent reports on endoscopic gastroduodenal findings in known IBD patients, bamboo joint-like appearance of the gastric mucosa has been described as a typical, although not exclusive lesion of CD, as it has been described in some UC patients too23,24. In the present series no such lesion has been observed possible because no standard dye- spraying technique was used or, more likely due to a lower incidence of bamboo joint- like appearance in untreated patients with early stage of IBD. Up till now the presence of granulomatous inflammation in gastroduodenal biopsies appeared to be the only histological finding in IBD patients restricted to CD. The reported prevalence of granuloma formation in upper GI biopsies of adult CD patients

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lies between 4-11 %16,20-22. However these results originate from studies in patients with long-standing CD on immunosuppressive treatment. In children at first presentation of IBD, granulomas in upper tract mucosal biopsies were described in 26% to 40% of the cases29-32. The prevalence of upper GI lesions at primary diagnosis in pediatric IBD patients is high, such as mild macroscopic inflammation in up to 64% of the patients, both in CD as well as in UC29-31. Granulomatous inflammation, assuming that other causes are being excluded, was the only diferentiating feature consistent with the exclusive diagnosis of CD29-33. Therefore upper GI endoscopy at primary diagnosis was proposed as a standard procedure in the diagnostic work-up of pediatric IBD patients, allowing for diagnostic diferentiation using microscopic granulomatous inflammation as a guide34. In the present study a high prevalence of upper GI endoscopic lesions in newly diagnosed, treatment-naive CD patients has been observed. It comprised mostly mild aphthous gastritis and duodenitis, but the majority of these patients (68%) reported no upper GI related symptoms. In our experience, the clinical relevance of upper GI findings in asymptomatic, adult CD patients at the time of primary diagnosis is therefore limited, restricting the use of routine upper endoscopy in newly diagnosed, adult CD patients. Whether upper GI disease location of CD at the time of primary diagnosis may predict a complicated disease course has not yet been properly prospectively studied. There- fore, at this moment the use of upper endoscopy should be limited to symptomatic patients and patients with IBD Unclassified in order to discriminate between CD and UC, specifically in case of granulomatous inflammation at histopathological examination.

Acknowledgments

We thank Dr. A. A. van Bodegraven for his helpful suggestions during writing the manuscript.

50 upper gastrointestinal lesions at primary diagnosis of ibd

References

1 Van Assche G, Dignass A, Panes J et al. The second European evidence based Consensus on the diagnosis and management of Crohn’s disease: Definitions and diagnosis. Journal of Crohn’s Colitis 2010; 4:7-27. 2 Lichtenstein GR, Hanauer SB, Sandborn WJ and The Practice Parameters Committee of the Ameri- can College of Gastroenterology. Management of Crohn’s disease in adults. Am J Gastroenterol on- line publication 2009; doi:10.1038/ajg.2008.168. 3 Van Bodegraven AA, van Everdingen JJ, Dijkstra G et al. Guideline ‘Diagnosis and treatment of inflammatory bowel disease in adults’. I Diagnosis and treatment. Ned Tijdschr Geneeskd 2010; 154, A 1899. 4 Annese V, Daperno M, Rutter MD et al. European evidence based consensus for endoscopy in inflammatory bowel disease. J Crohn’s Colitis 2013; 7:982-1018. 5 Feuerstein JD, A.M., Giford AE, Cullen G et al. Systematic review: the quality of the scientific evidence and conflicts of interest in international inflammatory bowel disease practice guidelines. Aliment Pharmacol Ther. 2013 May;37(10):937-46. doi: 10.1111/apt.12290. Epub 2013 Apr 2., 2013. 6 Lennard-Jones JE. Classification of inflammatory bowel disease. Scand J Gastroenterol 1989; 24: 2-6. 7 Rutgeerts P, Onette E, Vantrappen G et al. Crohn’s disease of the stomach and duodenum: a clinical study with emphasis on the value of endoscopy and endoscopic biopsies. Endoscopy 1980; 12:288-294. 8 Nugent FW, Roy MA. Duodenal Crohn’s disease: an analysis of 89 cases. Am J Gastroenterol 1989; 84:249- 254. 9 Kleer CG, Appelman HD. Ulcerative colitis: patterns of involvement with colorectal biopsies and changes with time. Am J Surg Pathol 1998; 22:983-9. 10 Dignass A, Eliakim R, Magro F et al. Second European evidence based consensus on the diagnosis and management of ulcerative colitis part1: definitions and diagnosis. J Crohns Colitis 2012; 6: 965-990. 11 Turner D, Grifths AM. Esophageal, Gastric and Duodenal Manifestations of IBD and the Role of Upper Endoscopy in IBD Diagnosis. Curr Gastroenterol Rep 2007; 9:475-478. 12 Feakins RM. Ulcerative colitis or Crohn’s disease? Pitfalls and problems. Histopathology 2014; 64: 317-335. 13 Hori K, Ikeuchi H, Kakano H et al. Gastroduodenitis associated with ulcerative colitis. J Gastroenterol 2008; 43:193-201. 14 Hisabe T, Matsui T, Miyaoka M et al. Diagnosis and clinical course of ulcerative gastroduodenal lesion associated with ulcerative colitis: possible relationschip with pouchitis. Digestive Endoscopy 2010; 22:268-274. 15 Lin J, McKenna BJ, Appelman HD. Morphologic findings in Upper Gastrointestinal Biopsies of Pa- tients With Ulcerative Colitis. Am J Surg Pathol 2010; 34:1672-1677. 16 Annunziata ML, Caviglia R, Papparella LG et al. Upper Gastrointestinal Involvement of Crohn’s Dis- ease: A Prospective Study on the Role of Upper Endoscopy in the Diagnostic Work-Up. Dig Dis Sci 2012; 57:1618-1623. 17 Satsangi J, Silverberg MS, Vermeire S, et al. The Montreal classification of inflammatory bowel disease: controversies, consensus and implications. Gut 2006; 55 (6): 1295-1301. 18 Tontini GE, Vecchi M, Pastorelli L et al. Diferential diagnosis in inflammatory bowel disease colitis: State of the art and future perspectives. World J Gastroenterol 2015; 7:21-46. 19 Wright CL, Riddell RH. Histology of the stomach and duodenum in Crohn’s disease. Am J Surg Pathol 1998; 22:383-390. 20 Korelitz BI, Waye JD, Kreuning J et al. Crohn’s disease in endoscopic biopsies of the gastric antrum and duodenum. Am J Gastroenterol 1981; 76:103-109.

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21 Maeda K, Okada M, Seo M et al. Evaluation of gastroduodenal mucosal lesions in patients with Crohn’s disease and Ulcerative colitis. Digestive Endoscopy 2004; 16: 199-203. 22 Sakuraba A, Iwao Y, Matsuoka K et al. Endoscopic and pathologic changes of the upper Gastrointes- tinal tract in Crohn’s disease. Bio Med Research International 2014; Article ID 610767. 23 Fujiya M, Sakatani A, Dokoshi T et al. A Bamboo Joint-Like Appearance is a Characteristic Finding in the Upper Gastrointestinal Tract of Crohn’s Disease Patients. A Case-Control study. Medicine (Baltimore) 2015 Sep; 94 (37): e1500. 24 Kuriyama M, Kato J, Morimoto N et al. Specific gastroduodenoscopic findings in Crohn’s disease: comparison with findings in patients with ulcerative colitis and gastroesophageal reflux disease. Dig Liver Dis 2008; 40:468-475. 25 Oberhuber G, Puspok A, Oesterreicher C et al. Focally enhanced gastritis: a frequent type of gastritis in patients with Crohn’s disease. Gastroenterology 1997; 112:698-706. 26 Parente F, Cucino C, Bollani S et al. Focal gastric Inflammatory Infiltrates in Inflammatory Bowel Diseases: Prevalence, Immunohistochemical Characteristics and Diagnostic Role. Am J Gastroen- terol 2000; 95:705- 711. 27 Sharif F, McDermott M, Dillon M et al. Focally enhanced gastritis in children with Crohn’s disease and ulcerative colitis. Am J Gastroenterol 2002; 97:1415-1420. 28 Ushiku T, Moran CJ, Lauwers GY. Focally enhanced gastritis in newly diagnosed pediatric inflammatory bowel disease. Am J Surg Pathol 2013; 37: 1882-1888. 29 Cameron DJ. Upper and lower gastrointestinal endoscopy in children and adolescents with Crohn’s disease: a prospective study. J Gastroenterol Hepatol 1991; 6: 355-358. 30 Lemberg DA, Clarkson CM, Bohane TD et al. Role of esophagogastroduodenoscopy in the initial assessment of children with inflammatory bowel disease. J Gastroenterol Hepatol 2005; 20:1696-1700. 31 Sawczenko A, Sandhu BK. Presenting features of inflammatory bowel disease in Great Britain and Ireland. Arch Dis Child 2003; 88:995-1000. 32 Tobin JM, Sinha B, Ramani P et al. Upper Gastrointestinal Mucosal Disease in Pediatric Crohn Dis- ease and Ulcerative Colitis: A Blinded, Controlled Study. J of Pediatric Gastroenterol and Nutrition 2001; 32:443-448. 33 De Bie CI, Paerregaard A, Kolacek S et al. Disease Phenotype at Diagnosis in Pediatric Crohn’s disease: 5-year Analyses of the EUROKIDS Registry. Inflamm Bowel Dis 2013; 19: 378-385. 34 Bousvaros A, Antonioli DA, Colleti RB et al. Diferentiating Ulcerative Colitis from Crohn’s Disease in Children and Young Adults: Report of a Working Group of North American Society for Pediatric Gastroenterology, Hepatology and Nutrition and the Crohn’s and colitis Foundation of America. Journal of Pediatric Gastroenterol and Nutrition 2007; 44:653-674.

52 chapter 4

MR Enterography in addition to ileocolonoscopy in newly diagnosed adults with Crohn’s disease

submitted

CS Horjus Talabur Horje1

W Geerts1

L Roovers2

FBM Joosten3

MJM Groenen1

PJ Wahab1

1 Department of Gastroenterology, Rijnstate Hospital, Arnhem, the Netherlands 2 Department of Epidemiology and Statistics, Rijnstate Hospital, Arnhem, the Netherlands 3 Department of Radiology, Rijnstate Hospital, Arnhem, the Netherlands

Abstract background The value of Magnetic Resonance Enterography (MRE) in determining the extent and severity of Crohn’s disease (CD) in addition to a complete ileocolonoscopy at the time of the primary diagnosis has been advocated in recent guidelines though not yet been studied. The aim of this study was to assess the additional value of MRE following initial diagnostic ileocolonoscopy of CD. methods Patients who underwent MRE within 3 months of primary ileocolonoscopy were retrospectively included in the analysis. Additional findings on MRE next to ileocolonoscopy were assessed including strictures, entero-enteral fistula, abscesses and extended small bowel disease activity. MRE findings were subsequently analyzed in dif- ferent CD phenotypes. results A total of 163 newly diagnosed CD patients were included in the analysis. MRE revealed additional findings in 28% (45/163) of all patients. Prevalence of additional MRE findings was 67% (21/32) in those cases in which ileum could not been intubated, compared to 18% (24/131) in patients with a complete ileocolonoscopy. In patients without ileitis at ileocolonoscopy no additional lesions were seen on MRE. Strictures, enteroenteral fistula and abscesses were mostly observed during MRE examination in patients with severely active ileitis at endoscopy. conclusions Prevalence of MRE findings next to ileocolonoscopy in newly diagnosed CD patients is high in endoscopically assessed stricturing disease and severe disease activity of the terminal ileum, corroborating the use of standard diagnostic MRE in these clinical circumstances.

55 chapter 4

Introduction

Crohn’s disease (CD) is a chronic transmural inflammatory disease of the gastro-intestinal tract, which widely varies in presentation, extent and location. A single gold standard for the diagnosis of CD is not available but is confirmed by clinical evaluation and a combination of endoscopic, histological, radiological, biochemical investigations and occasionally surgery. For diagnostically assumed CD, ileocolonoscopy with biopsies is a well-established first line procedure to confirm the diagnosis1. Disease localization at primary diagnosis is reported in 33% of patients to be limited to the terminal ileum, in 47% of the patients limited to the colon and in 20% ileocolonic lesions have been reported2. Current diagnostic guidelines recommend additional routine small bowel imaging in all newly diagnosed CD patients, irrespective of the findings at ileocolonoscopy. How- ever, this recommendation is solely based on the expert opinion of a panel, as studies on this matter are lacking3. Magnetic Resonance Enterography (MRE) is the recommended radiological technique for determining the extent and complications of CD, considering its diagnostic accuracy, lack of ionizing radiation and patient acceptance compared to Ultrasound, Com- puted Tomography Enterography and Magnetic Resonance Enteroclysis3-16. MRE has a high sensitivity in the assessment of terminal ileum disease activity, fistulas, strictures, and abscesses as well as the extent of small bowel activity17-18. However, the complementary value of MRE in addition to ileocolonoscopy in newly diagnosed CD patients remains yet unclear, as it has not yet been studied. In patients with a non-complicated CD in the colon and/or terminal ileum, small bowel imaging is suspected to provide little extra information compared to ileocolonoscopy alone, especially in isolated colonic disease19. On the other hand an additional MRE could provide vital information in more elaborate and penetrating forms of small bowel CD, such as the detection of strictures, extended small bowel activity, fistulas or abscesses. Therefore, the aim of this study was to assess the value of MRE regarding new clinical relevant findings in addition to ileocolonoscopy at primary diagnosis of CD. More- over, we wanted to identify CD patients that would benefit the most from an additional MRE at the time of primary diagnosis.

Methods

Study population and period All patients labeled with the diagnosis treatment combination (DTC) code for CD at a large regional hospital in Arnhem, The Netherlands (Rijnstate Hospital) between 2009 and 2015 were retrospectively reviewed Figure 1. The CD diagnosis was established and

56 mr enterography at primary diagnosis of cd

Figure 1 Inclusion diagram of the patients

CD patients identiﬁed by DTC (N=545)

Did not meet inclusion criteria (N= 380): • Age <16 years (N=15) • No primary CD diagnosis (N=326) • No MRE within 3 months (N=39)

Inclusion criteria (N=165): • Established CD • Age >16 years • MRE within 3 months

Excluded (N=2) • Insufcient MRE quality

Included in the analysis (N=163)

registered by the treating physician and was based on a combination of endoscopic, histological, radiological and/or biochemical investigations according to the Lennard-Jones criteria. Patients of 16 years and older, who underwent a MRE within 3 months of ileocolonoscopy at primary diagnosis were included. Patients primarily diagnosed before 2009 were not included due to possible changes in MRE protocol. Patients with a diagnosis of ulcerative colitis (UC), inflammatory bowel disease unclassified (IBD-U) or an otherwise doubtful CD diagnosis and in case of inadequate MRE quality, were excluded from the analysis. From the medical records of the included patients the following data were retrieved: patient age, gender, date and findings of ileocolonoscopy at the time of primary diagnosis, date and findings of MRE, time to follow-up, disease location and behavior at the time of primary diagnosis according to the Montreal classification. Subgroups were made based on Montreal classifications, intubation of terminal ileum during endoscopy (yes or no) and severity of the ileitis (mild or severe). The cause for not intubating the ileum was identified and these patients were analyzed separately. The Montreal classification was used to classify CD location [ileal (L1), colonic (L2), ileocolonic (L3)] and disease behavior [non-stricturing, non-penetrating (B1), stricturing (B2), and penetrating (B3)] based on primary ileocolonoscopy20,21. Gastroesophageal in-

57 chapter 4

volvement was not considered in this study as not all patients underwent upper endoscopy. Subjects with both stricturing and penetrating disease were classified as having penetrating disease (B3). Perianal fistulizing disease was recorded as a separate modifier (+P) and this localization did not constitute penetrating disease (B3) on its own.

Ileocolonoscopy Patients underwent endoscopy under conscious sedation. The ileocolonoscopy was performed afer standard bowel preparation with 4 liter of polyethylene glycol electro- lyte solution. The ileocolonoscopy was scored for presence of ileitis and/or colitis, severity of inflammation in the terminal ileum, stenosis and fistulas based on the report made by the performing endoscopist. Severity of inflammation in the terminal ileum was either classified as mild or severe, while colitis was only noted as present or absent. The ileitis was scored as mild in the presence of small aphthous ulcers (estimated diameter 0.1-0.5 centimeters) separated by normal mucosa and as severe when large ulcers (estimated diameter >0.5 centimeters) and/or difuse inflamed mucosa was seen. Stenosis was defined, as a localized narrowing of the intestinal lumen reported by the endoscopist, either passable or not passable with the scope.

Magnetic Resonance Enterography MRE was performed according to the standard local protocol. Patients fasted for at least 4 hours and were asked to drink 1.0 liter of a water-based oral preparation fluid containing 2,5% mannitol and 0,5% locust bean gum, in a time span of 45 minutes before the MRI, without any particular preparation of the colon. MRI scans were performed with the patient in prone position, using a body coil on a 1.5T MRI unit (Intera 1.5 T, Philips medical systems, Best, Holland). Patients were given butylscopolamine 20 mg intravenously, as intestinal motility inhibitor immediately before the examination to avoid bowel peristalsis artifacts. Patients were scanned in the prone position to avoid wall movement of the abdomen. The scanning protocol included a coronal breath-hold T2-weighted single-shot (SSh) turbo spin-echo and a coronal T2 breath-hold balanced fast field echo (FFE). The field of view of these T2 weighted studies was the whole abdomen, from the xyphoid process to the pubic bone. Slice thickness was 5 mm consecutive, which resulted in an average of 30 images in the coronal plane. Afer 60 seconds of manual administration of the intravenous contrast medium (Gadoteric acid, Dotarem) (0.5 mmol/ml) 0.2 ml per kg body weight), T1 weighted sequences were applied in the transverse and coronal plane with a fat saturation spectrally attenuated inversion recovery (SPAIR). The MRE report was scored for the presence of findings such as terminal ileitis (terminal ileum afected adjacent to the ileocecal valve), preterminal ileitis (more than 10 cm normal ileum between the ileocecal valve and the afected ileum), extended ileitis

58 mr enterography at primary diagnosis of cd

(more than 40cm of small bowel afected), stenosis, fistulas and abscesses. Stenosis was defined as a narrowing of a thickened bowel segment with a proximal area of dilation. Fistulae were defined as tubular tracts with or without high signal intensity, between two small bowel loops. Abscesses were described as a fluid collection, with or without associated air and a well-defined wall. Mural contrast enhancement was considered present if there was a clear visual enhancement of a small bowel segment compared to adjacent loops of the terminal ileum, at visual assessment. All these scans were re-examined by an experienced abdominal radiologist who was blinded to the patient data.

Outcome measures Primary outcome measures were the percentage of patients with additional small bowel findings on MRE and the descriptive characteristics of these findings. The findings of ileocolonoscopy and MRE were analyzed and compared between diferent subgroups: intubation of the terminal ileum during endoscopy (yes or no), diferent Montreal classification afer primary ileocolonoscopy and endoscopic severity of the ileitis (mild or severe).

Statistical analysis Statistical analysis of the data was performed using IBM SPSS statistics (SPSS 21.0). Cat- egorical variables were described using frequencies and percentages. Continuous variables were summarized using medians and range. The Pearson’s chi-squared or Fisher- Exact test was used to determine statistical signiﬁcance between proportions. P values less than 0.05 were considered to indicate statistical signiﬁcance.

Ethical considerations Approval of the local ethics and feasibility committee for medical research from the Rijnstate hospital Arnhem, in The Netherlands was obtained in August 2015. The study was performed according to the principles of the Declaration of Helsinki. Furthermore, there was no conﬂict of interest or sponsoring involved.

Results

The study population consisted of 163 patients with CD who underwent MRE within 3 months of primary diagnosis by ileocolonoscopy. The main baseline characteristics and endoscopic ﬁndings of our patients are summarized in Table 1 and Table 2. Two CD patients were excluded because the MRE quality was insufcient to examine the terminal ileum due to lack of sufcient distension of the ileum.

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Table 1 Baseline characteristics of patients at presentation

Baseline characteristics Number of patients Median (spread)/Percentage

Age at diagnosis 163 28 years [16-72] A2 (16-39 years) 116 71.2% A3 (>40 years) 47 28.8% Male 61 37.4% Female 102 62.6% Perianal disease (+p) 23 14.1%

Days between MRE and ileocolonoscopy 163 11 days [0-92] < 1 week 64 39.3% 1 week - 1 month 70 42.9% 1 month - 3 months 29 17.8%

A number of 39 newly diagnosed CD patients did not undergo MRE within 3 months of initial ileocolonoscopy and were therefore excluded from the analysis Figure 1. Out of these patients, 10 (26%) had colonic CD with normal ileum on ileocolonoscopy, 16 (41%) had mild ileitis on ileocolonoscopy, 12 (30%) had severe ileitis and 1 patient had an incomplete ileocolonoscopy because of stricture. The patient with incomplete colonoscopy and two other patients with severe disease underwent an CT of the abdomen next to ileocolonoscopy.

Endoscopic findings A total of 131/163 (80%) patients had successful endoscopic intubation of the terminal ileum during endoscopy. In 32/163 (20%) patients the terminal ileum was not intubated due to stenosis (16.5%) or technical limitations (3.1%) The findings of ileocolonoscopy, including disease location and endoscopic severity are described in Table 2. From the 20 patients with no endoscopic signs of active ileal inflammation, 17 patients had isolated colonic CD with a normal endoscopic and histologic aspect of the terminal ileum. In three patients ileal disease was present in the form of fibrotic scars, post-inflammatory polyps and stricturing of the terminal ileum without signs of active inflammation at the time of the primary ileocolonoscopy.

Magnetic Resonance Enterography findings The Magnetic Resonance Enterography findings in addition to ileocolonoscopy findings are summarized in Table 3. In 28% of all patients additional small bowel lesions were found on MRE afer ileocolonoscopy. Additional findings on MRE were present in 18% of the patients following complete ileocolonoscopy compared to 66% of the patients without ileum intubation (p<0.05) Table 3.

60 mr enterography at primary diagnosis of cd

Table 2 Findings at ileocolonoscopy in all patients

Ileocolonoscopy characteristics Number of patients (%)

Procedure (N=163) Ileum intubated 131(80.4%) Ileum not intubated 32 (19.6%) → due to technical limitations 5 (3.1%) → due to stenosis 27 (16.5%) Location of stenosis → colon 7 (4.3%) → ileocecal valve 20 (12.3%)

Ileum inﬂammation severity (N=131) → no active inﬂammation 20 (15.3%) → mild 48 (36.6%) → severe 63 (48.1%)

Localization at endoscopy (N=131) → isolated ileum activity (L1) 66 (50.4%) → isolated colonic activity (L2) 17 (13.0%) → ileocolonic activity (L3) 48 (36.6%)

Behavior at endoscopy (N=131) Non-stricturing/Non-penetrating (B1) 100 (76.3%) Stricturing (B2) 29 (22.1%) → passable with scope 15 (11.5%) colon 1 (0.8%) ileocecal valve 5 (3.8%) ileum 9 (6.9%) → impassable with scope 14 (10.7%) ileum Entero-enteral ﬁstulas (B3) 2 (1.5%)

No statistically significant diference was found between patients with isolated ileum (L1) and ileocolonic (L3) endoscopic disease. Patients with isolated colonic disease during ileocolonoscopy (L2) showed no additional small bowel findings on MRE, precluding Chi-square testing in this group. Patients with endoscopic stricturing disease (B2) had more new lesions on MRE compared to non-stricturing/non-penetrating disease (B1) (p<0.05). In the patients with signs of entero-enteral fistula on primary endoscopy (B3), the MRE confirmed the presence of fistula and revealed new lesions such as stenosis or abscess Table 3. The additional MRE findings in mildly active endoscopic ileitis were restricted to five cases showing ileitis in the proximal ileum (skip lesions) without small bowel complications and one case of extended ileitis (>40 cm).

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Table 3 Additional ﬁndings at MRE next to ileocolonoscopy

Magnetic Resonance Enterography findings Additional New terminal Preterminal Length >40cm New stenosis New entero- Abscesses Change in Montreal findings (%) ileitis (%) ileum activity (%) (%) enteral (%) classification (%) fistula (%) (Behavior/Location)

All patients (N=163) 45 (27.6%) 21 (12.9%) 16 (9.8%) 6 (3.7%) 3 (1.8%) 9 (5.5%) 6 (3.7%) 11 (6.7%)/14 (8.5%) Ileum intubated (n= 131) 24 (18.3%)* 1 (0.8%) 12 (9.2%) 5 (3.8%) 3 (2.3%) 7 (5.3%) 5 (3.8%) 8 (6.1%)/0 (0.0%) Ileum not intubated (n=32) 21 (65.6%)* 20 (62.5%) 4 (12.5%) 1 (3.1%) 0 (0%) 2 (6.3%) 1 (3.1%) 3 (9.4%)/13 (40.6%)

Clinical phenotype afer ileocolonoscopy (N=131) Ileal (L1) (n=66) 14 (21.2%)** 0 (0.0%) 6 (9.1%) 3 (4.5%) 2 (3.0%) 5 (7.6%) 2 (3.0%) 5 (7.5%)/0 (0.0%) Colonic (L2) (n=17) 0 (0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)/0 (0.0%) Ileocolonic (L3) (n=48) 10 (20.8%) ** 1 (2.1%) 6 (12.5%) 2 (4.2%) 1 (2.1) 2 (4.2%) 3 (6.3%) 3 (6.3%)/0 (0.0%) Non-stricturing/non-penetrating (B1) (n=100) 12 (12.0%)* 0 (0.0%) 8 (8 %) 3 (3.0%) 0 (0.0%) 1 (1.0%) 2 (2.0%) 2 (2.0%)/0 (0.0%) Stricturing (B2) (n=29) 10 (34.5%)* 0 (0.0%) 3 (10.3%) 1 (3.4%) 2 (6.9%) 4 (13.8%) 2 (6.9%) 6 (20.7%)/0 (0.0%) Penetrating (B3) (n=2) 2 (100%) 1 (50.0%) 1 (50.0%) 1(50.0%) 1 (50.0%) 2 (100%) 1 (50.0%) 0 (0%)/0 (0.0%)

Ileum inﬂammation severity (N= 114) Fibrotic lesions and stenosis without active inﬂammation (N=3) 3 (100%) 1 (33.3%) 1 (33.3%) 2 (66.6%) 3 (100%) 1 (33.3%) 1 (33.3%) 2 (66.6%)/0 (0.0%) Mild (N=48) 6 (12.5%) 0 (0.0%) 5 (10.4%) 1 (2.1%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)/0 (0.0%) Severe (N=63) 15 (23.8%) 0 (0.0%) 6 (9.5%) 2 (3.2%) 0 (0.0%) 6 (9.5%) 4 (6.3%) 8 (12.7%)/0 (0.0%)

* p <0.05 ** p=0.11

Discussion

In the present study, additional findings on MRE following primary ileocolonoscopy were observed in 28% of newly diagnosed CD patients. The highest percentage of additional MRE small bowel lesions was found in patients with incomplete ileocolonoscopy (67%) and in patients with stricturing endoscopic disease (35%). In isolated colon CD at ileocolonoscopy no additional findings were seen on the MRE. Patients with mild endoscopic ileitis had no small bowel complications on MRE. Current guidelines recommend additional imaging of the small bowel in all newly diagnosed CD patients, irrespective of the findings at ileocolonoscopy. The underlying reason for this recommendation may be that sensitivity of ileocolonoscopy in determining the location and extent of disease activity, especially in penetrating and stricturing forms of small bowel CD is limited3. However a majority of the patients experience no CD complications at time of first presentation. Previous studies have shown that MRE has a high sensitivity for both assessing the terminal ileum disease activity, fistulas, strictures, and abscesses as well as the extent of small bowel activity of Crohn’s dis- ease17,18. In most studies the accuracy of MRE has been compared with ileocolonoscopy but the complementary diagnostic value of MRE was disregarded22,23. It has recently been stated that nearly half of the Inflammatory Bowel Disease guideline recommendations are based on expert opinion or lack any solid scientific evidence necessitating corroborating (or denying) investigations24.

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Table 3 Additional ﬁndings at MRE next to ileocolonoscopy

* p <0.05 ** p=0.11

In a previous subgroup analysis of the value of MRE next to primary ileocolonoscopy in 49 patients was stated that small bowel imaging provides little extra information in non-complicated CD19. Moreover, it was suggested that in isolated colon CD during complete ileocolonoscopy, small bowel imaging adds no information to endoscopy. An important role for MRE was mentioned in case of incomplete ileocolonoscopy or suspicion of complicated disease. However predictive factors of complicated disease were not addressed19. Predictive factors for complicated CD might include extensive small bowel disease, young age at diagnosis, perianal disease activity, early stricturing/ penetrating disease and smoking but the clinical value of these factors has not yet been prospectively studied in a large patient cohort25,26. One of the strengths of the present study lies in refining the indication for additional small bowel imaging based on macroscopically findings of the primary ileocolonoscopy. Furthermore, the period between the primary ileocolonoscopy and MRE was short, as 82.2% underwent MRE within a month, with an overall a median period of 11 days. Moreover, the number of patients included was sufciently high to draw robust conclusions. The main limitation of this study was the retrospective design. The prognostic value and clinical impact of the additional MRE findings at the time of the primary diagnosis could not be reliably determined in this study. Furthermore, the inclusion criteria might have induced a selection bias, because only patients who underwent MRE were included. The isolated colonic disease group could therefore be underrepresented in our study.

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Assessment of disease extension and activity in patients with CD is required in order to determine the therapeutic strategy, and may have major prognostic implica- tions27. The extent of the prognostic impact of small bowel imagining afer initial ileocolonoscopy is unclear as a combined disease severity index capturing diferent prognostic factors, such as clinical phenotype, mucosal lesions and bowel damage, is missing25,28. However, imaging of the small bowel by using MRE is a costly procedure and the indication for imaging should be appropriately argued based on the disease characteristics. Our results confirmed that in case of an incomplete primary ileocolonoscopy, MRE might reveal important new findings regarding small bowel extent and complications of CD. High prevalence of additional findings was also present in case of stricturing of the terminal ileum during a complete ileocolonoscopy. MRE findings in case of mildly active endoscopic ileitis were of limited clinical value and no small bowel complications were seen in this group of patients. In conclusion, MRE is a valuable addition to ileocolonoscopy in determining the location and extent of CD, in some well-defined groups of newly diagnosed patients. Additional MRE is indicated in all cases of incomplete endoscopy, stricturing and severely active ileitis at primary endoscopy. Furthermore MRE should also be performed in all cases of endoscopic suspicion of entero-enteral fistula in order to assess the extent of the lesions and presence of concomitant complications such as stenosis and abscesses. In cases of mildly active endoscopic ileitis the additional value of MRE following complete primary ileocolonoscopy was limited and therefore MRE is not routinely indicated in these patients. Moreover, when the disease activity is isolated to the colon during a complete ileocolonoscopy, MRE could be omitted from the diagnostic work-up.

Acknowledgement

We want to thank dr. A. A. van Bodegraven for his helpful suggestions and thoughtful review of the manuscript.

64 mr enterography at primary diagnosis of cd

References

1 Coremans G, R.P., Geboes K, Van den Oord J, Ponette E, Vantrappen G. The value of ileoscopy with biopsy in the diagnosis of intestinal Crohn’s disease. . Gastrointest Endosc. 1984 Jun;30(3):167-72., 1984. 2 Sjöberg, H., Larsson, Nielsen, Holmquist, Ekbom, Rönnblom. Incidence and clinical course of Crohn’s disease during the first year- results from the IBD cohort of the Uppsala Region. J Crohns Colitis. 2014 Mar;8(3):215-22, 2014. 3 van Assche G, D.A., Panes J, Beaugerie L, Karagiannis J, Allez M, Ochsenkühn T, Orchard T, Rogler G, Louis E, Kupcinskas L, Mantzaris G, Travis S, Stange E; European Crohn’s and Colitis Organisa- tion (ECCO). ECCO guideline: the second European evidence-based consensus on the diagnosis and management of Crohn’s disease: definitions and diagnosis. J Crohns Colitis. 2010 Feb;4(1):7-27., 2010. 4 Cipriano LE, L.B., Zaric GS, Lofus EV Jr., Sandborn WJ. Cost-efectiveness of imaging strategies to reduce radiation-induced cancer risk in Crohn’s disease. Inflammatory bowel diseases 2012;18:1240– 1248. PMID: 21928375, 2012. 5 Israeli E, Y.S., Henderson B, Mottola J, Strome T, Bernstein CN. The impact of abdominal computed tomography in a tertiary referral centre emergency department on the management of patients with inflammatory bowel disease. Alimentary pharmacology & therapeutics 2013; 38:513–521, 2013. 6 Lee SS, K.A., Yang SK, Chung JW, Kim SY, Park SH, et al. Crohn disease of the small bowel: comparison of CT enterography, MR enterography, and small-bowel follow-through as diagnostic techniques. Radiology 2009; 251:751–761. doi: 10.1148/radiol.2513081184 PMID: 19276325, 2009. 7 Martinez MJ, R.T., Paredes JM, Blanc E, Marti-Bonmati L. Assessment of the extension and the inflammatory activity in Crohn’s disease: comparison of ultrasound and MRI. Abdominal imaging 2009, 2009. 8 Miao YM, K.D., Amin Z, Healy JC, Chinn RJ, Zeegen R, et al. Ultrasound and magnetic resonance imaging assessment of active bowel segments in Crohn’s disease. Clinical radiology 2002, 2002. 9 Negaard A, P.V., Sandvik L, Berstad AE, Borthne A, Try K, et al. A prospective randomized comparison between two MRI studies of the small bowel in Crohn’s disease, the oral contrast method and MR enteroclysis. European radiology 2007; 17:2294–2301. PMID: 17483955, 2007. 10 Panes J, B.R., Chaparro M, Garcia-Sanchez V, Gisbert JP, Martinez de Guerenu B, et al. Systematic review: the use of ultrasonography, computed tomography and magnetic resonance imaging for the diagnosis, assessment of activity and abdominal complications of Crohn’s disease. Alimentary pharmacology & therapeutics 2011; 34:125–145., 2011. 11 Peloquin JM, P.D., Sandborn WJ, Fletcher JG, McCollough CH, Schueler BA, et al. Diagnostic ionizing radiation exposure in a population-based cohort of patients with inflammatory bowel disease. The American journal of gastroenterology, 2008. 12 Leyendecker, MR Enterography in the management of patients with Crohn disease. Radiograph- ics. 2009 Oct;29(6):1827-46, 2009. 13 Fidler, MR imaging of the small bowel. Radiographics. 2009 Oct;29(6):1811-25, 2009. 14 Horsthuis K, B.S., Bennink RJ, Stoker J. Inflammatory bowel disease diagnosed with US, MR, scintig- raphy, and CT: meta-analysis of prospective studies. Radiology 2008;247(1):64–79., 2008. 15 Horjus Talabur Horje, B., Roovers, Groenen, Joosten, Wahab., Contrast Enhanced Abdominal Ultra- sound in the Assessment of Ileal Inflammation in Crohn’s Disease: A Comparison with MR Enterogra- phy. PLoS One. 2015 Aug 31;10(8):e0136105. doi: 10.1371/journal.pone.0136105. eCollection 2015., 2015. 16 Maccioni F, A.A.N., Mazzamurro F, Civitelli F, Viola F, Cucchiara S, Catalano C. Detection of Crohn disease lesions of the small and large bowel in pediatric patients: diagnostic value of MR enterography versus reference examinations. Am J Roentgenol. 2014 Nov;203(5):W533-42. doi: 10.2214/ AJR.13.11792., 2014.

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17 Masselli G, C.E., Polettini E, Lanciotti S, Bertini L, Gualdi G. Assessment of Crohn’s disease in the small bowel: Prospective comparison of magnetic resonance enteroclysis with conventional enteroclysis. Eur Radiol 2006;16(12):2817–27., 2006. 18 Gourtsoyiannis NC, G.J., Papamastorakis G, Koutroumabakis J, Prassopoulos P, Rousomoustakaki M, et al., Imaging of small intestinal Crohn’s disease: comparison between MR enteroclysis and conventional enteroclysis. Eur Radiol 2006;16(9):1915–25., 2006. 19 Jensen MD, N.T., Rafaelsen SR, Kjeldsen J., Ileoscopy reduces the need for small bowel imaging in suspected Crohn’s disease. Dan Med J. 2012 Sep;59(9):A4491., 2012. 20 Silverberg MS, S.J., Ahmad T, Arnott ID, Bernstein CN, Brant SR, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005;19(Suppl A): 5–36., 2005. 21 Satsangi J, S.M., Vermeire S, Colombel JF. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut 2006;55:749–53., 2006. 22 Florie J, H.K., Hommes DW, Nio CY, Reitsma JB, van Deventer SJ, Stoker J. Magnetic resonance imaging compared with ileocolonoscopy in evaluating disease severity in Crohn’s disease. Clin Gastroen- terol Hepatol. 2005 Dec;3(12):1221-8., 2005. 23 Silverstein J, G.D., Kawatu D, Shah SA, Steinkeler J, LeLeiko N. Feasibility of using MR enterography for the assessment of terminal ileitis and inflammatory activity in children with Crohn disease. J Pediatr Gastroenterol Nutr. 2012 Aug;55(2):173-7. doi: 10.1097, 2012. 24 Feuerstein JD, A.M., Giford AE, Cullen G, Lefer DA, Sheth SG, Cheifetz AS. Systematic review: the quality of the scientific evidence and conflicts of interest in international inflammatory bowel disease practice guidelines. Aliment Pharmacol Ther. 2013 May;37(10):937-46. doi: 10.1111/apt.12290. Epub 2013 Apr 2., 2013. 25 Fiorino G, P.-B.L., Bonifacio C, Naccarato P, Malesci A, Danese S, MRE and colonoscopy findings in early Crohn’s disease predict the course of the disease: a prospective observational cohort study. Gastroenterology; May 2013Volume 144, Issue 5, Supplement 1, Page S-639, 2013. 26 Zallot C, P.-B.L. Clinical risk factors for complicated disease: how reliable are they? Dig Dis 2012;30(Suppl 3):67–72, 2012. 27 Rimola J, R.S., García-Bosch O, Ordás I, Ayala E, Aceituno M, Pellisé M, Ayuso C, Ricart E, Donoso L, Panés J. Magnetic resonance for assessment of disease activity and severity in ileocolonic Crohn’s disease. Gut 2009;58;1113-1120, 2009. 28 Danese S, P.-B.L., Fiorino G. Predicting future disease course in Crohn’s disease by colonoscopy or magnetic resonance: which is the crystal ball? Gut. 2015 Sep;64(9):1347-8. doi: 10.1136/ gutjnl-2014-308919. Epub 2015 Jan 28., 2015.

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Contrast Enhanced Abdominal Ultrasound in the Assessment of Ileal Inﬂammation in Crohn’s Disease: a Comparison with MR Enterography

plos one. 2015 aug 31; 10(8):e0136105

CS Horjus Talabur Horje1

R Bruijnen2

L Roovers3

MJM Groenen1

FBM Joosten2

PJ Wahab1

1 Department of Gastroenterology, Rijnstate Hospital, Arnhem, the Netherlands 2 Department of Radiology, Rijnstate Hospital, Arnhem, the Netherlands 3 Department of Epidemiology and Statistics, Rijnstate Hospital, Arnhem, the Netherlands.

Abstract background and aims To prospectively examine the feasibility and accuracy of Con- trast Enhanced Ultrasound (CEUS) in the assessment of Crohn’s disease (CD) activity in the terminal ileum in comparison to Magnetic Resonance Enterography (MRE), using endoscopy as a reference standard. methods 105 consecutive patients with alleged clinically active CD were assessed by MRE and CEUS. CEUS of the terminal ileum was performed using an intravenous microbubble contrast enhancer. Accuracy values of CEUS and MRE for the presence of active terminal ileitis were evaluated using the Receiver Operating Characteristic method, using endoscopic findings as a reference standard. Sensitivity and specificity values of MRE and CEUS were compared by the McNemar test. results CEUS was feasible in 98% of patients, MRE in all. Optimal diagnostic accuracy in CEUS was obtained at a peak intensity value of 10%, showing 100% sensitivity, 92% specificity and an accuracy of 99% in demonstrating ileal mucosal inflammation. For MRE, overall sensitivity, specificity and accuracy were, 87%, 100%, and 88%, respectively. CEUS and MRE were highly correlated in assessing length and wall thickness of the terminal ileum. CEUS identified 11 of 16 MRE-detected strictures, but no fistulae. conclusion The accuracy of CEUS is comparable to that of MRE in the assessment of active, uncomplicated terminal ileal CD and therefore a valuable bedside alternative to MRE in the follow-up of these patients.

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Introduction

Crohn’s disease (CD) is a chronic transmural inflammatory disease of the gastrointestinal tract characterized by a widespread variation in severity and relapsing episodes of disease activity. Exclusive ileum disease is observed in approximately 30% of patients, whereas the ileocecal segment is involved in more than 50% of all CD patients1. Assessment of the severity of CD-associated inflammatory activity is used to monitor therapeutic results and plan further medical or surgical therapy. Clinical markers and laboratory tests are suboptimal indicators of disease activity2,3. Additional methods to reliably estimate disease activity include endoscopy and radiological imaging techniques. Ileocolonoscopy is considered the gold standard for CD disease activity in the colon and terminal ileum, allowing tissue sampling. This however is an invasive and, from patients’ perspective, burdensome method4. Complete visualization of the small bowel and of extramural disease manifestations may be performed by CT Enterography and MR Enterography (CTE and MRE)5-7. These two techniques have a similar diagnostic accuracy for CD disease extension and activity, as reported by Panes et al8. MRE is not widely available as it is a rather costly technique, whilst repeated CTE induces cumula- tive ionizing radiation exposure, particularly important in this group of relatively young patients9-11. The sensitivity of grey scale ultrasound (US) for the detection of CD disease activity of the terminal ileum ranges from 63% to 100%, with a specificity in the range of 77-100%8,12-15. Contrast enhanced ultrasound (CEUS) with new intravenous contrast agents allows visualisation of bowel wall perfusion. Quantitative evaluation of enhancement during CEUS improves the accuracy of ultrasound for the presence and severity of active inflammation in ileal CD16-22. Ultrasound is less suitable in the assessment of CD activity located in the large bowel, because of the limited ability to accurately localise the afected bowel segments. The diagnostic value of bowel wall enhancement with CEUS in the evaluation of CD disease activity, in comparison to MRE, was assessed in two studies23-24. Both of these used clinical disease activity scores as a reference, showing no correlation with either contrast enhancement by CEUS or by MRE. Therefore, these studies did not provide a clear assessment of the diagnostic value of CEUS as compared to MRE in daily clinical practice. The aim of this study was to assess the feasibility and accuracy of CEUS in the detection of CD activity in the terminal ileum, as compared to MRE, using endoscopy as a reference standard for active inflammation.

Materials and Methods

Study population From October 2009 to December 2012 a total of 107 consecutive adult patients with established active CD, who attended the Department of Gastroenterology of a large

70 ceus in the assessment of ileal cd

regional hospital (Rijnstate Hospital, Arnhem, NL), were prospectively enrolled in the study. The initial diagnosis of CD located in the terminal ileum, colon or both was con- ﬁrmed in all patients according to the usual clinical, endoscopic and histological cri- teria25. The inclusion criteria were: age over 18 years and a clinical indication for an ileocolonoscopy, to assess localization, extent and severity of the presumptive activity of CD (primary diagnosis, clinical relapse or recurrence). Patients who did not agree to participate in the study were excluded from the study, as were pregnant women. Further exclusion criteria were: contraindications for MRE such as claustrophobia, MR- unsafe devices and implants, renal insufciency and conditions potentially related to the toxicity of Sonovue® (Bracco, Italy); also a history of ischemic coronary disease and cardiac failure. The clinical disease activity at inclusion was scored using the Harvey Bradshaw In- dex26 and blood samples were taken for the measurement of C-reactive protein (CRP). The study was performed according to the principles of the Declaration of Helsinki. Approval of the regional ethics committee for medical research from Arnhem-Nijme- gen, in The Netherlands (CMO Arnhem-Nijmegen) was obtained on September 2009 and all patients provided written informed consent.

Endoscopy All patients underwent endoscopy under conscious sedation. The ileocolonoscopy was performed afer standard bowel preparation with 4 liter of polyethylene glycol elec- trolyte solution. The disease activity was scored using the Simple Endoscopy Score for CD (SES-CD)27, adjusted to score ileal disease activity. The SES-CD score was originally designed by Daperno et al. to score the mucosal disease activity in Crohn’s disease, through the whole colon and the terminal ileum for the length that has been examined by endoscopy. The four variables selected were: the size of ulcers, proportion of ulcer- ated surface, the surface with any other lesions and the presence of stenosis. Each vari- able was scored from 0 to 3 in each of the 5 segments (ileum, right colon, transverse colon, lef colon, rectum) with a maximal total score for the SES-CD of 48. When these variables are only scored for the terminal ileum the maximal score is 12. A score of 0 equals no endoscopic lesions and a score of 1 implies the presence of erythematous mucosa over less than 50% of the surface without any aphthous lesions or ulcerations. Before the start of the study, we defined a score of 0-1 in the terminal ileum, as no active inflammation and a score of 2 or higher as active inflammation.

Magnetic Resonance Enterography Within a period of maximally two weeks from the reference ileocolonoscopy and before any therapeutic changes, the patients underwent MRE followed by CEUS on the same day. The MRE studies were performed according to a standardized protocol. Patients fasted for at least 6 hours and were asked to drink 1.0 liter of a water-based oral preparation ﬂuid containing 2,5 % mannitol and 0,5% locust bean gum, in a time span of 45

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minutes before the MRI, without any particular preparation of the colon. MRI scans were performed with the patient in prone position, using a body coil on a 1.5T MRI unit (Intera 1.5 T, Philips medical systems, Best, Holland). All patients were given butylscopolamine 20 mg intravenously, as intestinal motility inhibitor immediately before the examination to avoid bowel peristalsis artifacts. Patients were scanned in the prone position to avoid wall movement of the abdomen. The scanning protocol included a coronal breath-hold T2-weighted single-shot (SSh) turbo spin-echo and a coronal T2 breath-hold balanced fast field echo (FFE). The field of view of these T2 weighted studies was the whole abdomen, from the xyphoid process to the pubic bone. Slice thickness was 5 mm consecutive, which resulted in an average of 30 images in the coronal plane. Afer 60 seconds of manual administration of the intravenous contrast medium (Gadoteric acid, Dotarem®) (0.5 mmol/ml) 0.2 ml per kg body weight), T1 weighted sequences were applied in the transverse and coronal plane with a fat saturation spectrally attenuated inversion recovery (SPAIR). The terminal ileum was the center of the field of view of these last three sequences. The MRE images were analysed by two experienced abdominal radiologists. They were aware of the diagnosis of CD and blinded for clinical, endoscopic and CEUS data. Parameters scored included the length of afected bowel, wall thickness measured at the point of maximum diameter of the afected small bowel wall, stratified wall appearance and perivisceral fibrofatty proliferaton, stenosis, fistulae, abscesses and enhancement afer administration of gadolinium (Table 1). Stenosis was defined as a narrowing of a thickened bowel segment with a proximal area of dilation (>3.0 cm). Fistulae were defined as tubular tracts with high signal intensity, between two small bowel loops. Abscesses were described as a fluid collection, with or without associated air and a well-defined wall. Mural contrast enhancement was considered present if there was a clear visual enhancement of a small bowel segment compared to adjacent loops of the terminal ileum, at visual assessment.

Grey scale Ultrasound and Contrast enhanced Ultrasound Ultrasound was performed with a Philips US device (Philips IU22®), using a convex 3-5 MHz probe. All studies started with a grey scale US examination to find the terminal ileum and assess wall thickness, induration of surrounding fatty tissue and enlarged lymphnodes. Stenosis and length of the pathologic bowel wall was estimated, as well as complications such as abscess formation and fistulae. The thickest segment of the terminal ileum was identified and used to perform analysis of contrast enhancement. We injected the second generation, ultrasonic contrast agent (SonoVue®, Bracco, Milan, Italy) as a bolus of 2.5 ml through a three-way 20-gauge catheter in an antecubital vein, followed by a bolus of 10 ml saline solution (0.9% NaCl)28. CEUS was performed with a 7.5MHz linear probe and contrast-tuned technology, based on a low mechanical index

72 ceus in the assessment of ileal cd

Table 1 Radiological parameters assessed by MR Eenterography (MRE), Grey scale Ultrasound (US) and Contrast enhanced Ultrasound (CEUS)

Radiologic parameters MRE Grey scale US

Wall thickness + + Length + + Stratiﬁed wall appearance + + Perivisceral ﬁbrofatty proliferation + + Stenosis + + Fistulae + + Abscesses + +

MRE CEUS

Maximum Enhancement + + Time to peak − + Regional Blood Volume − +

and a real-time scan to ensure the preservation of the contrast agent. A low acoustic power setting was used, expressing a low mechanical index (MI), 0.09-0.14, of a 7.5 MHz linear probe. Disease parameters analyzed during the grey scale US included identification of the terminal ileum, wall thickness measurement at the maximal diameter of the af- fected terminal ileum (pathological mural thickening defined as a wall thickness above 3 mm), evaluation of the afected bowel length, stratified wall appearance, perivisceral findings such as creeping fat and stenosis, fistula or abscess (Table 1). Stenosis was described as narrowing of a thickened, rigid bowel lumen with a proximal area of dilation (>3.0 cm). Fistulae were defined as tubular hypoechoic tracts between two small bowel loops. Abscesses were described as a fluid collection, with or without associated air and a well-defined wall19. CEUS images were assessed directly afer injection of the contrast agent, by re- cording a videoclip at the level of the most afected terminal ileum loop. Afer primary imaging, quantification sofware Qontrast® (Bracco, Italy) was applied to obtain contrast-enhanced sonographic perfusion maps, allowing immediate evaluation of the peak intensity (PI), time to peak (TTP), and the regional blood volume (RBV), being pro- portional to the area under the curve (Figure 1). These parameters, measured within the region of interest (ROI) selected by the operator, have formerly been described as indicators for disease activity16-19. Qontrast sofware is able to quantify the contrast enhancement from a sequence of the perfusion frames and to generate a chromatic map that allows immediate evaluation of the perfusion properties of the selected region (region of interest, ROI). The quantification procedure is being performed for each pixel encompassed by the ROI on the frame sequence. The ultrasound video intensity of the

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Figure 1 Perfusion map obtained by Qontrast® sofware.

Lef side: contrast uptake based on increased microvascularization of the terminal ileum in a patient with severe endoscopic disease activity. Right side: contrast uptake of the terminal ileum in a patient with normal endoscopic appearance.

pixels comprised in each ROI is being measured in gray-scale levels, from 0 (black pixels) to 255 (white pixels), and expressed in mean ± SD through histogram analysis. Further- more a virtual color image of the bowel can be obtained, composed of a primary color scale varying from red (maximum signal intensity) to blue (minimum signal intensity) and correlating with the numeric value of the signal intensity expressed in percentages (maximum intensity =100%). In each patient two diferent regions of the color map with the highest echo-signal intensity afer contrast injection, were analyzed and the mean value was used for the statistical analysis. Within the brightest zone of the intestinal wall, which represents the highest degree of vascularization, we made two measurements per injection. The kinetic parameters (PI, TTP, RBV) calculated for both ROI were then averaged for each patient18. The radiolo-

74 ceus in the assessment of ileal cd

gist performing the ultrasonography (grey scale and CEUS) was aware of the diagnosis of Crohn’s disease, but blinded to the patient’s present clinical, endoscopic and MRE data.

Statistical Analysis

Categorical variables were described using frequencies and percentages. Continuous variables were summarized using medians and range. A Receiver Operating Characteristic (ROC) analysis was performed to determine the optimal cut-of value for diferent CEUS variables in order to diferentiate active from inactive endoscopic disease. The value of contrast enhancement at MRE and CEUS in diagnosing active luminal CD was assessed by calculating the sensitivity, specificity, positive and negative predictive values and accuracy with 95% confidence intervals (95% CI), using endoscopic disease activity as reference standard. The sensitivity and specificity of the two modalities were compared by the McNemar test. The correlations between diferent radiological parameters assessed by MRE and ultrasound were determined by Spearman’s nonparametric correlation coefcient. We additionally applied the plot of the diference between the values measured by the two techniques against the mean of the measurements. This method described, by Bland and Altman, allows the visualization of the concordance between two tests for diferent measured values, in order to examine the actual agreement more closely. The smaller the diference between the values measured by the two tests, the better the correlation29. P values less than 0.05 were considered to indicate statistical significance. Statistical analysis of the data was performed using the Statistical Package for the Social Sciences (SPSS 21.0).

Results

Study population Over a 37-month period, 107 consecutive patients were included. The main demographic and clinical data are shown in Table 2. Two CD patients (2%) were not included in the analysis because their terminal ileum could not be visualized by B mode US and therefore no contrast assessment was done. Both patients had no active ileal disease on endoscopy. The endoscopically determined location and disease behavior of all patients is described in Table 2 according to the Montreal classiﬁcation. Intubation of the terminal ileum was attempted in all patients. However, 11 (10%) of the 105 patients had a stenosis (colon or ileocecal valve) disallowing endoscopic intubation of the terminal ileum. An incomplete stricture of the terminal ileum that could be passed by the endoscope was

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Table 2 Baseline demographic and clinical characteristics of the CD patients (n=105)

Variables Absolute frequency (%)

Male 46 (44 %) Female 59 (56%) Age [median (range)] 33 (18-65)

Disease location

Ileal 56 (53%) Colonic 12 (12%) Ileocolonic 37 (35%)

Disease behavior

Non-stricturing/non-penetrating 65 (62%) Stricturing 34 (32%) Penetrating 6 (6%) Postoperative status (ileocecal resection) 16 (15%) Serum CRP > 5 mg/l 63 (60%) Clinical remission (HBI <4) 15 (14%)

present in 19 (18%) patients. Twelve patients (11%) had a normal appearance of the terminal ileum next to an inflamed colon and these were classified as L2 according to the Montreal classification30. Endoscopic disease activity at the site of the terminal ileum was present in 82 (79%) of the patients.

Table 3 Statistical values for MR Enterography (MRE) and Contrast enhanced Ultrasound (CEUS) in detection of active endoscopic inﬂammation in the terminal ileum.

Contrast enhancement Contrast enhancement at MRE (95% CI) at CEUS (peak>10%) (95% CI)

Positive true 71 82 False positive 0 1 Negative true 12 11 False negative 11 0 Sensitivity * 87% (77%-93%) 100% (96%-100%) Speciﬁcity # 100% (73%-100%) 92% (62%-99%) PPV 100% (95%-100%) 99% (93%-100%) NPV 52% (31%-73%) 100% (71%-100%) Accuracy 88% (80%-94%) 99% (94%-100%)

* P value for pairwise McNemar test was 0,01 for MRE versus CEUS. # P value for pairwise McNemar test was 1.0 for CEUS versus MRE.

76 ceus in the assessment of ileal cd

MR Enterography No adverse events from MRE, performed as per protocol in all patients, were reported. The signal intensity afer administration of gadolinium was scored as normal in 27 patients (26%) and increased in 78 (74%) of the patients. Overall sensitivity of gadolinium enhancement in detecting the presence of any active inflammation of the terminal ileum was 87%, with a specificity of 100% and an accuracy of 94% (Table 3). The median length of the afected terminal ileum was 7 cm (range 3-50 cm). The median measured wall thickness was 5.5 mm (range 3-13 mm). MRE detected a total of five enteroenteral fistulae (Figure 2) and sixteen cases of ileal stenosis with prestenotic dilation (Figure 3).

Figure 2 Forty- two-year-old male patient with Crohn’s disease and enteroenteral ﬁstula of the terminal ileum

A C

A Coronal MR Enterography images (T2-weighted single-shot turbo spin-echo, on the lef image; T1-weighted fat saturated, spectrally attenuated inversion recovery afer Gadoteric acid administration, on the right image) of a patient with enteroenteral fistula and increased enhancement afer contrast administration (white arrow). B Longitudinal Contrast enhanced ultrasound of the afected ileum of the same patient (Gray scale ultrasound image on the right side; Contrast enhanced ultrasound image on the lef side), showing severe wall thickening on the Gray scale ultrasound (white arrow) and strong enhancement afer Sonovue® administration on the Contrast enhanced ultrasound (white arrow), but no fistula. C Endoscopic image of the same patient, with a fistula opening in the terminal ileum.

Grey scale US and Contrast enhanced US A large majority of patients (98%) were suitable for grey scale US and CEUS and no adverse efects were reported. The median value of the peak intensity (PI) was 33.5% (4-62%) and the median value of the regional blood volume (RBV) was 646.1 cm³ (18- 1769,2 cm³). PI was registered afer a median time to peak (TTP) of 20,5 seconds (2-42,8 seconds).

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Figure 3 Thirty-one-year-old female patient with Crohn’s disease and stenosis of the ileocolic anastomosis afer ileocecal resection

A C

A Coronal MR Enterography images (T2-weighted single-shot turbo spin-echo on the lef image; T1-weighted fat saturated, spectrally attenuated inversion recovery afer Gadoteric acid administration, on the right image) of a patient with stenosis (white arrow) and increased enhancement of the ileocolic anastomosis (white arrow). B Longitudinal Contrast enhanced ultrasound (Gray scale ultrasound image, on the right side; Contrast enhanced ultrasound image, on the lef side), of the same patient with a short stenosis of the ileocolic anastomosis (white arrow) and increased enhancement afer Sonovue® administration (white arrow). C Endoscopic image of the same patient showing an ulcerative stenosis of the neoterminal ileum.

Figure 4 Receiver Operating Characteristic curve for performance of Contrast enhanced Ultrasound for the diagnosis of active ileitis at endoscopy. Peak intensity (solid line) and Regional Blood Volume (dotted line).

1,0 peak rbv

0,8

0,6

Sensitivity 0,4

0,2

0,0 0,0 0,2 0,4 0,6 0,8 1,0 Speciﬁcity

78 ceus in the assessment of ileal cd

The cut-of value for PI that best depicted the presence of active endoscopic disease, indicated by the area under the ROC curve, was 10% (Figure 4). This value provided a sensitivity of 100% in detecting the presence of active inﬂammation at the site of the terminal ileum, with a speciﬁcity of 92% and an accuracy of 99% (Table 3).

The cut-of value for RBV that best detected active endoscopic disease was 200 cm³, achieving a sensitivity of 93%, speciﬁcity of 83% and accuracy of 88%. The area under the curve for PI was 0.96 (95%CI 0.89-1.00) and for RBV 0.93 (95% CI 0.82-1.00). The median length of the afected bowel was 6 cm (range 3-20 cm). The median measured wall thickness was 5.5 mm (range 3-13 mm) and a stratiﬁed wall appearance was detected in 53 patients (50%) (Figure 5). Furthermore, grey scale US detected stenosis of the terminal ileum with prestenotic dilation, in 11 patients.

Figure 5 Twenty-ﬁve-year-old male patient with Crohn’s disease of the terminal ileum

A C

A Coronal MR Enterography images (T2-weighted single-shot turbo spin-echo on the lef image; T1-weighted fat saturated, spectrally attenuated inversion recovery afer Gadoteric acid administration, on the right image ) of a patient with ileitis terminalis demonstrating marked wall thickening of the terminal ileum (white arrow) and mild enhancement afer contrast administration (white arrow). B Longitudinal Contrast enhanced ultrasound (Gray scale ultrasound image, on the right side; Contrast enhanced ultrasound image, on the lef side), of the same patient showing wall thickening on Gray scale ultrasound (white arrow) and strong enhancement afer Sonovue® administration on Contrast enhanced ultrasound (white arrow). C Endoscopic image of the same patient with an ulcerative ileitis terminalis.

Correlation between MRE and US ﬁndings The length of the lesion measured by MRE correlated with the measured length of the pathologic bowel wall by US (r=0.92, P<0.001). In order to more closely examine the agreement of measurement between the two imaging techniques, we constructed a Bland and Altman plot. There was a high agreement for diseased segments below 10 cm, but a rather poor agreement for diseased segments above 15 cm (Figure 6).

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Figure 6 The relation between the mean length and the diference in length of the afected small bowel measured by MR Enterography and Contrast Enhanced Ultrasound.

50.00

40.00

30.00

20.00

10.00 length di f erence (MRE-CE) 1.16 0.00

-10.00

0.00 10.00 20.00 30.00 40.00 mean length

There was also a high correlation between the wall thickness of the afected terminal ileum, between MRE and US (r=0.97, P<0.001). When looking at the Bland and Altman plot, the agreement between the two techniques was least in patients with a wall thickness between 6 and 10 mm (Figure 7). In assessing the wall stratiﬁcation and perivisceral ﬁndings, the two methods agreed in 89 and 90 out of the 105 patients, respectively.

In diagnosing stenosis, grey scale US showed a sensitivity of 63% compared to MRE (11 out of the 16 strictures similarly diagnosed). None of the five enteroenteral fistulae on MRE could be identified by US.

Discussion

This study has demonstrated a high accuracy of CEUS combined with grey scale US for detecting mucosal disease activity in ileal CD. Grey scale US identiﬁed the (neo)terminal ileum in 105 out of 107 CD patients.

80 ceus in the assessment of ileal cd

Figure 7 The relation between the mean wall thickness and the diference in wall thickness measured by MR Enterography and Contrast Enhanced Ultrasound.

3.00

2.00

1.00

0.00 0.07

-1.00 di f erence in thickness (MRE-CE) -2.00

-3.00

2.50 5.00 7.50 10.00 12.50 mean thickness

When compared to endoscopic disease activity, enhancement by means of SonoVue® administration during CEUS had a high sensitivity in diagnosing active inflammation located in the terminal ileum. For assessing active intestinal inflammation, a higher accuracy for the peak intensity (PI) value than for the regional blood volume (RBV) was confirmed by CEUS-generated time-intensity curves. Regarding morphology, the US and MRE showed a high correlation for a diseased segment under 10 cm and for a wall thickness under 6 mm or above 10 mm, with slightly less accuracy for thicknesses between these two measurements. US seemed less accurate in identifying afected bowel segments longer than 10 cm. Furthermore, grey scale US identified 63% of the stenosis but none of the five enteroenteral fistulae. In a former study, semi-quantitative findings from CEUS were found to correlate with dynamic contrast-enhanced MRE (r=0,623, P=0,003). CEUS and MRI findings, however, did not correlate with clinical and laboratory parameters measuring disease activity (CDAI and CRP). Therefore, the authors concluded that CEUS and MRE were not able to evaluate disease activity23. In a more recent study, CEUS was compared with MRE in the assessment of small bowel CD, again using clinical parameters as reference standard. In

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about one third of the 30 patients included, CEUS or MRE were able to detect active disease. Additionally MRE and CEUS were weakly correlated with clinically assessed disease activity (CDAI), with a Spearman’s coefcient r=0.39824. Comparison of our results with findings in the literature is difcult. Study protocols and definitions of disease activity vary considerably and standardized semi-quantitative measurements to compare outcome are lacking. In the present study, cut-of values for PI and RBV values of contrast enhancement at CEUS were calculated using ROC analysis and therefore could difer from other studies. Increased wall microvascularization and proliferation of microvessels in the severely afected bowel of CD patients has been reported in several histopathological stud- ies31-33. Furthermore, a strong relation between changes in the vascularization of the diseased bowel loop and inflammatory activity has been suggested31,34. Color and power Doppler ultrasonography have also been used to assess the wall hypervascularization of inflamed loops31,34,35. Findings detected by Doppler US are related to large arteries of the bowel wall and therefore this method is probably less accurate in predicting mild parietal inflammation35. Nonetheless, parietal contrast enhancement afer intravenous gadolinium administration during MRE has been used to indicate transmural inflammation and was shown to be helpful in diferentiating active from inactive CD7,36-38. At present, MRE is recommended as the preferred imaging technique in CD because of its safety and its accurate, reproducible visualization of the entire small bowel tract39. In this study, quantitative assessment of the terminal ileum enhancement by CEUS was more sensitive in detecting active endoscopic inflammation than the qualitative assessment of enhancement afer gadolinium administration during MRE. This diference may be explained by diferences in the assessment technique and by the diferent phar- macodynamics of both contrast agents. Gadolinium chelates migrate and accumulate in the interstitium, including the fibrous tissue of chronic lesions. US contrast agents (gas microbubbles) remain in the microcirculation and degrade quickly in the vascular system. These blood-pool contrast agents theoretically lead to enhancement restricted to structures with an increased microvascularization and are not retained in the fibrous tissue19. This could explain a higher sensitivity of CEUS in distinguishing between active and inactive intestinal lesions. A limitation of the diagnostic performance of US in this study was the lack of accurate detection of fistulae and abscesses that may be due to underrepresentation in this series of patients. Recently, a retrospective analysis demonstrated good accuracy of CEUS with grey scale US in diagnosing abscesses and phlegmons in CD40. Another limitation was that contrast enhancement at MRE was not quantitatively scored. However, quantitative scoring of contrast-uptake during MRE is not standard procedure in daily practice, being rather complex and time-consuming. Besides, several recent papers have described the value of qualitative assessment of contrast enhancement during MRE compared to endoscopic disease activity41,42.

82 ceus in the assessment of ileal cd

One of the strengths of the present series was the size of the study population, which enabled a reliable statement with respect to feasibility and sensitivity. However, the specificity may be less reliable due to the limited number of patients with a normal terminal ileum during endoscopy. Furthermore, the use of endoscopically assessed disease activity authorized an objective grading of active inflammation and therefore a reliable reference standard. Among the cross sectional imaging techniques available for the assessment of patients with ileal CD during follow-up, the addition of CEUS to a standard grey scale US study may constitute an attractive, practical imaging modality: there is a lack of radiation exposure, it is readily available and it comes at low cost. Furthermore, three diferent recent studies in CD patients argue the value of CEUS in predicting outcome afer treatment with immunomodulators and/or TNF blockers43-45 and diagnosing postoperative recurrence46. Therefore, we believe, although not replacing MRE as an imaging technique in all patients, CEUS may well be used as an additional technique in the follow-up of patients with known ileal CD. In conclusion, CEUS is feasible and accurate in detecting the presence and activity of CD in the terminal ileum. The performance of this imaging modality is comparable to MRE in uncomplicated ileitis, defined as a short afected terminal ileum not complicated by fistulae or abscesses. CEUS represents a valuable, easily applicable bedside tool in the follow-up of terminal ileum involvement in CD.

Acknowledgements

We thank Dr. A A van Bodegraven for his helpful suggestions during the writing of the manuscript.

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References

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19 Ripolles T, Martinez-Perez MJ, Blanc E, Delgado F, Vizuete J, Paredes JM, et al. Contrast-enhanced ultrasound (CEUS) in Crohn’s disease: technique, image interpretation and clinical applications. Insights into imaging 2011;2:639-652. 20 De Franco A, Marzo M, Felice C, Pugliese D, Veronica AD, Bonomo L, et al. Ileal Crohn’s disease: CEUS determination of activity. Abdominal imaging 2012;37:359-368. 21 Ripolles T, Rausell N, Paredes JM, Grau E, Martinez MJ, Vizuete J. Efectiveness of contrast-enhanced ultrasound for characterisation of intestinal inflammation in Crohn’s disease: a comparison with surgical histopathology analysis. Journal of Crohn’s & Colitis 2013;7:120-128. 22 Ripolles T, Martinez MJ, Paredes JM, Blanc E, Flors L, Delgado F. Crohn disease: correlation of findings at contrast-enhanced US with severity at endoscopy. Radiology 2009;253:241-248. 23 Pauls S, Gabelmann A, Schmidt SA, Rieber A, Mittrach C, Haenle MM, et al. Evaluating bowel wall vascularity in Crohn’s disease: a comparison of dynamic MRI and wideband harmonic imaging contrast-enhanced low MI ultrasound. European radiology 2006;16:2410-2417. 24 Malago R, D’Onofrio M, Mantovani W, D’Alpaos G, Foti G, Pezzato A, et al. Contrast-enhanced ultrasonography (CEUS) vs. MRI of the small bowel in the evaluation of Crohn’s disease activity. La Radiologia medica 2012;117:268-281. 25 Lennard-Jones JE. Classification of inflammatory bowel disease. Scandinavian Journal of Gastroenter- ology Supplement 1989;170:2-6; discussion 16-19. 26 Harvey RF, Bradshaw JM. A simple index of Crohn’s-disease activity. Lancet 1980;1:514. 27 Daperno M, D’Haens G, Van Assche G, Baert F, Bulois P, Maunoury V, et al. Development and validation of a new, simplified endoscopic activity score for Crohn’s disease: the SES-CD. Gastrointestinal endoscopy 2004;60:505-512. 28 Girlich C, Jung EM, Iesalnieks I, Schreyer AG, Zorger N, Strauch U, et al. Quantitative assessment of bowel wall vascularisation in Crohn’s disease with contrast-enhanced ultrasound and perfusion analysis. Clinical hemorheology and microcirculation 2009;43:141-148. 29 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307-310. 30 Satsangi J, Silverberg MS, Vermeire S, Colombel JF. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut 2006;55:749-753. 31 Brahme F, Lindstrom C. A comparative radiographic and pathological study of intestinal vaso-archi- tecture in Crohn’s disease and in ulcerative colitis. Gut 1970;11:928-940. 32 Wakefield AJ, Sawyerr AM, Dhillon AP, Pittilo RM, Rowles PM, Lewis AA, et al. Pathogenesis of Crohn’s disease: multifocal gastrointestinal infarction. Lancet 1989;2:1057-1062. 33 Drews BH, Barth TF, Hanle MM, Akinli AS, Mason RA, Muche R, et al. Comparison of sonographi- cally measured bowel wall vascularity, histology, and disease activity in Crohn’s disease. European radiology 2009;19:1379-1386. 34 Maconi G, Parente F, Bollani S, Imbesi V, Ardizzone S, Russo A, et al. Factors afecting splanchnic haemodynamics in Crohn’s disease: a prospective controlled study using Doppler ultrasound. Gut 1998;43:645-650. 35 Karoui S, Nouira K, Serghini M, Ben Mustapha N, Boubaker J, Menif E, et al. Assessment of activity of Crohn’s disease by Doppler sonography of superior mesenteric artery flow. Journal of Crohn’s & colitis 2010;4:334-340. 36 Sinha R, Murphy P, Hawker P, Sanders S, Rajesh A, Verma R. Role of MRI in Crohn’s disease. Clinical radiology 2009;64:341-352. 37 Negaard A, Paulsen V, Sandvik L, Berstad AE, Borthne A, Try K, et al. A prospective randomized comparison between two MRI studies of the small bowel in Crohn’s disease, the oral contrast method and MR enteroclysis. European radiology 2007;17:2294-2301.

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38 Leyendecker JR, Bloomfeld RS, DiSantis DJ, Waters GS, Mott R, Bechtold RE. MR enterography in the management of patients with Crohn disease. Radiographics: a review publication of the Radiological Society of North America, Inc 2009;29:1827-1846. 39 van Bodegraven AA, van Everdingen JJ, Dijkstra G, de Jong DJ, Oldenburg B, Hommes DW, volwassenen CB-wIb. [Guideline ‘Diagnosis and treatment of inflammatory bowel disease in adults’. I. Diagnosis and treatment]. Nederlands tijdschrif voor geneeskunde 2010;154:A1899. 40 Ripolles T, Martinez-Perez MJ, Paredes JM, Vizuete J, Garcia-Martinez E, Jimenez-Restrepo DH. Con- trast-enhanced ultrasound in the diferentiation between phlegmon and abscess in Crohn’s disease and other abdominal conditions. European journal of radiology 2013;82:e525-531. 41 Takenaka K, Ohtsuka K, Kitazume Y, Nagahori M, Fujii T, Saito E, et al. Comparison of magnetic resonance and balloon enteroscopic examination of the small intestine in patients with Crohn’s disease. Gastroenterology 2014;147:334-342 e333. 42 Steward MJ, Punwani S, Proctor I, Adjei-Gyamfi Y, Chatterjee F, Bloom S, et al. Non-perforating small bowel Crohn’s disease assessed by MRI enterography: derivation and histopathological validation of an MR-based activity index. European journal of radiology 2012;81:2080-2088. 43 Moreno N, Ripolles T, Paredes JM, Ortiz I, Martinez MJ, Lopez A, et al. Usefulness of abdominal ultrasonography in the analysis of endoscopic activity in patients with Crohn’s disease: Changes following treatment with immunomodulators and/or anti-TNF antibodies. Journal of Crohn’s & colitis 2014;8:1079-1087. 44 Saevik F, Nylund K, Hausken T, Odegaard S, Gilja OH. Bowel Perfusion Measured with Dynamic Contrast-enhanced Ultrasound Predicts Treatment Outcome in Patients with Crohn’s Disease. In- flammatory bowel diseases 2014. 45 Quaia E, Cabibbo B, De Paoli L, Toscano W, Poillucci G, Cova MA. The value of time-intensity curves obtained afer microbubble contrast agent injection to discriminate responders from non-responders to anti-inflammatory medication among patients with Crohn’s disease. European radiology 2013;23:1650-1659. 46 Paredes JM, Ripolles T, Cortes X, Moreno N, Martinez MJ, Bustamante-Balen M, et al. Contrast- enhanced ultrasonography: usefulness in the assessment of postoperative recurrence of Crohn’s disease. Journal of Crohn’s & colitis 2013;7:192-201.

S1 Supporting Information: STARD checklist.

86 part II Immunological characteristics in early Inﬂammatory Bowel Disease

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Naïve T cells in the gut of newly diagnosed, untreated, adult inﬂammatory bowel disease patients

inflamm bowel dis. 2014 nov; 20(11):1902-9

Carmen S. Horjus Talabur Horje1

Sabine Middendorp2

Elly van Koolwijk3

Lian Roovers4

Marcel JM Groenen1

Peter J Wahab1

Ellen van Lochem3

1 Department of Gastroenterology, Rijnstate Hospital, Arnhem 2 Department of Pediatric Gastroenterology, Wilhelmina Children’s Hospital, UMC Utrecht, The Netherlands 3 Department of Immunology, Rijnstate Hospital, Arnhem 4 Department of Epidemiology and Statistics, Rijnstate Hospital, Arnhem

Abstract background The phenotype of the T-cell subpopulations and their related cytokine networks in the gastrointestinal mucosa of IBD patients can potentially be used as a predictive value for clinical course and response to therapy. Here, we analyzed T-cell subpopulations in newly diagnosed, untreated, adult patients and correlated them with clinical presentation. methods Mucosal biopsies from duodenum, ileum and colon mucosa of patients with Crohn’s disease (CD), Ulcerative Colitis (UC) and controls were obtained. The Simple En- doscopy Score in CD and the full Mayo score in UC were used to score disease activity. Mucosa-infiltrating T cells were characterized by flowcytometric immunophenotyping and stimulated to assess cytokine secretion. results Based on the expression of the maturation and activation markers CD45RA and CD27, we identified four diferent profiles. Profile A contained mainly CD45RA+CD27+ naïve T cells; profile B: mainly CD45RA-CD27+ central memory T cells; profile C: mainly CD45RA-CD27- efector memory T cells, and profile D consisted of similar percentages of these afore mentioned subpopulations. Profile A was only observed in ileum/colon of IBD patients, associated with upper gastrointestinal location and perianal disease in CD and expressed more TNFα and less IFNg. In contrast, profile D was restricted to controls. There was no correlation between the diferent T-cell profiles and endoscopic disease activity. conclusions Newly diagnosed IBD patients display diferent T-cell maturation profiles in the gut mucosa, corresponding to distinct cytokine responses. Follow-up studies are needed to determine whether the profiles associate with clinical course and response to therapy.

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Introduction

Crohn’s disease (CD) and ulcerative colitis (UC) are complex, multifactorial disorders, which result from aberrant immune responses to gut luminal antigens in genetically susceptible hosts1,2. From the clinical point of view, inflammatory bowel disease (IBD) represents a heterogeneous group of conditions regarding disease location, disease behavior and clinical course. In an attempt to explain this diversity, clinical parameters such as young age at diagnosis, early need for steroid treatment and perianal involvement in CD, or various blood-derived biomarkers proved to be disappointing in predicting disease behavior3-5. Similarly, several international collaborative genetic studies have so far identified 163 IBD-associated susceptibility loci6. This knowledge underlined the immunological phe- nomena related to IBD but did not elucidated its pathogenesis and cannot be used as predictor in treating individual patients3,6,7. Next to the genetic background of the patient, alterations in T-cell subsets within the gut mucosa appear to play an important role in the pathogenesis of IBD8. Bone marrow-derived lymphoid progenitor T cells undergo positive and negative selection in the thymus, resulting in the release of immunocompetent naïve T cells in the circula- tion9. These naïve T cells have previously been thought to circulate exclusively between secondary lymphoid organs, such as peripheral and mesenteric lymph nodes9. However, several reports suggest that naïve T cells also enter non-lymphoid organs such as the intestinal mucosa, especially at sites of inflammation10-12. Besides an increased amount of naïve CD45RA+ T cells, which has been described in both CD and UC13, diferentiated T-cell subpopulations seem to difer between these two clinical entities. In CD, the major cytokines interferon y (IFNy) and IL17 are derived from T-helper (Th) 1 and Th17 cell diferentiation, respectively 14. Whereas UC has been shown to be predominantly driven by a Th2-like diferentiation process, leading to the expansion of natural killer T (NKT) cells and production of the cytokines IL4 and IL131,2. Fur- thermore, other cytokine profiles have been associated with both forms of IBD, such as the pro-inflammatory cytokines TNFα and IL6, and regulatory cytokines such as IL108,14. Notably, these immunological typologies were mainly based on experimental studies in murine models15,16, whereas studies on T cells in the intestinal mucosa of humans have typically been performed in IBD patients that were under treatment with immune modulating therapies17-19. Here, we have determined the distribution of diferent T-cell subpopulations and their related cytokine profiles in the gastrointestinal mucosa of newly diagnosed, untreated, adult IBD patients. We show that variations in T-cell subpopulations in the gut mucosa, early in the course of disease, were associated with diferent clinical subtypes of IBD and may in future help to stratify patients that are likely to respond to a certain therapy.

92 naïve t cells in the gut of ibd patients

Materials and methods

Patients and controls Patients with symptoms of chronic diarrhoea, rectal blood loss, abdominal pain or weight loss, underwent combined ileocolonoscopy and upper endoscopy. From this group, fify-one newly diagnosed CD patients and fourteen newly diagnosed UC patients were included in the study. The diagnosis of IBD was established and certified by clinical, endoscopic and histological standard criteria20. Endoscopic severity of disease activity was assessed using the Simple Endoscopy Score for CD (SES-CD) and the full MAYO score for UC21. Following the standardized diagnostic procedures, comprising laboratory tests, endoscopy and if needed additional imaging techniques, all IBD patients were classified according to the clinical phenotype at diagnosis using the Montreal classification, although this usual is confidently established afer 5 years follow-up22. Controls were recruited from a patient population who underwent endoscopy for other reasons. Control biopsies were taken from 14 individuals who underwent ileocolonoscopy for polyp/cancer screening and 10 individuals who underwent upper endoscopy because of functional dyspepsia, showing no signs of gastrointestinal pathology during endoscopic and histological examination. Baseline clinical characteristics according to Montreal classification at initial presentation and demographics data of all included patients and controls are presented in Table 1.

Tissue samples and cell preparation Mucosal biopsy specimens were sampled before any anti-inflammatory (IBD-)treatment. During colonoscopy, biopsies were harvested from the macroscopically most inflamed region of the colon and ileum. In addition to biopsies taken for histopathological examination, four biopsies from the terminal ileum and four biopsies from the colon were taken for immunological determination. During upper endoscopy, four mucosal biopsies of normal gastric corpus and antrum and two biopsies of normal duodenal mucosa were taken for histopathological examination and two additional biopsies for immunological examination. When endoscopic lesions were observed, biopsies for immunologic examination were specifically harvested from macroscopic lesions such as erosions or ulcerations. All biopsy specimens from apparently healthy individuals were sampled from macroscopically normal mucosa. Biopsy samples were kept in phosphate-bufered saline solution (PBS) at 2-8 oC and processed within 8 hours. All samples were finely minced in Hanks/1% BSA using a 70mm gaze and spatula followed by Ficoll density gradient centrifugation. Afer washing, the homogenate was resuspended in 0,5 ml Hanks/1% BSA and the cell number was

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Table 1 Baseline characteristics and Montreal classiﬁcation of patients and controls at presentation

Patient characteristics CD UC C

N of patients 51 14 14

Age at diagnosis 33(±13) 35(±14) 45(±16)

Gender (male/female) 19/32 5/9 4/10

Disease location (N(%)) • L1 (small bowel only) 20 (39%) • L2 (large bowel only) 12 (24%) • L3 (small and large bowel) 19 (37%) • +L4 (upper GI) 19 (37%) • +P (perianal disease) 4 (8%) • E1 (proctitis) 1 (7%) • E3 (pancolitis) 13 (93%)

Disease behavior (N(%)) • B1 (non-stricturing, non-penetrating) 39 (76%) • B2 (structuring) 9 (18%) • B3 (penetrating) 3 (6%)

Simple Endoscopy Score – CD (N(%)) • Mild (4-10) 21 (41%) • Moderate (11-19) 17 (33%) • High (≥20) 13 (26%) • MAYO Score – UC (N(%)) • 1 (mild) 2 (14%) • 2 (moderate) 7 (50% • 3 (severe 5 (36%)

estimated by microscopic counting using a Kova cell chamber. From the total cell suspension, 200ml was used for ﬂowcytometric analysis disregarding absolute cell number. When total cell number was more than 1 × 106/ml, the remaining cell suspension was incubated over night at 37oC in the presence of 1 µg/ml phorbol myristate acetate (PMA)/1µM ionomycin to assess cytokine secretion.

Flow cytometry and cytokine assays Flow cytometric immunophenotyping encompassed three six-color analyses: Mix 1: CD19-FITC, CD7-PE, CD45-PerCP/CY5.5, CD56-PE/CY7, CD3-APC and HLA-DR-APC-H7/ CY7. Mix 2: CD103-FITC, CD45RA-PE, CD27-PerCP/CY5.5, CD4-PE/CY7, CD3-APC and CD8-APC-H7/CY7. Mix 3: TCRγ∂-FITC, FoxP3-PE, CD45-PerCP/CY5.5, CD4-PE/CY7, CD25- APC, CD3-APC-H7/CY7 (all from Becton Dickinson, USA) using a FACS Canto (BD Biosci- ences, USA). Within the lymphocyte population (CD45+SSClow), B cells (CD19+), NK cells (CD56+CD3-), T cells (CD3+CD7+) and T-cell subpopulations like Tregs (CD25highFoxP3+),

94 naïve t cells in the gut of ibd patients

mucosal T cells (CD103+) and CD4+ or CD8+ T cells were expressed as percentage of the whole lymphocyte population. Naïve and memory T-cell profiles were studied in colon/ileum biopsies by analyzing the combination of CD45RA and CD27 expression23. The CD45RA+CD27+ population was defined as the naïve T-cell subset, the CD45RA-CD27+ as the central memory T-cell subset and the CD45RA-CD27- as the efector memory T-cell subset23. Additionally to the phenotypic identification, mucosal T cells were functionally characterized, by stimulating the cell suspensions from biopsies with PMA/ionomycin. Afer overnight stimulation, the supernatants were frozen for flowcytometric cytokine analysis using BDTM Cytometric Bead Array (CBA, BD Biosciences; USA). Levels of IL2, IL4, IL6, IL10, TNFα, IFNy en IL17 were reflected to standard concentrations of the respective pro- teins and initially expressed in pg/ml. However, as cytokine levels measured in the su- pernatant of overnight incubated cell suspensions were dependent of the cell number in the culture and the relative T-cell number, the individual cytokine levels were expressed as a percentage of the total excreted cytokine level instead of pg/ml.

Statistical analysis Data are shown as mean ± standard deviation (SD), as median in case of non-Gaussian distribution, or as values for individual specimens in the figures. The Mann-Whitney non-parametric test was used for determination of statistical significance. Values of P ≤0.05 were considered to be significant.

Results

Distribution of lymphocytes in mucosa of duodenum and ileum/colon To assess the distribution of lymphocyte subsets in duodenum and ileum/colon, we determined their numbers in IBD patients and healthy controls. In the lymphocyte population of the duodenum of IBD patients, T cells were predominant: 74% in CD and 75% in UC, with low frequencies of B cells: 4% in CD and 9% in UC. The median CD4/CD8 ratio in duodenum was 0,3 in CD and 0,2 in UC patients. The majority of the duodenal T cells were of mucosal origin, (CD103+): 81% in CD and 84% in UC (Figure 1 and Table 2). Furthermore, B cells were more numerous in the ileum and colon of IBD patients than in duodenum, with a median of 34% and 38% of the lymphocyte population in CD and UC, respectively. In both CD and UC, T cells represented

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Figure 1 Distribution of lymphocyte subsets in mucosa of duodenum and ileum/colon of IBD patients and controls. Representative ﬂow cytometry dot plots show the distribution of (A) CD19+ B cells and CD3+ T cells, (B) mucosal CD103+ within the CD3+ T cells, (C) CD4 and CD8 T cells and (D) CD25highFoxP3+ regulatory T cells.

Duodenum control Duodenum IBD Ileum/colon control Ileum/colon IBD

A CD19 CD3

B CD103 CD3

C CD8 CD4

D CD25 FoxP3

56% of the lymphocytes, of which a minority was of mucosal origin (CD103+): 15% in CD and 11% in UC. The mean CD4/CD8 ratio in ileum/colon was 2,3 in CD and 3,2 in UC. The frequencies of CD25highFoxP3+ Tregs in ileum/colon were 14% in CD and 13% in UC (Figure 1 and Table 2). In healthy control samples, the distribution of B and T-cell numbers in duodenum and ileum/colon was comparable to that of IBD patients. However, the numbers of Tregs (CD- 25highFoxP3+), non-mucosal (CD103-) T cells and the CD4/CD8 ratio in the ileum/colon were signiﬁcantly higher in IBD patients compared to controls (Figure 1 and Table 2).

96 naïve t cells in the gut of ibd patients

Table 2 The median values of gut-derived lymphocytes (CD19+ B cells, CD3+ T cells, CD4/CD8 ratio, CD103+ mucosal T cells and CD25highFoxP3+ Tregs) in the duodenum and ileum/colon mucosa of controls, CD and UC patients. Numbers (with exception of CD4/CD8 ratio) are percentages of lymphocyte population, characterized by light scatter characteristics and CD45 expression. Representative FACS plots for each staining are shown in Figure 1.

CD UC C p value

Duodenum

B cells (CD19+) 4 (1-34) 9 (1-36) 6 (1-20) Ns

T cells (CD3+) 74 (12-95) 75 (55-88) 78 (35-97) Ns

Mucosal T cells (CD103+CD3+) 81 (13-97) 84 (34-96) 83 (17-96) Ns

CD4/CD8 ratio 0,3 (0,03-2,8) 0,2 (0,06-0,75) 0,2 (0,04-2,8) Ns

Tregs (CD25highFoxP3+) 2 (0-17) 3 (0-14) 2 (0-8) Ns

Ileum/colon

B cells (CD19+) 34 (1-66) 38 (14-56) 26 (5-49) Ns

T cells (CD3+) 56 (11-80) 56 (41-84) 64 (28-84) Ns

Mucosal T cells (CD103+CD3+) 15 (2-65) 11 (3-62) 30 (4-78) 0,02

CD4/CD8 ratio 2,3 (0,38-7,5) 3,18(0,47-8) 1,2 (0,4-4,59) Ns

Tregs (CD25highFoxP3+) 14 (6-28) 13 (5-20) 5 (3-14) 0,002

Naïve and memory T-cell profiles in ileum/colon mucosa of IBD patients and controls Within the CD3+ T-cell population present in ileum/colon, naïve and memory T-cell profiles were studied by analyzing expression of CD45RA and CD27 (23). The CD45RA+CD27+ - + population was defined as the naïve (TN), the CD45RA CD27 as the central memory (TCM) - - and the CD45RA CD27 as the efector memory (TEM) T-cell subset.

IBD patients showed a higher frequency of TN cells compared to controls (31% in CD and 28% in UC vs 19% in controls). Additionally, IBD patients also showed an increased

number of TCM cells (37% in CD and 42% in UC vs 25% in controls). In contrast, the num-

ber of the TEM cells was lower in IBD patients compared to controls (20% in CD and 19% in UC vs 33% in controls). Within the ileum/colon mucosa of IBD patients, we observed a remarkable variability in the frequencies of naïve and memory T cells. We identified four diferent profiles (profile A-D), which consisted mainly (>40%) of either CD45RA+CD27+ naïve T cells (profile A), CD45RA-CD27+ central memory T cells (profile B), CD45RA-CD27- efector memory T cells (profile C), or an equal (each ~30%) distribution of these before mentioned

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Figure 2 Naïve and memory T-cell profiles in ileum/colon mucosa of IBD patients and controls. (A) Representative flow cytometry dot plots of the four diferent maturation T-cell profiles: Profile A: >40% TN cells, CD45RA+CD27+, profile B: >40% TCM cells, CD45RA-CD27+; profile C: >40% TEM cells CD45RA-CD27- and profile D: an equal distribution of TN, TCM and TEM cells. (B) Percentage of IBD patients and controls displaying the various T-cell maturation profiles.

A B 100 D C 80 B A 60 Proﬁle A Proﬁle B

Percentage of proﬁles (%) 20

CD45RA 0 Proﬁle C Proﬁle D Control CD UC (n=14) (n=51) (n=14) CD27

subpopulations (profile D, Figure 2A). The TN and TCM cells were mostly non-mucosal - + + (CD103 ) CD4 T cells, whereas the TEM cells were mostly CD8 T cells of mucosal origin (CD103+) (Figure 3). Notably, the mainly naïve T-cell profile A was only observed in IBD patients, while the mixed T-cell profile D was only present in controls. In IBD patients, the T-cell profile

Figure 3 Distribution of lymphocytes within diferent maturation T-cell proﬁles. Distribution of (A) CD103+ mucosal T cells, (B) CD4/CD8 ratio and (C) CD25+FoxP3+ regulatory T cells within the diferent maturation proﬁles (as indicated in Figure 2) in ileum/colon samples of IBD patients and controls.

80 8 30 Patients (%) + Controls (%) + 25 CD 60 6 FOXP3 20 UC high CD103 + 40 4 15 CD25 + 10

20 CD4/CD8 ratio 2 5

Percentage CD3 0 0 0

A B C D A B C D Percentage CD4 A B C D Profiles Profiles Profiles

98 naïve t cells in the gut of ibd patients

Figure 4 Cytokine levels in diferent maturation T-cell proﬁles of patients and controls. Cells derived from colon/ileum biopsies of IBD patients and controls were stimulated overnight with PMA/Ionomycin to assess cytokine production. The levels of TNFa, IFNg, IL17 and IL10 are indicated for the diferent T-cell maturation proﬁles.

60 100 Patients Controls CD 50 80 UC 40 60 30 of total cytokines (%) of total cytokines (%) 40 20

10 20

0 0 Percentage TNF Percentage TNF A B C D A B C D Proﬁles Proﬁles 20 2,5

2 15

1,5 10 1

5 0,5

0 Percentage IL10 of total cytokines (%) 0 Percentage IL17 of total cytokines (%)

A B C D A B C D Proﬁles Proﬁles

B with mostly central memory T cells, was the most predominant. However, we did not observe signiﬁcant diferent proﬁles between CD and UC patients (Figure 2B).

Cytokine levels in diferent maturation T-cell proﬁles of patients and controls In addition to the phenotypic identiﬁcation, cytokine production by mucosal T cells from ileum/colon was characterized by stimulating biopsy-derived cell suspensions for 24h with PMA/ionomycin (Figure 4). The number of lymphocytes in duodenum was too low to perform stimulation assays.

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Supernatants from ileum/colon biopsies containing high numbers of naïve T cells (pro- ﬁle A and B) produced more TNFα compared to the supernatants of biopsies containing

predominantly TEM cells (profile C). TNFα levels as percentage of total measured cytokines from biopsies with T-cell profiles A and B were 30% and 26%, respectively vs. 17 % from biopsies with T-cell profile C (P=0,004) in CD; A 32%, B 20% vs. C 19%; P=0,12 in UC (Figure 4A). In addition, these cells produced lower amounts of IFNγ (A and B 47% vs. C 66%, P=0,003 in CD; A 47%, B 37% vs. C 63% P=0,34 in UC) (Figure 4B). In contrast,

IBD patients with mainly TEM cells (proﬁle C) produced more IL17 and less IL10 compared to the more naïve A and B proﬁles (Figure 4CD).

T-cell maturation profile in CD patients is associated with the clinical phenotype at presentation In order to determine the clinical value of immunophenotypic T-cell profiling in mucosa of IBD patients, we correlated the observed profiles to the clinical phenotype. We found that in CD patients profile A was associated with upper gastrointestinal tract involvement (65%) and perianal disease at presentation (75%). However, there was no association between the immunologic profiles and the disease extension in patients with UC or with the endoscopic disease activity scored by SES-CD and endoscopic MAYO score.

In summary we describe three immunologic subgroups in early stage, untreated, adult IBD patients characterized by diferent numbers of naïve and memory T-cell subsets in the gut mucosa. Remarkably, cytokine levels difer signiﬁcantly between these proﬁles and we observed an association with clinical phenotype at presentation

Discussion

In this study, we have analyzed T-cell subsets in the gut mucosa of a large cohort of newly diagnosed, untreated, adult IBD patients. CD19+ B cells were particularly located in ileum/colon, while CD3+ T cells were the major lymphocytic cell population in duodenum. The T-cell compartment in the ileum/colon samples of the IBD patients was dominated by T cells of non-mucosal origin (CD103-), corroborating the concept of increased recruitment of systemic activated T cells to the inflamed mucosa. Furthermore, high frequencies of CD45RA+CD27+ naïve T cells were identified in the intestinal mucosa of colon/ileum of a subgroup of CD as well as UC patients. + + The subgroup of patients with a high frequency of TN cells (profile A, CD45RA CD27 ) in ileum/colon, showed higher levels of TNFa and lower levels of IFNγ, compared to the - - subgroup with predominant TEM cells (profile C, CD45RA CD27 ).

100 naïve t cells in the gut of ibd patients

Cells derived from IBD patients with mainly TEM cell phenotype (profile C), secreted increased levels of IFNγ and IL17 compared to the other profiles, reflecting a predominant Th1/Th17 response in this subgroup of IBD patients. Furthermore, higher frequencies of naïve T cells in CD patients correlated with more extended and penetrating disease, reflecting a more aggressive presentation. + + In healthy controls, lower frequencies of TN cells (CD45RA CD27 ) and relative higher number of T cells from mucosal origin (CD 103+) were found. These findings, as well as higher frequencies of B cells in colon versus duodenum, are in line with a recent study of human T and B cell compartmentalization in healthy subjects10. Although the paradigm is that only activated and memory T cells have access to non-lymphoid tissues, such as the gut mucosa, it has been shown that naïve T cells are also able to access parenchymal tissues24. Previously, it has been demonstrated that transfer of naïve T cells (CD4+CD45RB+) in T cell-deficient mice can cause an IBD-like syndrome15,16. In humans, there have been a limited number of studies investigating the recruitment of naïve T cells to the gut mucosa of IBD patients, which were all under immunosuppressive treatment. A relative high number of naïve T cells (CD4+CD45RBhigh CD45RA+), producing a high amount of TNFα and a low amount of IFNγ, has been described in the intestinal mucosa of both CD and UC13. This has been confirmed by demonstrating the presence of CD45RA+ T cells in the inflamed mucosa of five UC patients25. Increased levels of truly naïve T cells, which have not encountered cognate antigen for their specific T-cell receptor (also known as recent thymic emigrants (RTE)) have also been described in the mucosa of treated UC patients, irrespective of disease activity26. In contrast, these RTE’s were not found in CD patients. Furthermore, it was recently reported that in IBD patients frequencies of mucosal CD4+CD45RA+ T cells were decreased during treatment with corticosteroids or biologicals, whereas frequencies of early activated (CD4+CD69+) and memory T cells (CD4+CD45RO+) remained unchanged17. This efect of immunosuppressive therapy may explain the discrepancy with the findings of an increased frequency of CD45RA+ cells in our cohort of newly diagnosed, untreated, IBD patients. Irrespective of disease phenotype and T-cell maturation profiles, we have detected a higher number of regulatory T cells (Tregs, CD25highFoxP3+) in colon/ileum of IBD patients compared to controls. In other studies, similar frequencies have been observed in IBD patients during immunosuppressive therapy27,28, suggesting that the frequency of Tregs is not influenced by such treatment. In mouse models it has been shown that Tregs are capable to regulate Th1- and Th17-derived colitis by producing high levels of IL10, which is not yet confirmed in humans29-31. Additionally to IL-10 secreting Tregs, a distinct population of regulatory T cells (FoxP3+) producing IL17 and IFNγ was described in inflamed colon mucosa of CD patients32. Recent data suggest that Th17 and FoxP3+ T cells could be related to or arise from the same precursor, dependent on a distinct

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cytokine environment33-37. Therefore, the exact role of Tregs in the pathogenesis of IBD remains to be resolved. In our cohort, cells derived from ileum/colon biopsies with a more naïve T-cell profile produced higher levels of TNFα compared to biopsies with a more efector memory T-cell profile. This finding corroborates the role of TNFα in the initiation of the immune response, T-cell proliferation and diferentiation described by others38,39. Diferent numbers of naïve and memory T cells in the gut mucosa of IBD patients could reflect diferent stages of the inflammatory process or on diferent T-cell migration and maturation pathways characterizing diferent IBD phenotypes. The efect of neutralizing TNFα during priming of naïve CD4+ T cells was recently studied in vitro. It was shown that anti-TNFα treatment inhibited T-cell activation and polarized naïve T cells towards higher IL-10 and lower IFNγ production40. These findings raise the question if early anti-TNFa therapy in patients with a predominant naïve T-cell maturation profile could beneficially influence the course of the disease. Our data suggest that there are diferences in influx of naïve T cells in the gut mucosa associated with diferent levels of pro-inflammatory cytokines during the early phase of untreated IBD. Further studies will elucidate if the described T-cell maturation profiles reflect diferent disease phenotypes and provide a rationale for the heterogenic clinical presentation of IBD. The long-term follow-up of this patient cohort may reveal the alleged clinical value of these mucosal T-cell profiles, both with respect to disease progression as well as response to immunosuppressive therapy.

Acknowledgements

A. A. van Bodegraven participated in the writing process. C. Smids contributed to creation of artwork.

102 naïve t cells in the gut of ibd patients

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23 Larbi A, Fulop T. From “truly Naive” to “exhausted senescent” T cells: When markers predict func- tionality. Cytometry A. 2013 24 Cose S, Brammer C, Khanna KM, et al. Evidence that a significant number of naive T cells enter non- lymphoid organs as part of a normal migratory pathway. Eur J Immunol. 2006;36:1423-1433 25 Weninger W, Carlsen HS, Goodarzi M, et al. Naive T-cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis. Journal of immunology. 2003;170:4638-4648 26 Elgbratt K, Kurlberg G, Hahn-Zohric M, et al. Rapid migration of thymic emigrants to the colonic mucosa in ulcerative colitis patients. Clin Exp Immunol. 2010;162:325-336 27 Yu QT, Saruta M, Avanesyan A, et al. Expression and functional characterization of FOXP3+ CD4+ regulatory T cells in ulcerative colitis. Inflamm Bowel Dis. 2007;13:191-199 28 Saruta M, Yu QT, Fleshner PR, et al. Characterization of FOXP3+CD4+ regulatory T cells in Crohn’s disease. Clin Immunol. 2007;125:281-290 29 Schmitt EG, Haribhai D, Williams JB, et al. IL-10 produced by induced regulatory T cells (iTregs) controls colitis and pathogenic ex-iTregs during immunotherapy. J Immunol. 2012;189:5638-5648 30 Chaudhry A, Samstein RM, Treuting P, et al. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity. 2011;34:566-578 31 Huber S, Gagliani N, Esplugues E, et al. Th17 cells express interleukin-10 receptor and are controlled by Foxp3(-) and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner. Immunity. 2011;34:554-565 32 Hovhannisyan Z, Treatman J, Littman DR, et al. Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases. Gastro- enterology. 2011;140:957-965 33 Grundstrom J, Linton L, Thunberg S, et al. Altered immunoregulatory profile during anti-tumour necrosis factor treatment of patients with inflammatory bowel disease. Clin Exp Immunol. 2012;169:137-147 34 Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic efector TH17 and regulatory T cells. Nature. 2006;441:235-238 35 Mangan PR, Harrington LE, O’Quinn DB, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006;441:231-234 36 Marie JC, Letterio JJ, Gavin M, et al. TGF-beta1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J Exp Med. 2005;201:1061-1067 37 Koenen HJ, Smeets RL, Vink PM, et al. Human CD25highFoxp3pos regulatory T cells diferentiate into IL-17-producing cells. Blood. 2008;112:2340-2352 38 Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol. 2003;3:745-756 39 Aspalter RM, Eibl MM, Wolf HM. Regulation of TCR-mediated T-cell activation by TNF-RII. J Leukoc Biol. 2003;74:572-582 40 Boks MA, Kager-Groenland JR, Mousset CM, et al. Inhibition of TNF receptor signaling by anti-TN- Falpha biologicals primes naive CD4(+) T cells towards IL-10(+) T cells with a regulatory phenotype and function. Clin Immunol. 2014;151:136-145

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On naivety of T cells in Inﬂammatory Bowel Disease: A review

inflamm bowel dis. 2015 jan; 21(1):167-72

Carolijn Smids1

Carmen S. Horjus Talabur Horje1

Peter J. Wahab1

Marcel J.M. Groenen1

Sabine Middendorp2

Ellen G. van Lochem3

1 Department of Gastroenterology and Hepatology, Rijnstate Hospital, Arnhem, The Netherlands 2 Pediatric Gastroenterology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands 3 Department of Microbiology and Immunology, Rijnstate Hospital, Arnhem, The Netherlands

105

Abstract

Little is known about diferent phases of T-cell maturation in gut mucosa. Based on current knowledge about the migratory pathways of naïve and memory T cells, it is believed that access to peripheral, non-lymphoid tissues is restricted to memory T cells. Surpris- ingly, there is increasing evidence of high numbers of naïve T cells in the chronically inflamed gut tissue of patients with inflammatory bowel disease. This could partially be explained by new formation of ectopic lymphoid organs. Ongoing recruitment of naïve T cells at inflammatory sites might play a role in the immunopathogenesis of inflammatory bowel disease.

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Introduction

Inflammatory bowel diseases (IBD) are believed to result from aberrant inflammatory responses to the intestinal bacterial antigens in a genetically susceptible individual. Available evidence argues an important role for the adaptive immune system in IBD. Most research has focused on increased migration and proliferation of activated or memory T cells (Th1, Th2, Th17 and regulatory T cells) in the inflamed gut mucosa1,2. As

naïve T cells (TN) are normally thought to be excluded from inﬂamed tissue, the maturation process at extra-lymphoid efector sites like the inﬂamed gut mucosa in IBD, has

received less attention. However, TN are the initiators of the inﬂammatory cascade of activated T-cell expansion and diferentiation, and may therefore play a central role in inﬂammatory processes. Lymphoid progenitors originating from bone marrow stem cells undergo maturation and selection in the thymus to become functional CD4+CD8- or CD4-CD8+ (single positive) T lymphocytes. In this thymic selection process, T cells that can recognize peptide in the context of self-major histocompatibility complex (MHC) molecules are positively selected whereas auto-reactive T cells are deleted in the thymus3. Of all T cells enter- 4 ing the thymus, the stringent thymic selection leaves only 3% . The TN cells leaving the thymus still have to experience antigen driven post-thymic maturation before they 5 proliferate and diferentiate into memory T cells . The classical view states that TN cells exclusively recirculate between secondary lymphoid organs (SLOs, e.g. lymph nodes, Peyer’s patches (PPs) and the spleen) through lymph and blood, whereas only memory T cells are able to enter non-lymphoid organs6. However, recent evidence has shown that

TN cells routinely trafc through non-lymphoid organs and might be activated outside 7-16 the conﬁned lymphoid complex . These ﬁndings shed new light on the role of TN cells in autoimmune disease.

This review summarizes the current knowledge of the involvement of TN cells in IBD. We focus on the migration and activation of these cells in the non-lymphoid, healthy and inﬂamed gut.

T-cell subsets: maturation and activation Human T cells can be subdivided into diferent sets, either according to their immunophenotype (expression of leukocyte diferentiation markers) or to their functional- ity (production of chemokines). Considering phenotypic characterization, the two main

categories are TN cells, which have not yet encountered any (foreign) antigen, and memory T cells, which are antigen-experienced. The phenotypic changes during the antigen driven maturation and activation process are depicted in Table 1 and Figure 1.

TN cells can be subdivided in two subsets: recent thymic emigrants (RTEs), also called 4 ‘truly naïve’ and mature naïve T cells (TMN) . RTEs have not yet met the antigen for their

speciﬁc T-cell receptor (TCR) and have no proliferation history. TMN cells are also ‘antigen

108 on naivety of t cells in inflammatory bowel disease: a review

Table 1 Diferent maturation stages of human T cells are distinguished by various cell surface markers.

Surface markers RTE TMN TCM TEM TEMRA

CD31 + − − − −

CD45RA + + − − +

CCR7 + + + − ±

CD62L + + + − −

CD27 + + + ± −

CD28 + + + ± −

CD45RO − − + + −

Immunophenotypical subsets of T cells: RTE, recent thymic emigrant; TMN, mature naïve T cells; TCM, central memory T

cells; TEM, efector memory T cells; TEMRA, efector memory T cells re-expressing CD45RA. Surface markers: CD = cluster of diferentiation; CCR7, chemokine receptor 7, CD62L, L-selectin. Normal expression (+), moderate expression (±), no expression (-).

-inexperienced’ but underwent proliferation in response to homeostatic survival sig- nals17. RTEs can be identiﬁed by the expression of CD45RA, CD31, CD27, CD28, CCR7, CD62L18 and are enriched with TCR excision circles (TRECs)4. TRECs are non-replicative circular excision DNA products of TCR gene rearrangement17,19. Post-thymic proliferation of RTEs causes diminishing of TRECs, downregulation of a set of surface markers 4 (CD31, PTK7) generating ‘proliferative-experienced’ TMN cells .

Figure 1 Human T-cell maturation subsets.

CD45RA CD27 L-selectin

CD45RO CD28 47 CCR7 CD31

RTE TMN TCM TEM TEMRA

Peripheral tissue homing Lymphoid homing Immunophenotypical subsets of T cells by expression of several cell surface markers. Intensity of color of markers indicates level of expression. RTE, recent thymic emigrant; TMN. mature naïve T cells; TCM, central memory T cells; TEM, efector memory T cells; TEMRA efector memory T cells re-expressing CD45RA; CCR7, chemokine receptor 7; CD62L, L-selec tin.

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T cells that have encountered antigen, can immunophenotypically be subdivided

into central memory T cells (TCM), efector memory T cells (TEM) and efector memory

T cells re-expressing CD45RA (TEMRA). During maturation, TCM express CD45RO instead of CD45RA, but retain CCR7 and CD62L, which are important homing markers for lymphoid

tissue. During further maturation, TEM cells downregulate both CCR7 and CD62L and acquire new adhesion markers such as the gut-homing markers α4β7 and CCR920-22. A

subset of efector memory T cells, known as TEMRA cells (re)express CD45RA and occa-

sionally also CCR7, but remain distinguishable from TN cells by their lack of CD27, CD28

and CD62L. When deﬁning TN cell subsets by immunophenotype, the use of at least two markers is recommended18. Diferent combinations have been suggested: CD45RA and CD27/CD2818, or CCR7 and CD2723. Unfortunately, this is not yet common practice and T cells are ofen deﬁned as naïve or memory solely by the expression of CD45RA/

RO. This makes the distinction between TN cells and TEMRA cells practically impossible.

Contamination of the TN cell subset with TEMRA cells may lead to inaccurate conclusions and hamper comparability between studies.

Migration of naïve T lymphocytes during homeostasis Migration of immune cells is a process referred to as homing. The general belief before 1964 was that lymphocytes randomly circulate through the body. The first signs hinting otherwise were found in rats and mice24,25, showing that certain lymphocytes only homed to the lymph node or the spleen. Thirty years later, these findings were confirmed in sheep. Additionally distinct migratory routes for naïve and memory T cells were suggested6. Most of our further understanding on T-cell migration and proliferation is still based on research in animal models although it is obvious that these models (with a lifespan of several months up to a few years, in pathogen-free conditions) cannot reflect the human exposure to multiple pathogens over decades26. Human studies are still limited, as only peripheral blood or tissues from surgical resection material are easily accessible. The formerly accepted migration model with distinct routes for naïve and memory

T cells turns out not to be as segregated as suggested before. TN cells do not only circulate through blood and lymph between SLOs but also enter non-lymphoid organs,

which were first thought to be exclusively confined to memory T cells. TN cells have recently been identified in non-lymphoid tissue of healthy individuals, representing approximately 10% of the T cells at the intestinal sites (jejunum, ileum and colon)14. Ad- ditionally, up to 50% of the T cells in non-lymphoid organs of the gut in steady-state 10 mice were shown to express CCR7 (TN, TCM, and TEMRA) . An immune-surveillance or

tolerance-induction role of TN cells entering the non-lymphoid tissues has been advo- cated11. Although central tolerance during thymic selection eliminates most of the auto- reactive T cells, some of them might escape and migrate to the periphery. In this way,

naïve auto-reactive T cells (RTEs and TMN) could enter healthy tissues; become activated

110 on naivety of t cells in inflammatory bowel disease: a review

and initiate chronic inflammation. On the other hand, influx of TN cells in non-lymphoid tissue possibly induces tolerance to organ-specific antigens within the tissue itself17.

Antigen-presentation to TN cells takes place primarily in the SLOs. Following antigen

recognition, further maturation of TN cells evolves towards memory T cells, which ex-

press tissue-speciﬁc surface receptors. TCM cells express the same lymph node homing

molecules as TN cells, namely L-selectin (CD62L) and chemokine receptor 7 (CCR7, also 27 known as CD197), and therefore also have the potential to home to SLOs . TEM cells are CCR7-CD62L- and express tissue homing molecules like α4β7 which binds to mucosal adressin-cell adhesion molecule-1 (MAdCAM-1), and CCR9 which interacts with chemokine (C-C motif) ligand 25 (CCL25) in the gut endothelium12.

TN cells usually do not express tissue-speciﬁc homing receptors on their surface, with exception of moderate levels of α4β7. They enter the lymph node and PPs across specialized post-capillary venules called high endothelial venules (HEV). HEV endothelial cells are cuboidal and are surrounded by multiple layers of ﬁbroblastic reticular cells that produce various extracellular matrix components forming a thick basal lamina. Micro-

Figure 2 Migration of mature naïve (TMN) and central memory (TCM) T cells during homeostasis

TMN TMN TCM TCM

L-selectin LFA-1 L-selectin LFA-1

CCR7 CCR7 1

2 3 47 2 3 1b 1a

PNAd CCL21 ICAM-1 MAdCAM-1 CCL21 ICAM-1 a. High Endothelial Venule in lymph node b. High Endothelial Venule in Peyer’s patch

a TMN and TCM cells migrate into a lymph node by interaction with surface receptors on high endothelial venules (HEV). (1) CD62L binds with peripheral lymph node adressin (PNAd); (2) chemokine receptor 7 (CCR7) binds with chemokine (c-c motif) ligand 21 (CCL21) and (3) lymphocyte function-associated antigen 1 (LFA-1) binds with intercellular adhesion molecule 1 (ICAM-1). b TMN and TCM cells migrate into Peyer’s patches by interaction with surface receptors on HEV. Tethering and rolling is mediated by interaction of mucosal adressin-cell adhesion molecule 1 (MAdCAM-1) and CD62L (1a) and αβ7 integrin (1b). Further adhesion and migration occurs through CCR7 with CCL21 (2) and LFA-1 with ICAM-1 (3).

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scopically they can easily be distinguished from normal venules, which have ﬂat endothelium and a thin basal lamina28. HEV endothelium expresses molecules that function in lymphocyte trafcking. This occurs through interaction between surface molecules on the lymphocytes with their HEV-expressing ligand counterparts: CD62L on the T cells with peripheral lymph node addressin (PNAd) and MAdCAM-1; CCR7 on the T cells with CCL21; and lymphocyte function-associated antigen 1 (LFA-1, also known as CD11aCD18) on the T cells with intercellular adhesion molecule 1 (ICAM-1 also known as CD54) Fig- ure 2a12,29. In contrast to HEV in peripheral lymph nodes, HEV in PPs do not express PNAd. In PPs, the tethering/rolling step is mediated by the interaction of CD62L and α4β7 with MAdCAM, while further adhesion and migration of T cells happens through LFA-1 and ICAM-1 Figure 2b30. The binding of CD62L to MAdCAM-1 is dependent on the presence of speciﬁc carbohydrate ligands on the MAdCAM-131,32. Evidence supporting this interaction is demonstrated in mice studies, but is still missing for human.

Migration of naïve T lymphocytes in IBD

Tertiary Lymphoid Organs There is increasing evidence for primary T-cell responses independent of SLOs. Chronic inflammation and solid tumors have been found to induce formation of HEV-like vessels in non-lymphoid inflamed tissue and tumor tissue, organizing around so called tertiary lymphoid organs (TLOs)33-37. These TLOs are ectopic accumulations of lymphoid cells, arising at a non-lymphoid location, through a process called lymphoid neogenesis. Contrarily, primary and secondary lymphoid organs are found at specific, predefined, sites that seem to be genetically fixed and develop before birth38. TLOs morphologically resemble SLOs, since they also have T and B cell compartments, lymphoid chemokines, antigen presenting cells (like dendritic cells) and HEV-like vessels and are therefore also

able to recruit TN cells. How and why TLO formation occurs is unclear. HEV-like vessels presumably facilitate

lymphocyte influx into TLOs, but it is also possible that TN cells gain initial access to non-lymphoid tissues via regular blood vessels and that HEVs are formed later in the 8 process . Lymphotoxin α1β2 (LTα1β2, TNF family member) may have a prominent role in regulating the formation of (secondary and tertiary) lymphoid organs and is crucial 39,40 + for HEV development and maintenance . Afer priming, TN CD4 cells induce LTα1β2 expression on their surface41,42. The presence of ectopic CCL21 on blood vessels dur- + ing inflammation may trigger the influx of CCR7 T cells (TN and TCM). This provides a

source of LTα1β2, perpetuating CCL21 expression and facilitating the formation of HEV- like vessels and TLOs. Notably, blocking the Lymphotoxin (LT) pathway in colitis mouse models reduced the severity of inﬂammation and was equally efcacious as anti-TNFα therapy43,44.

112 on naivety of t cells in inflammatory bowel disease: a review

Clinical signiﬁcance of TLOs has already been described in other diseases like colorectal cancer and rheumatoid arthritis. In early-stage colorectal cancer, TLOs are associated with a better prognosis45 and are suggested as a possible marker for prognosis46. In patients with rheumatoid arthritis TLOs were present in 49% of synovial tissue and its presence coincided with longer disease duration and lower response to therapy (more refractory disease)47. Reversal of TLOs was a good marker of response to therapy.

Tertiary lymphoid organs in IBD Recent studies have shown that TLOs might play a role in chronic inflammation of the gut in IBD. Ectopic CCL21 on blood vessels and PNAd-expressing HEV-like vessels has been detected in inflamed gut tissue of patients with ulcerative colitis (UC). A positive correla- 8 tion between CCL21 expression and influx of TN cells was observed . CCL21 production was demonstrated in inflamed tissue of patients with Crohn’s disease (CD) and UC, and lymphoid aggregates were found in 50% of their intestinal biopsy samples35. HEV-like vessels in the active phase of UC and CD were identified only in inflamed gut tissue, compared to a scarce presence of these vessels in non-inflamed colon or during remission of IBD48,49. Furthermore, the formation of ectopic lymphoid follicles or TLOs was found in diseased ileal segments of CD patients50. In contrast to the chronically inflamed IBD tissue, acute inflammation was not accompanied by the induction of lymphoid aggregates and HEV-like vessels48. MAdCAM-1+ intestinal lamina propria venules are increased in UC, both in active disease and remission, compared to normal controls51. There was great co-expression of PNAd among MAdCAM-1+ vessels in patients with active disease (66.5%) when compared to patients in remission (7.62%) and PNAd was not detected in the colonic mucosa of normal controls. In a recent study in mice with chronic ileitis, the induction of ectopic lymphoid tissue has been demonstrated in the inflamed intestine, with a statistically significant increase of CCL21 expression and accumulation of TN cells, 15 TEM and TCM cells in the terminal ileum, compared to healthy wild type mice . Thus far, it remains difcult to distinguish gut-associated lymphoid tissue (GALT) from TLOs and an unambiguous definition is lacking33. Nonetheless, chronically inflamed gut induces organized structures that cannot be observed in the gut of healthy controls. These structures, referred to as TLOs, are present in the deeper layers of the epithelium and contain PNAd-expressing HEV-like vessels, that distinguishes them from the submucosal PPs48,50,51.

Naïve T cells in IBD

Mice: the T-cell transfer model Immunodeficient mice (CB17 severely combined immunodeficient/recombinase acti- vating gene-1-/-) develop chronic, severe intestinal inflammation, afer transfer of na-

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ïve (CD4+CD45RBhigh) donor T cells into their peritoneal cavity52. Co-transferring memory (CD4+CD45RBlow) T cells inhibits the development of intestinal inﬂammation53 and single transfer of the memory subset does not cause inﬂammation54. One could expect that

the transferred TN cells are primed within GALT and mesenteric lymph nodes (MLNs)

before inducing colitis. However, the induction of colitis by transfer of TN cells was also demonstrated in mice deﬁcient of SLOs (GALT, MLN and spleen)55. This study underlines

that SLOs are not required for priming TN cells into colitogenic efector T cells to initiate colitis, leaving the site of priming in these models to be deﬁned. As the pattern of exacer- bation and remission of human IBD has not been perfectly phenocopied in these mouse 56 models, the presence and the role of TN cells in the gut of IBD remains to be elucidated .

The potential role of naïve T cells in human IBD

The ﬁrst suggestions indicating a potential role of (maturation of) TN cells in the pathogenesis of IBD came from a few (case-)reports showing the efectiveness of thymectomy in adult patients with IBD57-60. Thymectomy has been performed in UC patients with thymic hyperplasia and was successful for maintaining remission in patients refractory to conventional therapy58,60. Additionally, thymectomy was also performed in a CD patient developing myasthenia gravis, leading to remission of her CD activity59. No further publications have arisen on the potential of thymectomy in IBD. Already in 1995 it was revealed that chronic inﬂammation in CD induced migration

of TN cells to inflamed sites. The study involved ileal samples of CD patients and bowel samples of controls with colon carcinoma. In the intestines of controls, blood vessels were only found in the lamina propria and beneath the muscularis mucosa, and they exhibited the characteristics of normal (flat) venules. The inflamed intestines of CD patients showed numerous dilated and highly activated small vessels with HEV characteristics, extending from the superficial mucosa layers throughout the deep submucosa. These vessels and the perivascular and mucosal infiltrates all harbored mainly macrophages and T cells. The majority of T cells within the (sub)mucosal infiltrates were + + + + + - + + TN (CD45RA CD31 CD62L CD27 ) and TCM (CD45RO CD31 CD62L CD27 ) cells. The normal + - intestinal mucosa of controls was predominantly populated by TEM cells (CD45RO CD31 CD62L- CD27-)7. The expression of maturation markers on CD4+ T cells was analyzed in the intestinal mucosa of both CD and UC patients with active disease. Almost all patients were using corticosteroid medication and were included at the time they underwent partial resection of the intestine. Non-involved bowel segments of patients undergoing bowel resection for cancer treatment were used as control. The numbers of CD45RA+ T cells were increased in the intestine of IBD patients (49.0% in UC and 27.7% in CD) as compared to controls (4.5%). The CD45RA+ cells found in IBD patients were mainly located in ‘follicular structures’9.

114 on naivety of t cells in inflammatory bowel disease: a review

Another study aimed to characterize T cells present in the intestine of IBD patients before and afer therapy. Sigmoid-derived biopsy specimens of IBD patients (CD as well as UC) with distal active colitis were evaluated. Patients that were in remission with mucosal healing showed a reduction of >50% of CD4+CD45RA+ T cells, compared to inflamed mucosa of patients with active disease. There was no diference in the amount of memory T cells in the inflamed mucosa when compared to non-inflamed mucosa16. Regarding RTEs in IBD patients, increased TREC levels in mucosal but not peripheral blood lymphocytes were found in UC patients, indicating direct recruitment of RTEs into the inflamed mucosa13. However these results have not yet been duplicated. We recently studied the maturation of T cells in the gut mucosa in a large group of newly diagnosed, untreated IBD patients. Remarkably high frequencies of TN were identified in a subgroup of CD as well as UC patients (naïve profile). This group of patients expressed diferent pro-inflammatory cytokine levels (higher TNF-alpha and lower interferon-gamma) compared to the group of patients with high frequencies of TEM. Furthermore, there was an association between the naïve profile and extended disease location in CD61. We elaborated on the possible correlation between diferences in the maturation status of the T-cell subsets early in disease course and disease heterogeneity. An extended follow-up of this cohort may elucidate the prognostic value of diferent maturation profiles at primary diagnosis.

Conclusions

Contrarily to previous ideas, there is increased evidence that TN cells migrate to non- lymphoid organs in homeostasis and even more in chronic inﬂammation. During chronic inﬂammation, migration of TN cells could be facilitated by TLOs. Formation of these

TLOs appears dependent on LTα1β2; however the signals triggering these mechanisms are waiting to be characterized. Presence of TLOs implies an ongoing recruitment of TN and TCM cells to the gut mucosa, maintaining a continuous inﬂammatory process. The relevance of targeted therapy against this phenomenon has not yet been studied. It is conceivable that a process involving dissolution of TLOs could restore immunologic homeostasis.

Acknowledgement

We want to thank dr. A. A. van Bodegraven for his helpful suggestions during writing the manuscript.

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46 Pimenta EM, Barnes BJ. Role of Tertiary Lymphoid Structures (TLS) in Anti-Tumor Immunity: Po- tential Tumor-Induced Cytokines/Chemokines that Regulate TLS Formation in Epithelial-Derived Cancers. Cancers (Basel). 2014;6:969-997 47 Canete JD, Celis R, Moll C, et al. Clinical significance of synovial lymphoid neogenesis and its reversal afer anti-tumour necrosis factor alpha therapy in rheumatoid arthritis. Ann Rheum Dis. 2009;68:751-756 48 Suzawa K, Kobayashi M, Sakai Y, et al. Preferential induction of peripheral lymph node addressin on high endothelial venule-like vessels in the active phase of ulcerative colitis. Am J Gastroenterol. 2007;102:1499-1509 49 Kobayashi M, Fukuda M, Nakayama J. Role of sulfated O-glycans expressed by high endothelial venule-like vessels in pathogenesis of chronic inflammatory gastrointestinal diseases. Biol Pharm Bull. 2009;32:774-779 50 Sura R, Colombel JF, Van Kruiningen HJ. Lymphatics, tertiary lymphoid organs and the granulomas of Crohn’s disease: an immunohistochemical study. Aliment Pharmacol Ther. 2011;33:930-939 51 Kobayashi M, Hoshino H, Masumoto J, et al. GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitis. Inflamm Bowel Dis. 2009;15:697-706 52 Leach MW, Bean AG, Mauze S, et al. Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells. Am J Pathol. 1996;148:1503-1515 53 Morrissey PJ, Charrier K. Induction of wasting disease in SCID mice by the transfer of normal CD4+/ CD45RBhi T cells and the regulation of this autoreactivity by CD4+/CD45RBlo T cells. Res Immunol. 1994;145:357-362 54 Aranda R, Sydora BC, McAllister PL, et al. Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients. J Immunol. 1997;158:3464-3473 55 Takebayashi K, Koboziev I, Ostanin DV, et al. Role of the gut-associated and secondary lymphoid tissue in the induction of chronic colitis. Inflamm Bowel Dis. 2011;17:268-278 56 Maynard CL, Weaver CT. Intestinal efector T cells in health and disease. Immunity. 2009;31:389-400 57 Tsuchiya M. Immunological abnormalities involving the thymus in ulcerative colitis and therapeutic efects of thymectomy. Gastroenterol Jpn. 1984;19:232-246 58 Tsuchiya M, Hibi T, Watanabe M, et al. Thymectomy in ulcerative colitis: a report of cases over a 13 year period. Thymus. 1991;17:67-73 59 Finnie IA, Shields R, Sutton R, et al. Crohn’s disease and myasthenia gravis: a possible role for thymectomy. Gut. 1994;35:278-279 60 Okubo K, Kondo N, Okamoto T, et al. Excision of an invasive thymoma: a cure for ulcerative colitis? Ann Thorac Surg. 2001;71:2013-2015 61 Horjus Talabur Horje CS, Middendorp S, van Koolwijk E, et al. Naive T cells in the gut of newly diagnosed, untreated adult patients with inflammatory bowel disease. Inflamm Bowel Dis 2014;20:1902-9.

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High endothelial venules associated with naïve and central memory T cells in the inﬂamed gut of newly diagnosed Inﬂammatory Bowel Disease patients

submitted

Carmen S. Horjus Talabur Horje1

Carolijn Smids1

Jos W.R. Meijer2

Marcel J. Groenen1

Maaike K. Rijnders2

Ellen G. van Lochem3

Peter J. Wahab1

1 Department of Gastroenterology and Hepatology, Rijnstate Hospital, Arnhem, the Netherlands 2 Department of Pathology, Rijnstate Hospital, Arnhem, the Netherlands 3 Department of Microbiology and Immunology, Rijnstate Hospital, Arnhem, the Netherlands

119

Abstract

background Naive and central memory T lymphocytes (TN and TCM) can infiltrate the inflamed gut mucosa in Inflammatory Bowel Disease (IBD) patients. The homing of these subsets to the gut might be explained by ectopic formation of tertiary lymphoid organs (TLOs), containing high endothelial venules (HEVs). We aimed to evaluate the presence of HEVs and TLOs in inflamed intestinal mucosa of newly diagnosed, untreated, IBD patients in relation to the presence of TN and TCM lymphocytes. methods IBD patients (n=39) and healthy controls (n=8) were prospectively included. Biopsy samples of respectively inflamed and normal intestine were analyzed by immunohistochemistry for lymphocytes (CD3/CD20), blood vessels (CD31) and PNAd expression (MECA-79). TN and TCM lymphocyte subsets were identified by flowcytometric immunophenotyping. results A higher number of HEVs was found in the inflamed colon of patients with ulcerative colitis (median density 3.05 HEV/mm2; (IQR) 0-6.39) and ileum of Crohn’s disease patients (1.40 HEV/mm2; IQR 0-4.34) compared to healthy controls (both 0 HEV / mm2; P=0.033). A high density of colonic HEVs (HEVhigh) was associated with increased numbers of TN and TCM in the inflamed gut (median 87%; IQR 82-93% of T-cell population), compared to HEVlow patients (58%; IQR 38-81%; P=0.003). The number of colonic follicles was higher in HEVhigh patients (median 0.54/mm2; IQR 0.28-0.84) compared to HEVlow patients (median 0.25/mm2; IQR 0.08-0.45; P=0.031) and controls (0.31/mm2; IQR 0.23-0.45; P=0.043). conclusion The presence of extra-follicular HEVs and TLOs was strongly associated with TN and TCM cell mucosal infiltration in a subgroup of newly diagnosed IBD patients.

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Introduction

Lymphocyte activation and homing of lymphocytes to target organs are key phenom- ena in chronic inﬂammation, including Inﬂammatory Bowel Diseases (IBD). Lymphocyte activation is triggered by interaction of antigen receptors with cognate antigens. The

initial priming of naive T lymphocytes (TN) occurs in secondary lymphoid organs (SLOs; spleen, lymph nodes, gut associated lymphoid tissue (GALT; Peyer’s patches, isolated lymphoid follicles and appendix)). SLOs are genetically encoded specialized environ- ments for optimal antigen presentation and lymphocyte activation. Upon antigen priming in the SLO, activated T lymphocytes migrate to non-lymphoid tissue sites, where they may exercise efector (pro-inﬂammatory or regulatory) functions upon renewed

antigen encounter. It is well known that activated efector memory T lymphocytes (TEM) travel to the gut mucosa following α4β7/MAdCAM-1 and CCR9/CCL25 interaction at the level of post-capillary venules1,2.

In physiological conditions, TN and central memory T lymphocytes (TCM) are thought to migrate exclusively to SLOs1,3. Specialized vessels, so-called high endothelial venules

(HEVs), direct tissue migration of circulating TN and TCM into SLOs. This process is guided by diferent vascular addressins, such as MAdCAM-1 and peripheral lymph node addressin (PNAd). PNAd on endothelial cells is the ligand of L-selectin, which is expressed

on TN and TCM lymphocytes, while practically absent on the majority of TEM or efector

memory T cells re-expressing CD45RA (TEMRA). PNAd can be identiﬁed by the monoclonal antibody MECA-794,5. Distribution of these addressins difers between peripheral lymph node, mesenteric lymph node and Peyer’s patches. MAdCAM-1 is constitutively displayed on HEVs in Peyer’s patches and mesenteric lymph node and on ﬂattened ven- ular endothelial cells in the intestinal lamina propria6. PNAd is predominantly present on HEVs in peripheral lymph node, mesenteric lymph node and at very low levels on the abluminal side of HEVs in Peyer’s patches4.

Previously we have demonstrated the presence of TN and TCM lymphocytes in non- lymphoid gut tissue7-11. In our previous work we identiﬁed three subgroups of newly 7 diagnosed IBD patients with either increased percentage of mucosal TN, TCM or TEM . It

is still unclear how homing of these TN and TCM lymphocytes to non-lymphoid tissue is established. Processes occurring during chronic inflammation and formation of solid tumors were shown to induce ectopic formation of HEV-like vessels in non-lymphoid inflamed and tumor tissue, constituting the so-called tertiary lymphoid organs (TLOs)12-15. Little is known about HEVs in IBD pathogenesis (reviewed in16), since published data is limited to patients with long-standing disease who are commonly using anti-inflammatory treatment5,17. We aimed to investigate the presence of HEVs and their relation to lymph follicles in the inflamed intestinal mucosa of newly diagnosed, untreated IBD patients. Since 7,18,19 TN and TCM lymphocytes are known to express L-selectin , we aimed to determine

122 hevs associated with t-cell subsets in ibd

whether the inﬁltration of these cells in the inﬂamed intestinal mucosa of newly diagnosed, untreated, IBD patients is correlated with the presence of HEVs and TLOs.

Material and methods

Patients and samples At the department of Gastroenterology and Hepatology of the Rijnstate Hospital, Arn- hem, The Netherlands, 39 newly diagnosed adult IBD patients and eight healthy controls were prospectively included in the study. The diagnosis of IBD was based on a combination of clinical, endoscopic, histologic and radiologic internationally accepted criteria. Intestinal biopsy specimens of the macroscopically most inflamed ileal and colonic mucosa (when ulcerations were present these biopsy specimens were taken from the edge of this ulceration) were obtained at the moment of diagnosis, before the initiation of any medical treatment. Thus, when disease was located in the ileum, only ileal biopsies were analyzed; when disease was located in the colon, only colonic biopsies were analyzed and when the disease was located in ileum and colon both ileal and colonic biopsies were analyzed. Besides regular histopathological analysis, four samples were used to immunophenotype the mucosal lymphocyte population. Patient characteristics were documented and disease phenotype was assessed. Ileal and colonic biopsy specimens were obtained from eight control subjects undergoing ileocolonoscopy for iron deficiency anemia or polyp surveillance. Biopsy specimens of controls were taken from macroscopically non-afected, non-inflamed ileal and colonic areas. Regular histopathological examination confirmed that these areas had no signs of inflammation. Besides regular histopathological analysis, four samples were used to immunophenotype the mucosal lymphocyte population. Parallel venous blood samples were drawn following endoscopic evaluation at primary diagnosis. Serum samples were coded and stored at -20°C until analysis.

Immunohistochemistry Immunohistochemistry was carried out on 3 µm thick sections from formalin-fixed, parafn-embedded archived blocks of biopsied specimen of ileal and colonic mucosa. The following monoclonal antibodies were used for immunostaining: MECA-79 (identifies 6-sulfo sialyl Lewis on core-1 branched O-linked sugars (PNAd) which binds L- selectin, Sanbio, Uden, The Netherlands), CD31 (monoclonal, identifies human CD31 to demonstrate endothelial cells, Sanbio, Uden, The Netherlands), CD3 (clone LN10, identifies human CD3 to demonstrate T cells, Leica Microsystems B.V., Eindhoven, The Netherlands) and CD20 (clone L26, identifies human CD20 to demonstrate B-cells, Leica Microsystems B.V., Eindhoven, The Netherlands). Slides were incubated with the primary antibody in an automatic immunostainer (Bond, Leica biosystems Nussloch GmbH 2016, Eindhoven, The Netherlands).

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Stained slides were scanned with an Intellisite high-resolution scanner (Philips ultra- fast scanner 1.6 RA, Philips Digital Pathology, Best, The Netherlands) and analyzed with the Intellisite IMS image analyzer. Quantification was done by two independent observ- ers who were blinded to the clinical outcome and flowcytometric analyses. The total surface area of each slide was measured in square millimeter (mm2). Absolute numbers of CD31+ vessels and MECA-79+ HEV-like vessels were determined by optical counting of vessel numbers on all slides. The location of HEVs vessels relative to lymphoid follicles was determined on CD3 and CD20 immunostained sections and absolute numbers of intra- and extrafollicular HEVs were quantified. The density of extrafollicular HEVs vessels (HEVs/mm2 excluding the surface of follicles) and the ratio of HEVs vessels amongst all vessels (HEVs/CD31 vessels) were calculated. In malignancy, a cut-of point for increased density of HEVs has been described in patients with breast cancer.20 However, in chronic inflammation, no such approach has been undertaken. We discriminated patients with a high and a low density of HEVs using a cut-of point set at the median density of extrafollicular HEVs vessels/mm2 in the inflamed colon of UC patients. Presence of inflammation was established for all sections based on known architectural changes and inflammatory features such as crypt distortion, decreased crypt density, erosions, ulcers, increased lamina propria cellularity, crypt abscesses and epithelioid granuloma. Lymphoid follicles were defined as a dense nodular lymphocyte aggregate containing B and T-cell compartments. Their presence was assessed on CD3 and CD20 immunostained sections. The number of follicles was counted. In absence of a clear-cut definition of TLOs in the gut, we defined TLO as additional follicles in patients compared with the number of follicles in controls. Furthermore, it is our interpretation that the presence of extrafollicular HEVs represents an early phase of lymphoid neogenesis (TLOs).

Immunophenotyping the mucosal lymphocytes Intestinal biopsy specimen were sampled and analyzed as described earlier7. Briefly, besides the standard histopathological analysis of biopsy specimens, four samples of the most inflamed regions of colon and ileum were used for flowcytometric immunophenotyping. Biopsy specimen were kept in phosphate-bufered saline solution at 2-8°C and processed within 8 hours. Specimens were finely minced in Hanks’/1% bovine serum albumin using a 70-mm gaze and spatula followed by Ficoll density gradient centrifugation. The homogenate was resuspended, afer washing, in 0.5 mL Hanks’/1% bovine serum albumin. The cell number was estimated by microscopic counting with a KOVA glasstic slide (Hycor Biomedical Ltd., Penicuik, United Kingdom). For flow cytometric analysis 200 mL of the total cell suspension was used, disregarding absolute cell number. Single cell suspensions of the biopsies were analyzed using a FACS Canto (BD Biosciences). Mucosal lymphocyte subpopulations were diferentiated: B cells (CD19+), T cells (CD3+),

124 hevs associated with t-cell subsets in ibd

regulatory T cells (CD3+CD25highFoxP3+), mucosal T cells (CD3+CD103+), T-helper cells (CD3+CD4+) and cytotoxic T cells (CD3+CD8+) and expressed as percentages of the whole lymphocyte population (CD45+/low side scatter). The maturation state of T cells was + + assessed by using CD45RA and CD27, deﬁning naive T cells (TN: CD45RA CD27 ), central - + - - memory T cells (TCM: CD45RA CD27 ), efector memory T cells (TEM: CD45RA CD27 ) and + - T efector memory cells re-expressing CD45RA (TEMRA: CD45RA CD27 ). We also analyzed groups of T cells according to their known migration phenotypes. TN and TCM lymphocytes are known to enter HEVs through the ligand PNAd, as they are L-selectin+18,19. and were analyzed both independently as well as taken together. The same analysis was -18,19 performed for TEM and TEMRA lymphocytes, which are known to be L-selectin .

Multiplex immunoassay In a subgroup of the current patient cohort we analyzed serum chemokines using Mul- tiplex immunoassays, including CXCL-13 and CCL-19, which are known to be required for lymphoid organogenesis21. These assays were performed at the MultiPlex Core Facility of the Laboratory of Translational Immunology (UMC Utrecht, The Netherlands) using an in-house validated platform (ISO9001). Briefly, color-coded magnetic beads (MagPlex Microsperes, Luminex, Austin, Texas) were conjugated to analyte-specific antibodies and incubated with standard dilutions or sample for 1 hour during continuous shaking in the dark. Pre-treatment of samples with HeteroBlock (Omega biologicals, Inc., Boze- man, Montana, USA) was performed, to prevent interference by binding of heterophilic antibodies. Plates were washed (Bio-Plex Pro II Wash Station; Bio-Rad, Hercules, Califor- nia, USA) and a corresponding cocktail of biotinylated detection antibodies was added followed by streptavidin-phycoerythrin (PE) incubation. Fluorescence intensity of PE was measured with a Flexmap 3D system (Luminex) and analysed by BioPlex Manager Sofware (version 6.1; Bio-Rad, USA) using 5-parameter curve fitting.

Statistical analysis Data of ileal biopsy specimens of CD patients was compared to ileal biopsy specimens of healthy controls. The data of colonic biopsy specimens of UC and CD patients were compared to the colonic biopsy specimens of healthy controls. Ileum and colon were not mutually compared because of anatomical and physiological diferences, including the influence of the microbiome. Data was collected and analyzed with SPSS statistics (version 21.0.0.0; IBM Corp). Diferences between groups were tested using non-parametric tests. Continuous variables were described as median with interquartile range (IQR, 1st quartile-3rd quartile). Mann-Whitney U test was used to identify diferences in continuous variables. Categorical variables were described as absolute frequencies. Kruskal- Wallis analysis was used followed by the Dunn test when applicable. Spearman rank correlation was performed to measure the degree of association between variables. Statistical significance was defined as a P-value less than 0.05.

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Ethical Considerations Written informed consent was obtained from each participating patient. The procedures were performed in accordance with the Declaration of Helsinki. The regional medical ethics committee approved the protocol, according to Dutch Law (NL28761.091.09).

Results

Ten newly diagnosed patients with ulcerative colitis (UC) and 29 newly diagnosed patients with Crohn’s disease (CD) were included in this study. In addition eight healthy controls were studied. Baseline characteristics of patients are outlined in Table 1.

Table 1 Baseline characteristics

CD (n=29) CU (n=10)

Age at diagnosis in years, median (IQR) 28 (20-44) 28 (20-35)

Female, n (%) 21 (72%) 7 (70%)

Symptoms before diagnosis in months 0-3 10 (35%) 7 (70%) 3-6 9 (31%) 1 (10%) >6 10 (35%) 2 (20%)

Follow-up period in months, median (IQR) 55 (23-62) 44 (22-48)

Family with IBD, n (%) 4 (14%) 4 (40%)

Smoking status • Never 17 (59%) 8 (89%) • Current 10 (34%) 1 (11%) • Ceased 2 (7%) - • Unknown - -

HBI score, median (IQR) 9 (6-13) -

CD disease location (Montreal) - • Ileal, n (%) 8 (28%) • Colonic, n (%) 5 (17%) • Ileocolonic, n (%) 16 (55%)* • + Upper GI involved, n (%) 10 (39%) • + Perianal disease, n (%) 5 (17%)

CD disease behavior (Montreal) - • Inﬂammatory, n (%) 21 (72%) • Stricturing, n (%) 7 (24%) • Penetrating, n (%) 1 (4%)

126 hevs associated with t-cell subsets in ibd

SES-CD score, median (IQR) 14 (8-22) -

SES-CD severity - • Mild (4-10), n (%) 11 (38%) • Moderate (11-19), n (%) 9 (31%) • Severe (>19), n (%) 9 (31%)

UC clinical score (Montreal) - • Remission, n (%) - • Mild, n (%) 1 (10%) • Moderate, n (%) 4 (40%) • Severe, n (%) 5 (50%)

UC disease location (Montreal) - - • Proctitis, n (%) - • Lef-sided, n (%) - • Pancolitis, n (%) 10 (100%)

UC endoscopic severity (Mayo) - • Normal, n (%) - • Mild, n (%) 2 (20%) • Moderate, n (%) 4 (40%) • Severe, n (%) 4 (40%)

CD = Crohn’s disease, UC = ulcerative colitis, IQR = interquartile range, IBD = inﬂammatory bowel disease, HBI= Harvey-Bradshaw Index, SES-CD = simple endoscopic score for Crohn’s disease * One patient was excluded from the analyses of colon biopsies because of technical failure of MECA-79 staining.

Number of blood vessels and follicles was comparable in patients and controls In each sample, we determined the absolute number of blood vessels (CD31+ vessels and MECA-79+, hereafer denoted as HEVs) and follicles by immunohistochemical staining. HEV-like vessels were determined by optical counting of MECA-79+ vessel numbers on all slides. The location of HEVs relative to lymphoid follicles was determined on CD3 and CD20 immunostained sections and absolute numbers of intra- and extrafollicular HEVs were quantified. The density of extrafollicular HEVs (MECA-79+/mm2 excluding the surface of follicles) and the ratio of HEVs amongst all vessels (MECA-79+/CD31 vessels) were calculated. Overall we observed high heterogeneity in patient results. No statistical significant diference was found when comparing the entire patient group and controls for the number of vessels or follicles in biopsy specimen (see Table 2). In addition, the number of HEVs/CD31+ vessels within these follicles was also comparable. However, in UC there was a trend towards more HEVs in inflamed colon (4.80/mm2, (0.45-6.14)) than in the colon of healthy controls (0.71/mm2, (0-1.85), P=0.109).

Increased extrafollicular PNAd expression in patients compared to controls Next, we calculated the percentage and density of HEVs/CD31+ vessels outside the lymphoid follicles (extrafollicular) and found that control colon samples did not show any

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Table 2 Blood vessels and follicles in gut mucosa of IBD patients and controls

CD UC Healthy controls P-value (inflamed) (inflamed) (non-inflamed)

Number of CD31+ vessels/mm2 84 (60-107) 125 (63-139) 71 (43-89) NS colon, median (IQR)

Number of CD31+ vessels/mm2 93 (70-114) - 87 (69-102) NS ileum, median (IQR)

Number of HEVs/mm2 colon, 0.35 (0-5.17) 4.80 (0.45-6.14) 0.71 (0-1.85) NS median (IQR)

Number of HEVs/mm2 ileum, 1.58 (0.34-6.69) - 1.23 (0-5.19) NS median (IQR)

% PNAd expressing CD31+ vessels 0.85 (0-4.80) 4.63 (0.38-12.24) 0.79 (0-2.59) NS colon, median (IQR)

% PNAd expressing CD31+ vessels 2.02 (0.39-6.82) - 1.02 (0-8.99) NS ileum, median (IQR)

Colonic follicles/mm2 tissue, 0.27 (0.12-0.51) 0.51 (0-0.82) 0.31 (0.23-0.45) NS median (IQR)

Ileal follicles/mm2 tissue, 0.35 (0.15-0.57) - 0.37 (0-0.55) NS median (IQR)

CD = Crohn’s disease, UC = ulcerative colitis, IQR = interquartile range

Figure 1a Density of extra-follicular PNAd expressing HEV-like vessels/mm2 in colon

10,00 p=0.033

8,00

p=0.092 6,00

4,00

2,00 HEV-like vessels/mm2 in colon 0,00 Density of extra-follicular PNAd expressing Control Crohn’s Disease Ulcerative Colitis Type IBD

128 hevs associated with t-cell subsets in ibd

Figure 1b PNAD expressing HEV-like vessels in colonic tissue

B1 B2 B3

B4 B5

Density of extrafollicular PNAd expressing HEV-like vessels in colonic tissue (A), determined in the inflamed colonic tissue of CD and UC patients as well as in healthy controls. The density is higher for UC when compared with controls (P =0.033) and there is a trend between CD and controls (P =0.092). Immunohistochemical staining (B1-B5). Representative photomicrographs, with a magnification of 200x, of an immunostained colonic biopsy sample from a Crohn’s disease patient with MECA-79 staining (B1), CD20 staining showing B lymphocytes (B2) and CD3 staining showing T lymphocytes (B3). PNAd expressing HEVs are shown in closer detail in B4 (extrafollicular) and B5 (inside a follicle). Arrows in figure B1, B4 and B5 indicate PNAd expressing HEVs. Red arrows indicate HEVs inside a lymphoid follicle and black arrows indicate extrafollicular HEVs.

extrafollicular HEVs, whereas the inﬂamed colon of UC patients showed a high percentage (median 3.71% (IQR 0-6.23%), P=0.033, Figure 1a) and a high median density (3.05 /mm2 (IQR 0-6.39), P=0.033) of extrafollicular HEVs. In the inﬂamed colon of CD, we only observed a trend towards a higher number of extrafollicular HEVs/mm2 (median 0.10 (IQR 0-2.98), P=0.092) compared to controls (median 0 (IQR 0-0), P=0.092).

In the ileum, the percentage of extrafollicular HEVs/CD31+ vessels was higher in inﬂamed ileum of CD patients (median 1.17% (IQR 0-3.77%)) compared to controls (median 0% (IQR 0-0.4%)) (P=0.028). These samples showed a median density of 1.40 extrafollicular HEVs/mm2 (IQR 0-4.34) whereas in healthy controls only a few HEVs/mm2 (IQR 0-0.50) were observed (P=0.033). The presence of extrafollicular HEVs in the ileum and colon of inﬂamed gut mucosa of IBD patients might represent an early phase of TLO formation.

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HEV density is not associated with neovascularization To examine whether the diference in density of PNAd-expressing HEV-like vessels in patients could be attributed to neovascularization, we quantified the number of HEVs/ mm2 and correlated this with the total number of CD31+ vessels in patients and controls. We also compared inflamed colonic UC with colonic CD biopsy specimens. We did not find a correlation and no statistical diference was found between UC and CD patients (P=0.198), indicating that the diferences in HEVs density were not related or associated with neovascularization. To assess a potential correlation with several confounders, as listed in Table 1, we found that HEVs density in inflamed colon was negatively correlated with smoking (Spearman’s rho= -0.543, P=0.002) when calculating the results of all IBD patients. However, when subdivided into CD and UC, it was only significantly correlated in CD (Spearman’s rho -0.683, P=0.001) and not in UC (Spearman’s rho -0.139, P=0.721). Smok- ing was not correlated with HEVs density of inflamed ileum in CD patients. We did not find any correlations with other baseline characteristics (data not shown).

HEVhigh IBD patients have increased colonic lymphoid follicles and inﬁltration

of TN and TCM cells The immunohistochemical findings were compared with the maturation T-cell infiltrates, assessed by flowcytometric immunophenotyping of biopsies taken from the same area of inflamed tissue and harvested during the same ileocolonoscopy. The extrafollicular density of HEVs in inflamed colon of IBD patients (n=30, 10 UC patients, 20

CD patients) was positively correlated with the percentage of TN and TCM lymphocytes (Spearman’s rho= +0.544, P=0.002) and negatively correlated with the percentage of

TEM and TEMRA lymphocytes (Spearman’s rho= -0.545, P=0.002). Comparable percentages

were found in subsets of lymphocytes for UC and CD patients (TN and TCM lymphocytes

(P=0.406), TEM and TEMRA lymphocytes (P=0.451)). In malignancy, a cut-of point for increased density of HEVs has been described in patients with breast cancer20. However, in chronic inflammation, no such approach has been undertaken. We discriminated patients with a high and a low density of HEVs using a cut-of point set at the median density of extrafollicular HEVs/mm2 in the inflamed colon of UC patients. According to this cut of value, we identified 10 HEVhigh patients and 20 HEVlow patients (Table 3). Compared to HEVlow patients, HEVhigh patients displayed less cytotoxic T cells (P=0.00008), a higher CD4:CD8 ratio (P=0.0008), less mucosal T

cells (P=0.015), more TCM cells (P=0.0009), more TN and TCM cells (P=0.003), less TEM cells

(P=0.022), less TEMRA cells (P=0.008) and less TEM and TEMRA cells (P=0.004) in the inﬂamed colonic mucosa (Table 3 and Figure 2). The number of colonic follicles/mm2 was higher in HEVhigh IBD patients (median 0.54/mm2, (IQR 0.28-0.84)) compared with HEVlow patients (median 0.25/mm2, (IQR 0.08-0.45) P=0.031) and controls (0.31/mm2, (IQR 0.23-0.45), P=0.043). The number of

130 hevs associated with t-cell subsets in ibd

Table 3 Characteristics and FACS results of HEVhigh and HEVlow IBD patients

HEVhigh (n=10) HEVlow (n=20) P-value

CD/UC 5/5 15/5 0.169

Age at diagnosis in years, median (IQR) 21 (20-36) 28 (19-33) NS

Female, n (%) 9 (90%) 14 (70%) NS

Symptoms before diagnosis in months NS 0-3 4 (40%) 12 (60%) 3-6 5 (50%) 3 (15%) >6 1 (10%) 5 (25%)

Follow-up period in months, median (IQR) 34 (22-56) 50 (28-65) NS

Family with IBD, n (%) 3 (30%) 5 (25%) NS

Smoking status • Never 9 (90%) 11 (55%) 0.029 • Current - 8 (40%) • Ceased - 1 (5%) • Unknown 1 (10%) -

HBI score, median (IQR) 7 (5-14) (n=5) 10 (7-13) (n=15) NS

CD disease location (Montreal) NS • Ileal, n (%) - - • Colonic, n (%) 2 (40%) 3 (20% • Ileocolonic, n (%) 3 (60%) 12 (80%) • + Upper GI involved, n (%) 1 (10%) 7 (44%) • + Perianal disease, n (%) 2 (20%) 3 (17%)

CD disease behavior (Montreal) NS • Inﬂammatory, n (%) 5 (100%) 11 (73%) • Stricturing, n (%) - 3 (20%) • Penetrating, n (%) - 1 (7%)

SES-CD score, median (IQR) 14 (9-19) (n=5) 20 (12-34) (n=15) NS

SES-CD severity NS • Mild (4-10), n (%) 2 (40%) 2 (13%) • Moderate (11-19), n (%) 3 (60%) 5 (33%) • Severe (>19), n (%) 0 (0%) 8 (53%

UC clinical score (Montreal) NS • Remission, n (%) - - • Mild, n (%) 1 (20%) - • Moderate, n (%) 2 (40%) 2 (40%) • Severe, n (%) 2 (40%) 3 (60%)

UC disease location (Montreal) NS • Proctitis, n (%) - - • Lef-sided, n (%) - - • Pancolitis, n (%) 5 (100%) 5 (100%)

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UC endoscopic severity (Mayo) NS • Normal, n (%) - - • Mild, n (%) 2 (40%) - • Moderate, n (%) 1 (20%) 3 (60%) • Severe, n (%) 2 (40%) 2 (40%)

FACS analysis of inﬂamed colon* • CD3, median (IQR) 50 (45-63) 59 (52-72) 0.267 • CD19, median (IQR) 39 (29-49) 23 (18-42) 0.108 • CD4, median (IQR) 74 (38-83) 61 (36-75) 0.169 • CD8, median (IQR) 15 (11-19) 29 (22-37) 0.00008 • CD4/CD8 ratio, median (IQR) 4.3 (3.0-6.1) 2.2 (1.2-3.2) 0.0008 • CD103, median (IQR) 7 (4-12) 15 (8-43) 0.015 • TN, median (IQR) 23 (20-40) 23 (9-54) 0.779 • TCM, median (IQR) 58 (41-72) 33 (20-45) 0.001 • TEM, median (IQR) 12 (6-18) 26 (12-46) 0.024 • TEMRA, median (IQR) 2 (1-3) 6 (2-12) 0.010 • Treg, median (IQR) 12 (8-18) 14 (12-17) 0.379

CD = Crohn’s disease, UC = ulcerative colitis, HEV = high endothelial venule, IQR = interquartile range, IBD = inﬂammatory bowel disease, HBI= Harvey-Bradshaw Index, SES-CD = simple endoscopic score for Crohn’s

disease, TN = naïve T lymphocyte, TCM = central memory T lymphocyte, TEM = efector memory T lymphocyte, TEMRA = efector memory T lymphocyte re-expressing RA, Treg= regulatory T lymphocyte * Expressed as percentage of the whole lymphocyte population

colonic follicles/mm2 in the inﬂamed colon of patients was comparable between CD and UC (P=0.348). In a subgroup of the current patient cohort we analyzed a series of serum cytokines and chemokines of which CXCL-13 and CCL-19 (both associated with lymphoid organogenesis (22)) were present at higher concentrations in the HEVhigh group of patients. Patients in the HEVhigh group (n=4) had higher baseline levels of CXCL-13, with a median of 67 pg/ml, IQR 52-742 pg/ml) compared to patients in the HEVlow group (n=8) with a median level of 36 pg/ml (IQR 27-47 pg/ml) P=0.008 and to healthy controls (n=4,median 24 pg/ml (IQR 18-28 pg/ml) P=0.029, Figure 3). Furthermore, there was a trend towards higher CCL-19 levels in HEVhigh patients (median 176 pg/ml, IQR 126-199 pg/ml) compared to HEVlow patients (median 125 pg/ml, IQR 90-141 pg/ml, P=0.073).

Discussion

In the present study, increased percentages of extrafollicular PNAd expressing HEV-like vessels were observed in inﬂamed colon and ileum of newly diagnosed IBD patients compared to healthy controls. In controls, these HEVs were restricted to lymphoid follicles. A high heterogeneity was observed in the number of extrafollicular PNAd expressing vessels in IBD patients. We identiﬁed subgroups of patients according to the

132 hevs associated with t-cell subsets in ibd

Figure 2 Correlation between peripheral lymph node addressin (PNAd) expressing high endothelial venules (HEVs) and T-cell subpopulations

HighHigh or low or lowHEV HEV expression expression HighHigh or low or lowextrafollicular extrafollicular HEV HEV expression expression in colon in colon Low Low Low Low High High High High

100100A A 100100B B

80 80 80 80

50 50 60 60

40 40 40 40 Percentage Percentage

20 20 20 20

Percentage of total T cell population 0Percentage of total T cell population 0 0 0 TN TN TemTem TemTemTemraTemra CD3 CD3CD19 CD19CD4 CD4CD8 CD4:CD8CD8 CD4:CD8CD3 CD3Treg Treg ratio ratioCD103CD103 MaturationMaturation state state of T ofcells T cells LymphocteLymphocte subsets subsets

C1 C1 C2 C2

Lymphocyte subsets as well as maturation state of T lymphocytes was assessed with flowcytometric immunophenotyping of biopsies taken from the same inflamed colonic tissues during the same ileocolonoscopy. Patients are divided in HEVhigh (n=10) or HEVlow (n=20) group using the median density of extrafollicular PNAd expressing HEV like vessels in inflamed colon of ulcerative colitis (UC) patients as cut of value. Statistical significant results are indicated with an asterisk (*). - + - - A Shows more central memory T cells (TCM, CD45RA CD27 , p=0.0009), less efector memory T cells (TEM, CD45RA CD27 , + - high p=0.022) and less efector memory T cells re-expressing RA (TEMRA, CD45RA CD27 , p=0.008) in HEV patients when compared with HEVlow patients. There was no statistical significant diference for naive T cells + + (TN CD45RA CD27 ). B Shows less cytotoxic T cells (CD3+CD8+, p=0.00008), a higher CD4:CD8 ratio (p=0.0008) and less mucosal T cells (CD3+CD103+, p=0.015) in HEVhigh patients. There was no statistical significant diference for B cells (CD19+), T cells (CD3+), T-helper cells (CD3+CD4+) and regulatory T cells (CD3+CD25highFoxP3+). C Representative flow cytometric dot plots from inflamed colonic biopsy specimens of IBD patients which show + high the distribution of the maturation of (CD3 ) T lymphocytes. C1 shows a HEV patient with mostly (75%) TN + + - + low (CD45RA CD27 ) and TCM (CD45RA CD27 ) lymphocytes, whereas C2 shows a HEV patient with mostly (91%) TEM (CD45RA-CD27-) lymphocytes.

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Figure 3 Serum CXCL-13 concentrations in IBD patients and healthy controls

1000,00

Healthy HEVhigh controls versus 100,00 versus HEVlow HEVhigh P=0.008 P=0.029 serum CXCL-13 pg/ml

10,00

Healthy controls HEVhigh HEVlow (N=4) (N=4) (N=8)

HEVhigh patients had higher CXC:-13 concentrations compared to HEVlow patients (P=0.008) and compared to controls (P=0.029)

density of the extrafollicular HEVs in the inflamed colonic mucosa: HEVhigh and HEVlow. high In HEV patients, a higher percentage of TN and TCM lymphocytes was observed in the inflamed colonic mucosa compared to HEVlow patients. Patients in the HEVhigh group also displayed a higher number of lymphoid follicles (TLOs) in the inflamed colonic mucosa compared to patients in the HEVlow group and controls. Furthermore chemokines associated with TLO formation were upregulated in the serum of HEVhigh patients. In physiologic conditions, HEVs are known to be exclusively present within SLOs, whereas in the present study we demonstrated their presence outside follicles in the inflamed intestine of IBD patients. It is our interpretation that extrafollicular HEVs represent an early phase of lymphoid neogenesis, as they were virtually absent in healthy controls. In addition to Peyer’s patches, GALT also contains other forms of SLOs, such as isolated lymphoid follicles and submucosal lymphocyte aggregations isolated lymphoid follicles are mostly seen within the mucosa, while submucosal lymphocyte aggregations lie deeper within the muscularis mucosa and submucosa23,24. Both isolated lymphoid follicles and submucosal lymphocyte aggregations are accepted as normal components of healthy small intestinal mucosa, representing solitary Peyers patches follicles. Peyers patches are mostly present in jejunum and ileum while isolated lymphoid follicles have also been found in the human colon24. In accordance, we found HEVs within lymphoid follicles of the normal gut mucosa of both ileum and colon of healthy controls, whereas they did not display such vessels outside these follicles.

134 hevs associated with t-cell subsets in ibd

In IBD, HEVs were previously investigated in one study using MECA-79 immunostaining of biopsy specimens from UC patients and surgical specimens of CD patients5. Increased PNAd expressing HEV-like vessels were found in inflamed colonic mucosa of longstanding UC patients and (to a lesser extent) in CD patients, compared to non-inflamed colonic mucosa of other IBD patients. However, no healthy controls were included, T-cell maturation in the gut mucosa was not analyzed and no serial biopsy samples at dif- ferent time points and phases of disease within the same patients were analyzed. The numbers of PNAd expressing HEVs in the patients with non-inflamed colonic mucosa have not been investigated during active disease and therefore it might concern a subgroup of patients without ectopic HEV formation. Our results suggest diferences in HEVs density and TLOs between IBD patients with active disease.. Direct comparison with the currently presented results is difcult, since we did include healthy controls, studied mucosal T-cell maturation and both the presence of follicles as well as PNAd expression inside and outside these follicles. We demonstrated increased extrafollicular HEV formation in a subgroup of patients (HEVhigh) in the early phase of disease. The lack of extrafollicular PNAd expression in a subgroup of patients (HEVlow) might be explained by a diferent disease phenotype or the early phase of disease, assuming that formation of PNAd expressing vessels in inflamed mucosa is a characteristic of chronicity. Lymphoid follicles in the gut were previously studied in established CD patients, where enhanced presence of submucosal de novo lymphoid follicles in the surgical resection specimen of CD patients has been demonstrated17. The authors interpreted these lymphoid follicles as being TLOs, mostly based on their location in the submucosa, muscularis propria and subserosa, without performing MECA-79 immunostaining17. Theoretically these follicles might still represent SLOs. Potential diferences between patients and associated T-cell subsets were not investigated. The process of TLO development has not yet been clarified, nevertheless chemokines such as CXCL-13, CCL-19 and CCL-21 together with lymphotoxin α1β2 (LTα1β2, TNF 22 family member) were shown to be required for lymphoid organogenesis . LTα1β2 is im- plicated in generation of follicular dendritic cells and HEVs out of stromal cells, while CCL-19, CCL-21 and CXCL-13 control the organization of the T- and B-cell follicular re- gions12,22,25. Remarkably, we found increased serum CCL-19 and CXCL-13 in the HEVhigh patients compared to HEVlow patients (serum CCL-21 was not analyzed). This suggests a systemic reflection of the gut localized formation TLOs. Development of TLOs containing PNAd-expressing HEV-like vessels has also been reported in other diseases with chronic inflammation and in patients with solid tumors. In patients with rheumatoid arthritis, TLOs were associated with an inferior therapeutic response to anti-TNF treatment and reversal of TLOs was a good marker of beneficial therapeutic response26.

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In our study, the higher number of colonic follicles found in the HEVhigh group indicated that in a subgroup of IBD patients, de novo lymphoid follicles were being formed, representing TLOs. The patients in HEVhigh group not only had more follicles, they also exhibited more HEVs outside these follicles and this was associated with a higher percentage +18,19 of TN and TCM lymphocytes (known to be L-selectin ) and lower TEM and TEMRA lymphocytes (known to be L-selectin-18,19) in their mucosa. Remarkably, the patients in HEVlow group were comparable to controls regarding these findings. HEV-like vessels express diferent functional ligands (PNAd, MAdCAM-1) for L-selectin facilitating extravasation + 27 and entry of TN TCM lymphocytes into lymphoid organs . These findings suggest diferences between IBD patients, both UC as CD, regarding TLO formation and recruitment + of TN TCM lymphocytes to the inflamed gut mucosa. These results further expand on our

previous paper in newly diagnosed IBD patients with increased numbers of TN and TCM cells in the inﬂamed gut of a subgroup of patients7. Taken together, our data indicate that lymphoid neogenesis with PNAd expressing HEV-like vessels enhances homing of + TN TCM lymphocytes to the inﬂamed gut in a subset of IBD patients.

A limitation of the present study was that mucosal biopsy specimens precluded full examination of the deeper layers of the intestine wall (submucosa, muscularis propria and subserosa), which potentially contain PNAd expressing HEV-like vessels and TLOs in IBD patients, particularly in CD. However, the strengths of our study were the prospective design, the sampling of untreated patients early in the course of IBD, and the use of a standardized description of TLOs and HEVs in inflamed mucosa of IBD patients. Clinical and therapeutic relevance of these findings remain to be studied; presence of lymphoid follicles in initial biopsy specimens may however be predictive for colectomy due to drug refractory UC28. Furthermore, novel treatment strategies targeting T-cell migration to the inflamed gut mucosa of IBD patients, such as anti-α4β7 (vedolizumab (Entyvio®)) and anti-MAdCAM-1 monoclonal antibodies (PF-00547659) are emerging29, 30 low + . HEV patients with higher influx of α4β7 TEM lymphocytes might theoretically benefit more and thus respond more beneficially to Vedolizumab treatment than HEVhigh patients. Moreover, these findings may lead to further development of new treatment targets in IBD designed to influence HEVs and TLO formation by targeting the lymphotoxin pathway31.

Conclusion

Higher numbers of extrafollicular HEV-like vessels were demonstrated in IBD patients compared to controls. Increased density of extrafollicular HEVs and TLOs in patients

was strongly associated with TN and TCM cell mucosal inﬁltration, while low density of

HEVs correlated with higher numbers of mucosal TEM cells. This suggests that the hom-

136 hevs associated with t-cell subsets in ibd

ing of TN and TCM cells to non-lymphoid gut tissue in IBD patients might be facilitated by extrafolicullar HEVs and de novo TLO formation. Diferent “T-cell migration phenotypes” based on TLO formation in the early phase of IBD may allow risk-stratiﬁcation of patients and enable individualized treatment.

Acknowledgements

We thank Ilze van der Kolk for histology assistance. We thank dr. A.A. van Bodegraven and dr. S. Middendorp for thoughtful review of the manuscript.

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References

1 Mackay CR, Marston WL, Dudler L. Naive and memory T cells show distinct pathways of lymphocyte recirculation. The Journal of experimental medicine. 1990;171:801-817 2 Lewis M, Tarlton JF, Cose S. Memory versus naive T-cell migration. Immunology and cell biology. 2008;86:226-231 3 Sathaliyawala T, Kubota M, Yudanin N, et al. Distribution and compartmentalization of human circulating and tissue-resident memory T-cell subsets. Immunity. 2013;38:187-197 4 Streeter PR, Rouse BT, Butcher EC. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J Cell Biol. 1988;107:1853-1862 5 Suzawa K, Kobayashi M, Sakai Y, et al. Preferential induction of peripheral lymph node addressin on high endothelial venule-like vessels in the active phase of ulcerative colitis. Am J Gastroenterol. 2007;102:1499-1509 6 Rosen SD. Ligands for L-selectin: homing, inflammation, and beyond. Annu Rev Immunol. 2004;22:129-156 7 Horjus Talabur Horje CS, Middendorp S, van Koolwijk E, et al. Naive T cells in the gut of newly diagnosed, untreated adult patients with inflammatory bowel disease. Inflamm Bowel Dis. 2014;20:1902- 1909 8 Cose S, Brammer C, Khanna KM, et al. Evidence that a significant number of naive T cells enter non-lymphoid organs as part of a normal migratory pathway. European journal of immunology. 2006;36:1423-1433 9 Elgbratt K, Kurlberg G, Hahn-Zohric M, et al. Rapid migration of thymic emigrants to the colonic mucosa in ulcerative colitis patients. Clin Exp Immunol. 2010;162:325-336 10 Burgio VL, Fais S, Boirivant M, et al. Peripheral monocyte and naive T-cell recruitment and activation in Crohn’s disease. Gastroenterology. 1995;109:1029-1038 11 Weninger W, Carlsen HS, Goodarzi M, et al. Naive T-cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis. J Immunol. 2003;170:4638-4648 12 Aloisi F, Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases. Nat Rev Immunol. 2006;6:205-217 13 Drayton DL, Liao S, Mounzer RH, et al. Lymphoid organ development: from ontogeny to neogenesis. Nat Immunol. 2006;7:344-353 14 Carragher DM, Rangel-Moreno J, Randall TD. Ectopic lymphoid tissues and local immunity. Seminars in immunology. 2008;20:26-42 15 Goc J, Fridman WH, Sautes-Fridman C, et al. Characteristics of tertiary lymphoid structures in primary cancers. Oncoimmunology. 2013;2:e26836 16 Smids C, Horjus Talabur Horje CS, Wahab PJ, et al. On naivety of T cells in inflammatory bowel disease: a review. Inflamm Bowel Dis. 2015;21:167-172 17 Sura R, Colombel JF, Van Kruiningen HJ. Lymphatics, tertiary lymphoid organs and the granulomas of Crohn’s disease: an immunohistochemical study. Aliment Pharmacol Ther. 2011;33:930-939 18 Sallusto F, Lenig D, Forster R, et al. Two subsets of memory T lymphocytes with distinct homing potentials and efector functions. Nature. 1999;401:708-712 19 Mahnke YD, Brodie TM, Sallusto F, et al. The who’s who of T-cell diferentiation: human memory T-cell subsets. European journal of immunology. 2013;43:2797-2809 20 Martinet L, Garrido I, Filleron T, et al. Human solid tumors contain high endothelial venules: association with T- and B-lymphocyte infiltration and favorable prognosis in breast cancer. Cancer Res. 2011;71:5678-5687

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21 Larbi A, Fulop T. From “truly naive” to “exhausted senescent “ T cells: When markers predict func- tionality. Cytometry Part A: the journal of the American Society for Analytical Cytology 2014; 85: 25-35. 22 Neyt K, Perros F, GeurtsvanKessel CH, et al. Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol. 2012;33:297-305 23 Moghaddami M, Cummins A, Mayrhofer G. Lymphocyte-filled villi: comparison with other lymphoid aggregations in the mucosa of the human small intestine. Gastroenterology. 1998;115:1414-1425 24 Azzali G. Structure, lymphatic vascularization and lymphocyte migration in mucosa-associated lymphoid tissue. Immunological reviews. 2003;195:178-189 25 Drayton DL, Ying X, Lee J, et al. Ectopic LT alpha beta directs lymphoid organ neogenesis with concomitant expression of peripheral node addressin and a HEV-restricted sulfotransferase. The Journal of experimental medicine. 2003;197:1153-1163 26 Canete JD, Celis R, Moll C, et al. Clinical significance of synovial lymphoid neogenesis and its reversal afer anti-tumour necrosis factor alpha therapy in rheumatoid arthritis. Ann Rheum Dis. 2009;68:751-756 27 Rivera-Nieves J, Gorfu G, Ley K. Leukocyte adhesion molecules in animal models of inflammatory bowel disease. Inflamm Bowel Dis. 2008;14:1715-1735 28 Melson JE, Giusto D, Kwasny M, et al. Histopathology predictors of medically refractory ulcerative colitis. Dis Colon Rectum. 2010;53:1280-1286 29 Feagan BG, Rutgeerts P, Sands BE, et al. Vedolizumab as induction and maintenance therapy for ulcerative colitis. The New England journal of medicine. 2013;369:699-710 30 Vermeire S, Ghosh S, Panes J, et al. The mucosal addressin cell adhesion molecule antibody PF- 00547,659 in ulcerative colitis: a randomised study. Gut. 2011;60:1068-1075 31 Browning JL. Inhibition of the lymphotoxin pathway as a therapy for autoimmune disease. Immuno- logical reviews. 2008;223:202-220

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140 part III Discussion

141 chapter 9

Summary and general discussion

143

This thesis focused on clinical, radiological and immunological aspects of the early phase of IBD. First we studied the initial work-up for establishing the primary diagnosis including clinical, endoscopic and imaging techniques. Next we hypothesised that assessment of integrated clinical and immunological characteristics in the early phase of IBD might identify diferent immune-based phenotypes with potential value in prognosis and therapy choice. IBD is ofen regarded as a modern disease because of its increasing incidence, which might in part be attributed to advances in endoscopic and radiologic techniques, allowing for more accurate early diagnosis of patients. The clinical diagnostic strategy of IBD has changed considerably in the last decades as sophisticated imaging techniques such as endoscopy, MRI and Contrast enhanced US (CEUS) have emerged. The research described in the first part of the thesis illustrates the complexity of clinical decision- making at the time of primary diagnosis, which is inherent to the high heterogeneity in disease presentation. Knowledge of the endoscopic and histologic findings at upper GI endoscopy, as well as imaging by MRE and CEUS in diferent newly diagnosed adult CD phenotypes, is important in the development of a more individualized and efcient diagnostic strategy for clinical practice. We therefore chose to study early IBD patients before starting treatment in order to answer the research questions. Despite substantial progress in research, IBD still defies a clear definition, known cause and definitive cure. The disease concept of IBD might best be defined as a disease of disruption: disrupted immunology and physiology in a specific genetic, microbial and environmental context. Characterisation of immunological changes in the early phase before the influence of medication might help better understanding of the disrupted immunology of IBD. Therefore the second part of the thesis focused on the inflamed mucosa of newly diagnosed patients including identification of diferent T-cell subsets and possible mechanisms involved in their recruitment to the gut. In contrast to the current paradigm of T-cell migration through lymphoid and non-lymphoid tissues, we observed increased numbers of naive (TN) and central memory T (TCM) cells in the inflamed mucosa of a subgroup of patients, suggesting a direct entry into the gut. Fur- thermore, higher numbers of ectopic lymphoid organs were identified in the mucosa of patients with predominant TN and TCM cells, providing a plausible explanation for their homing to the inflamed gut.

Part I

Which diagnostic modalities are needed in an individual patient in order to establish a certain diagnosis of IBD as well as the severity and extent of the disease? Chapter 2 is an overview of the changes in the utilisation of diagnostic examinations for the primary diagnosis of IBD in the period between 1970 and 2007. There is

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an evident trend from a prevalent use of radiological procedures, such as small bowel follow-through or enteroclysis and colonography, in the seventies and eighties to predominantly endoscopic procedures, ileocolonoscopy, sigmoidoscopy and upper GI endoscopy, in the last two decades. These changes might be explained by the prominent technical innovations of flexible video-endoscopy in that period, access to biopsy specimens for histopathological examination and increasing interest in an accurate diagnostic classification of IBD patients, preferably based on nosology. This was also been illustrated in the Montreal classification of IBD (2006), which constitutes a revision of the Vienna classification (1998) and allows for the additional designation of upper GI tract (+ L4) and perianal (+ P) involvement in CD1,2. However, a definite consensus on classification is lacking, e.g. definition of upper GI disease location in adult patients is not available. Does it require a combination of endoscopic and histologic findings or are isolated histological findings sufcient for disease location? Does upper GI involvement refer solely to the oesophagus, stomach and duodenum of does it also include proximal jejunum? Despite all this, recent guidelines recommend routine upper GI endoscopy in all newly diagnosed CD patients3. In Chapter 3 we chose to define upper GI involvement (+ L4) as a combination of endoscopic and histopathological abnormalities identified by upper GI endoscopy that reached up to the duodenum pars horizontalis (pars III). A high prevalence of +L4 disease location was observed in newly diagnosed, adult CD patients but the majority of these patients were symptom-free despite small-sized endoscopic upper GI lesions. Furthermore, most histopathological findings, including focally enhanced gastritis, were common for both CD as well as UC patients, except for granulomas that were exclusively present in CD patients. Given the limited clinical impact of these findings we see no grounds for recommending routine upper GI endoscopy in all newly diagnosed CD patients. With respect to the predictive value of upper GI location of CD at baseline for a severe disease course, the current literature is inconclusive. Diferences between population-based studies may be explained by the use of diferent definitions of severely active disease course or lack of complete phenotypic characterization of the patients at baseline (no statement as to whether routine upper GI endoscopy was performed at primary diagnosis in all these patients)4. However, clinical predictors for disabling disease described in recent landmark studies4-6 were: initial requirement for steroid use, age below 40 years, stricturing disease behaviour, and perianal disease, while upper GI disease location reached no statistical significance. Furthermore, in a recent cross-sectional population based study was stated that CD patients with jejunal disease (jejunal L4) were more likely to have stricturing disease behaviour and a more severe disease course than patients with esophagogastroduodenal location (non-jejunal L4)7. In an attempt to achieve an accurate classification of diferent CD phenotypes, recent diagnostic guidelines recommend small bowel imaging in all newly diagnosed CD

146 summary and general discussion

patients irrespective of ileocolonoscopy findings3. The clinical impact and predictive value of small bowel imaging at the time of primary CD diagnosis has not yet been studied. In Chapter 4 we showed that in colonic CD patients no additional small bowel findings were depicted by MRE at the time of primary diagnosis. Small bowel imaging conveyed most information regarding skip lesions or small bowel complications in CD patients with incomplete ileocolonoscopy, severe endoscopic ileitis and stricturing phenotype. There- fore we believe that an individualized recommendation is more adequate, and routine additional small bowel imaging is not necessary in all newly diagnosed CD patients. Next to the well-known advantages of MRE, such as high diagnostic accuracy for small bowel disease location and complications, and lack of radiation, there are also some disadvantages. It is a costly procedure requiring expensive technical and (relatively) complicated protocols, which may impair the quantification of inflammation in daily practice. In Chapter 5 we demonstrated that Contrast Enhanced Ultrasound (CEUS) has a comparable diagnostic accuracy to MRE for terminal ileitis allowing for easy quantification of the inflammation. Quantitative measurement of bowel wall enhancement by time-intensity curve analysis correlates well with endoscopic disease activity. However, given the limitations of abdominal ultrasound in providing information on the whole small bowel and enteroenteral fistula, we believe CEUS is less suitable in the primary diagnosis of CD when any skip lesions or complications must be excluded. CEUS is an adequate, patient-friendly and less costly alternative to MRE during the follow-up of established terminal ileitis in CD patients. Moreover, there is evidence that CEUS allows a clear and rapid diferentiation of abscesses from inflammatory infiltrates or phlegmons in CD8.

Diagnostic procedures in IBD in 2016 Recent European diagnostic guidelines recommend routine upper GI endoscopy and MRE in all newly diagnosed CD patients irrespective of the findings at ileocolonoscopy3. At the same time, recent Dutch and British guidelines recommend upper GI endoscopy solely in newly diagnosed CD patients with upper GI symptoms but not in the case of IBDU diagnosis9,10. The discrepancy between guidelines relies mostly on limited data available up till now on upper GI findings or findings at MRE in newly diagnosed IBD patients. Based on the research presented in this thesis we advise upper GI endoscopy only in newly diagnosed patients with upper GI symptoms or when other ailments (coeliac disease) are considered and, for reasons of specifying classification, in patients diagnosed with IBDU. Endoscopic lesions accompanied by histologic granulomatous inflammation in the upper GI tract of patients with initial IBDU allow classification of these patients as having CD. With respect to small bowel imaging, we found no evidence in our research for performing routine MRE in all newly diagnosed CD patients irrespective of the findings at ileocolonoscopy.

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Figure 1 Individualized diagnostic strategy for endoscopy and imaging in adult IBD patients

Primary diagnosis All patients Ileocolonoscopy Upper GI symptoms; IBDU upper GI endoscopy Stricturing CD, severe ileitis, incomplete endoscopy MRE

Follow-up CEUS/MRE and/or Ileocolonoscopy

MRE is needed in new CD patients with an incomplete ileocolonoscopy and in patients with severe endoscopic ileitis and stricturing disease phenotype. At the same time we recommend no routine small bowel imaging in CD patients with normal aspect of the terminal ileum during ileocolonoscopy. Furthermore, we state that CEUS is a feasible and a less costly alternative to MRE for follow-up of disease activity in CD patients with terminal ileitis (Figure 1).

Part II

Can we identify diferent immunological phenotypes in the inﬂamed gut mucosa of early IBD?

Both innate and adaptive immune responses are highly dependent upon trafcking of immune cells from their sites of origin to target organs. The modern concept of T-cell

homing is based on the hypothesis that only activated and efector memory T (TEM) cells

can enter the non-lymphoid tissues, while TN and TCM are thought to recirculate exclusively between secondary lymphoid organs via lymph vessels and the blood stream11. Up till now, most research into IBD has concentrated on the homing of activated, efec-

tor memory and regulatory T cells to the inﬂamed gut. Interestingly, beforehand, TN cells are the initiators of the inﬂammatory cascade as they precede T-cell diferentiation and expansion, upon activation. To answer the research question above we performed a prospective study on newly diagnosed, untreated IBD patients and healthy controls to assess diferent T-cell sub-

148 summary and general discussion

sets in the inflamed gut mucosa, described in Chapter 6. Diferent subgroups of patients were identified according to the maturation profile of T-cell subsets. The most

“naive” subgroup contained mainly TN cells, the most “mature” group mainly TEM cells and the intermediate group displayed mostly TCM cells. In this pilot study, the “naive” group was associated with upper GI disease involvement (+L4) and perianal disease at the time of ﬁrst presentation in CD patients. Moreover, higher numbers of regulatory (CD4+FoxP3+CD25+) T cells and a lower numbers of mucosal (CD103+) T cells were identi- ﬁed in IBD patients when compared to healthy controls. In Chapter 7 a comprehensive review was provided on the migration pathways of

TN and TCM, putting together recent evidence that TN routinely access peripheral tissues and are present in high numbers in the inflamed gut tissue of IBD patients. The homing process of TN to peripheral non-lymphoid tissues is not completely elucidated, but it is likely that TN will tether and roll on vascular endothelium given the high levels of L-selectin (CD62L) on their surface 12. However, the ligand of CD62L, peripheral LN addressin (PNAd), is mainly present on high endothelial venules (HEV’s), which are normally found in peripheral lymph nodes. In chronic inflammation, such as rheumatoid arthritis, the formation has been demonstrated of ectopic lymphoid organs, so called tertiary lymphoid organs (TLO’s) containing HEV’s, presumably facilitating the homing 13 of TN to inflamed tissues . Data on the presence of TN and TCM in the inflamed gut of IBD patients is scarce and restricted to patients with longstanding disease usually using immunomodulation14.

In search of an explanation for the increased influx of TN and TCM into the inflamed mucosa of a subgroup of patients, we studied the presence of HEV-like vessels and TLO’s in the initial biopsies of these patients, before medication use, as reported in Chapter 9. Extra-follicular HEVs were almost exclusively present in the inflamed colon and ileum of patients when compared to healthy controls. Moreover, the patients exhibited high variation in the numbers of extra-follicular HEVs, and were classified into two groups: a “HEVhigh” and “HEVlow” group. The high numbers of HEV-like vessels in the “HEVhigh” group correlated with increased numbers of TN and TCM, while very low numbers of HEV- low like vessels in the “HEV ” group was associated with higher numbers of TEM cells. This data demonstrated a clear association between the new formation of ectopic HEV-like vessels in inflamed gut mucosa and the increased local influx of TN and TCM cells. The role of naïve T cells in gut inflammation has been demonstrated in animal models where the transfer of naïve T cells (CD4+CD45RB+) into T cell-deficient mice caused an IBD-like syndrome15,16. In humans, recruitment of naïve T cells to the gut mucosa has solely been studied in patients under immunosuppressive treatment. A relatively high number of naïve T cells (CD4+CD45RA+), producing a high amount of TNFα and a low amount of IFNγ, has been observed in the intestinal mucosa of both CD and UC14. Furthermore, it was recently reported that in IBD patients the number of mucosal CD4+CD45RA+ T cells decreased during treatment with corticosteroids or biologicals,

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whereas memory T cells (CD4+CD45RO+) remained unchanged17. Little is known about the homing mechanism of naïve T cells to the peripheral tissues, but a relationship has been suggested with lymphoid neogenesis in inflamed tissue18. Increased PNAd-expressing HEV-like vessels were described in the inflamed colonic mucosa of longstanding IBD compared to the non-inflamed tissue of other IBD patients19 and increased numbers of lymph follicles were seen in surgical resection specimens from CD patients20. Up till now, most research has focused on diferences between IBD and healthy controls while diferences between diferent clinical IBD phenotypes have received less attention. Based on the research described in this thesis we strongly believe that IBD’s heterogeneity in clinical presentation and course of disease might be due to diferent immunological pathways. One such a pathway could be that of TLO formation during chronic inflammation, leading to diferent “T-cell migration” phenotypes.

Diferences in T-cell migration pathways to the inﬂamed gut might have great impact on the response to new anti-integrin therapies. It is possible that inﬂammatory responses in patients with a “HEVhigh” phenotype are less dependent on the homing of alfa4beta7-

expressing TEM cells from the periphery and might rely more on the local recruitment,

activation and diferentiation of TN and TCM in the inﬂamed mucosa. These data suggest that patients with a “HEVlow” phenotype might beneﬁt more from anti-integrin therapy targeting alfa4beta7, than “HEVhigh” patients.

Conclusions and future perspectives

Considering the results of our studies we recommend that a complete primary diagnostic work-up of IBD patients should be individualized according to the clinical phenotype at presentation, including symptoms and ﬁndings by ileocolonoscopy. Next to the already known clinical phenotypes, diferent “ T-cell migration” phenotypes were identiﬁed in newly diagnosed, untreated patients, according to diferences in TLO formation and maturation status of diferent T-cell subsets in the mucosa. Future research should focus on additional experiments regarding mucosal immunological markers in diferent IBD phenotypes in the early phase of the disease. The heterogeneity of IBD and complexity of its pathogenesis preclude proper control of disease activity by modulating single components at sporadic time points in the course of the disease or by treating all patients in the same way, as we currently do in the management of IBD. We aim to extend our research to further elucidate the heterogeneity of IBD phenotypes with respect to integrated clinical and immunological features.

150 summary and general discussion

Future research should focus on the following points: − Search for new mucosal and serological immunological features in the early phase of IBD that will help better characterisation of diferent immunological phenotypes − Study the changes of these immunological features under therapy and in remission during long-term follow-up of diferent IBD phenotypes and identify early predictive factors for the course of the disease − Perform prospective randomized drug trials in a speciﬁc, well-deﬁned cohort of clinical and immunological phenotypes that will open the way for personalized medicine for IBD patients

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References

1 Satsangi J, Silverberg MS, Vermeire S and Colombel J-F. The Montreal classification of inflammatory bowel disease: controversies, consensus and implications. Gut, 2006; 55:749-753. 2 Gasche C, Scholmerich J, Brynskov J, et al. A simple classification of Crohn’s disease: report of the working party of the world congresses of gastroenterology, Vienna 1998. Inflamm Bowel Dis, 2000; 6:8–15. 3 Van Assche G, Dignass A, Panes J et al. The second European evidence-based Consensus on the diagnosis and management of Crohn’s disease: Definitions and diagnosis. J Crohn’s Colitis. 2010 Feb;4(1):7-27. 4 Wolters FL, Russel MG, Sijbrandij J et al. Crohn’s disease: increased mortality 10 years afer diagnosis in a European-wide population based cohort. Gut, 2006; Apr; 55(4):510-8. 5 Beaugerie L1, Seksik P, Nion-Larmurier I et al. Predictors of Crohn’s disease. Gastroenterology 2006 Mar;130 (3):650-6. 6 Romberg-Camps MJ1, Dagnelie PC, Kester AD et al. Influence of phenotype at diagnosis and of other potential prognostic factors on the course of inflammatory bowel disease. Am J Gastroenterol 2009 Feb; 104(2): 371-83. 7 Lazarev M, Huang C, Bitton A et al. Relationship between proximal Crohn’s disease location and disease behaviour and surgery: a cross-sectional study of the IBD Genetics Consortium. Am J Gas- troenterol 2013; Jan;108(1):106-12. 8 Ripolles T, Martinez-Perez MJ, Paredes JM, Vizuete J, Garcia-Martinez E, Jimenez-Restrepo DH. Con- trast-enhanced ultrasound in the diferentiation between phlegmon and abscess in Crohn’s disease and other abdominal conditions. European Journal of Radiology 2013;82:e525-531. 9 Richtlijn Diagnostiek en behandeling van inflammatoire darmziekten bij volwassenen 2009. ISBN 978-90-8523-196-7. 10 Mowat C, Cole A, Windsor A et al. Guidelines for the management of Inflammatory Bowel Disease in adults. Gut, 2011. doi:10.1136. 11 Lewis M, Tarlton JF, Cose S. Memory versus naive T-cell migration. Immunol Cell Biol. 2008;86:226-231. 12 Cose S. T-cell migration: a naive paradigm? Immunology. 2007;120:1-7. 13 Canete JD, Celis R, Moll C et al. Clinical significance of synovial lymphoid neogenesis and its reversal afer anti-tumour necrosis factor alpha therapy in rheumatoid arthritis. Ann Rheum Dis 2009; 68(5): 751-756. 14 Burgio VL, Fais S, Boirivant M, et al. Peripheral monocyte and naive T-cell recruitment and activation in Crohn’s disease. Gastroenterology. 1995;109:1029-1038 15 Powrie F, Leach MW, Mauze S, et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity. 1994;1:553-562 16 Kanai T, Kawamura T, Dohi T, et al. TH1/TH2-mediated colitis induced by adoptive transfer of CD4+CD45RBhigh T lymphocytes into nude mice. Inflamm Bowel Dis. 2006;12:89-99 17 Karlsson M, Linton L, Lampinen M, et al. Naive T cells correlate with mucosal healing in patients with inflammatory bowel disease. Scand J Gastroenterol. 2014;49:66-74 18 Weninger W, Carlsen HS, Goodarzi M, et al. Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis. Journal of immunology. 2003;170:4638-4648. 19 Suzawa K, Kobayashi M, Sakai Y, Hoshino H, Watanabe M, Harada O et al. Preferential induction of peripheral lymph node addressin on high endothelial venule-like vessels in the active phase of ulcerative colitis. Am J Gastroenterol 2007; 102(7): 1499-1509. 20 Sura R, Colombel JF, Van Kruiningen HJ. Lymphatics, tertiary lymphoid organs and the granulomas of Crohn’s disease: an immunohistochemical study. Alimentary pharmacology & therapeutics 2011; 33(8): 930-939.

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Dutch summary Samenvatting en algemene discussie

153

Dit proefschrif richt zich op klinische, radiologische en immunologische aspecten in de vroege fase van IBD. Ten eerste hebben we het initiële onderzoekstraject voor het vaststellen van de primaire diagnose, met inbegrip van klinische, endoscopische en beeldvormende technieken, onderzocht. Vervolgens werd de hypothese opgesteld dat de beoordeling van geïntegreerde klinische en immunologische kenmerken in de vroege fase van IBD, verschillende immuun gebaseerde subtypes zou kunnen identificeren met potentiële waarde in de prognose en therapiekeuze. IBD wordt vaak beschouwd als een moderne ziekte vanwege de toenemende in- cidentie, die deels toegeschreven kan worden aan de vooruitgang in endoscopische en radiologische technieken, waardoor nauwkeurigere vroege diagnose van patiënten mogelijk is. De klinische diagnostische strategie van IBD is in de afgelopen decennia aanzienlijk veranderd omdat geavanceerde beeldvormende technieken zoals endoscopie, MRI en contrast versterkte echografie (CEUS) naar voren zijn gekomen. Het onderzoek dat in het eerste deel van het proefschrif wordt gepresenteerd, illustreert de complexiteit van de klinische besluitvorming ten tijde van de primaire diagnose. Deze complexiteit is inherent aan de hoge heterogeniteit van de ziekte bij presentatie. Kennis van de endoscopische en histologische bevindingen bij gastroduodenoscopie, evenals de kennis van radiologische beeldvorming (MRE en CEUS) in verschillende fenotypen van nieuw gediagnosticeerde volwassenen met de ziekte van Crohn, is belangrijk voor de ontwikkeling van een geïndividualiseerde en efciënte diagnostische strategie voor de klinische praktijk. Daarom hebben wij ervoor gekozen om nieuw gediagnosticeerde patiënten te bestuderen, voorafgaande aan het starten van de behandeling, om zo de onderzoeksvragen te kunnen beantwoorden. Ondanks de aanzienlijke vooruitgang in het onderzoek, ontbreekt het in de IBD toch aan een duidelijke definitie, bekende oorzaak en definitieve genezing. De ziekte IBD kan het best worden gedefinieerd als een ziekte van storing: verstoorde immunologie en fysiologie in een specifieke genetische, microbiële en omgevingscontext. Karakterisatie van immunologische veranderingen in de vroege fase, zonder het invloed van immuno- suppressieve medicatie, kan leiden tot een beter begrip van de verstoorde immunologie in IBD. Daarom concentreert het tweede deel van het proefschrif zich op het ontstoken slijmvlies van nieuw gediagnosticeerde patiënten, met daarin de identificatie van verschillende T cel subpopulaties en mogelijke mechanismen, die betrokken zijn bij hun rekrutering in de darm. In tegenstelling tot het huidige paradigma van T cel migratie door lymfoïde en niet- lymfoïde weefsels, observeerden we een verhoogd aantal geheu- gen naïeve (TN) en centrale T (TCM) cellen in het ontstoken slijmvlies van een subgroep van patiënten, suggestief voor een directe toegang tot de darm. Verder werden er hogere aantallen ectopische lymfoïde organen gevonden in de mucosa van patiënten met overheersende TN en TCM cellen, die een plausibele verklaring vormt voor hun ‘homing’ naar de ontstoken darm.

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Deel I

Welke diagnostische instrumenten zijn nodig in een individuele patiënt om de diagnose van IBD, de ernst en omvang van de ziekte vast te stellen? In hoofdstuk 2 wordt een overzicht gepresenteerd van de veranderingen in het gebruik van diagnostische onderzoeken voor de primaire diagnose van IBD in de periode tussen 1970 en 2007. Er is een duidelijke trend zichtbaar, van een wijdverspreide gebruik van radiologische procedures in de jaren zeventig en tachtig, naar overwegend endoscopische procedures, in de laatste twee decennia. Deze veranderingen kunnen worden verklaard door de prominente technische innovaties van de flexibele video-endoscopie in die periode, met toegang tot biopten voor histopathologisch onderzoek en toenemende belangstelling voor een nauwkeurige diagnostische classificatie van IBD patiën- ten, bij voorkeur gebaseerd op nosologie. Dit is ook geïllustreerd in de Montreal indeling van IBD (2006), die een herziening van de Vienna classificatie (1998) vormde en zorgde voor bijkomende aanwijzing van de bovenste maagdarmkanaal (+ L4) en perianale (+ P) betrokkenheid bij ziekte van Crohn1,2. Maar een duidelijke consensus over de indeling ontbreekt, zo is een heldere definitie van de bovenste tractus digestivus lokalisatie van de ziekte van Crohn bij volwassen patiënten bijvoorbeeld niet beschikbaar. Vereist de bovenste tractus digestivus betrokkenheid een combinatie van endoscopische en histologische afwijkingen of zijn geïsoleerde histologische bevindingen voldoende voor de locatie van de ziekte van Crohn? Heef bovenste tractus digestivus betrokkenheid alleen betrekking op de slokdarm, maag en de twaalfvingerige darm of moet het ook proximale jejunum bevatten? Ondanks dit alles, recente diagnostische richtlijnen bevelen routine gastroduodenoscopie aan, voor alle nieuw gediagnosticeerde patiënten met de ziekte van Crohn3. In hoofdstuk 3 hebben wij bovenste tractus digestivus betrokkenheid (+ L4) gedefinieerd als een combinatie van endoscopische en histopathologische afwijkingen, geïden- tificeerd bij gastroduodenoscopie tot pars horizontalis (pars III) van de twaalfvingerige darm. Een hoge prevalentie van + L4 locatie van de ziekte werd waargenomen in nieuw gediagnosticeerde volwassen patiënten met de ziekte van Crohn, maar de meerderheid van deze patiënten waren symptoomvrij, ondanks milde endoscopische laesies. Boven- dien kwamen de meeste histopathologische bevindingen, inclusief focale gastritis, vaak voor, zowel in ziekte van Crohn als in Colitis ulcerosa patiënten, met uitzondering van de granulomen die uitsluitend aanwezig waren in patiënten met de ziekte van Crohn. Gezien de beperkte klinische impact van deze bevindingen zien we geen reden om routinematig gastroduodenoscopie aan te bevelen in alle nieuw gediagnosticeerde patiënten met de ziekte van Crohn. Met betrekking tot de voorspellende waarde van de bovenste tractus digestivus locatie (+L4) van de ziekte van Crohn, bij aanvang, voor een ernstige ziektebeloop, is de huidige literatuur niet overtuigend. Verschillen tussen studieresultaten uit het verle-

156 samenvatting en algemene discussie

den kunnen worden verklaard door het gebruik van verschillende definities voor ernstig ziektebeloop of gebrek aan volledige fenotypische karakterisering van de patiënten bij aanvang (geen mededeling of routine gastroduodenoscopie is uitgevoerd ten tijde van de primaire diagnose bij al deze patiënten)4. Echter, de klinische voorspellers voor een gecompliceerd ziektebeloop – beschreven in de meeste landmark studies4-6 – waren: het gebruik van steroïden, leefijd jonger dan 40 jaar, stenoserend ziekte gedrag en perianale ziekte bij eerste presentatie. Bovendien, in een recente, op populatie gebaseerde studie werd gesteld dat het risico op een ernstiger ziektebeloop met stenoserend ziekte- gedrag, hoger was in Crohn-patiënten met jejunale ziekte (jejunale L4) dan in patiënten met esophagogastroduodenale locatie (niet-jejunale L4)7. In een poging om tot een nauwkeurige classificatie van de verschillende fenotypes van ziekte van Crohn te komen, bevelen recente diagnostische richtlijnen aan om dunne darm beeldvorming te verrichten, in alle nieuw gediagnosticeerde patiënten ongeacht bevindingen bij ileocolonoscopie3. De klinische meerwaarde en de voorspellende waarde van de dunne darm beeldvorming ten tijde van de primaire diagnose van ziekte van Crohn is nog niet onderzocht. In hoofdstuk 4 hebben we aangetoond dat in Crohn-patiënten met uitsluitend colon lokalisatie geen additionele dunne darm bevindingen werden beschreven op MR Ente- rografie (MRE) ten tijde van de primaire diagnose. Dunne darm beeldvorming leverde de meeste aanvullende informatie over ‘skip lesions’ of dunne darm complicaties (stenose, fistels) op in Crohn-patiënten met een incomplete ileocolonoscopie, ernstige endoscopische ileitis en stenoserende fenotype. Daarom zijn wij van mening dat een individuele aanbeveling gerechtvaardigd is en routinematige dunne darm beeldvorming niet nodig in alle nieuw gediagnosticeerde Crohn-patiënten. Naast de bekende voordelen van MRE zoals hoge diagnostische nauwkeurigheid voor dunne darmziekte locatie en complicaties en gebrek aan straling, zijn er ook enkele nadelen. Het is een kostbare procedure en vereist relatief complexe protocollen om de kwantificering van de ontsteking toe te passen in de praktijk. In hoofdstuk 5 hebben we aangetoond dat contrast versterkte echografie (CEUS) een vergelijkbare diagnostische nauwkeurigheid heef met MRE voor ileitis terminalis, en daarnaast een gemakkelijke kwantificering van de ontsteking mogelijk maakt. Kwantitatieve meting van darmwand aankleuring, door de tijd-intensiteit curve analyse correleert goed met endoscopische ziekteactiviteit. Gezien de beperkingen van abdominale echografie in het beoordelen van de gehele dunne darm en enteroenterale fistels, vinden wij CEUS minder geschikt in de primaire diagnostiek van de ziekte van Crohn, waarbij eventuele skip laesies of complicaties moeten worden uitgesloten. CEUS is een adequaat, patiëntvriendelijk en minder kostbaar alternatief voor MRE tijdens de follow-up van een bekende ileitis terminalis bij Crohn-patiënten. Bovendien zijn er aanwijzingen dat CEUS een duidelijke en snelle diferentiatie tussen abcessen met inflammatoire infiltraten of phlegmons in ziekte van Crohn mogelijk maakt8.

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Diagnostische procedures in IBD anno 2016 Recente Europese diagnostische richtlijnen bevelen routinematige gastroduodenoscopie en MRE in alle nieuw gediagnosticeerde Crohn-patiënten, ongeacht de bevindingen bij ileocolonoscopie3. Tegelijkertijd bevelen de Nederlandse en Britse richtlijnen een gastroduodenoscopie uitsluitend in nieuw gediagnosticeerde Crohn-patiënten met klachten van het bovenste tractus digestivus, maar niet bij een initiële diagnose van niet-classiﬁceerbare IBD9,10. De discrepantie tussen de richtlijnen berust meestal op het gebrek aan beschikbare gegevens over de bevindingen bij gastroduodenoscopie of MRE in nieuw gediagnosticeerde patiënten, tot nu toe.

Op basis van het in dit proefschrif gepresenteerd onderzoek adviseren wij het uitvoeren van gastroduodenoscopie alleen in nieuw gediagnosticeerde Crohn-patiënten met klachten van het bovenste tractus digestivus, eventueel ter uitsluiting van andere aan- doeningen (coeliakie) en in het belang van een nauwkeurige classificatie bij patiënten met niet-classificeerbare IBD. Endoscopische laesies vergezeld van histologische granu- lomateuze ontsteking in het bovenste maagdarmkanaal van patiënten met niet-classificeerbare IBD vormt een sterke argument voor het vast stellen van de ziekte van Crohn. Met betrekking tot de dunne darm beeldvorming, hebben wij geen bewijs kunnen verzamelen voor het uitvoeren van routinematige MRE in alle nieuw gediagnosticeerde Crohn- patiënten, ongeacht de bevindingen van de ileocolonoscopie. MRE is geïndiceerd bij nieuwe Crohn-patiënten met een onvolledige ileocolonoscopie, bij Crohn-patiënten met een ernstige endoscopische ileitis en bij stenoserende ziekte fenotype bij presentatie. Te- gelijkertijd heef de routinematige dunne darm beeldvorming in Crohn-patiënten met een normaal aspect van het terminale ileum tijdens ileocolonoscopie, geen meerwaarde. Ver- der stellen we dat CEUS een haalbaar en goedkoper alternatief is voor MRE voor de follow- up van ziekteactiviteit ter hoogte van het ileum in Crohn-patiënten (Figuur 1).

Figuur 1 Individuele diagnostische strategie voor endoscopie en beeldvorming bij volwassen patiënten IBD

Primaire diagnose Alle patienten Ileocolonoscopie Klachten bovenste tr. dig. ; Niet classiﬁcerbare IBD gastroduodenoscopie Stenoserende zv Crohn, ernstige ileitis, incomplete endoscopie MRE

Follow-up CEUS/MRE en/of Ileocolonoscopie 158 samenvatting en algemene discussie

Deel II

Is het mogelijk om verschillende immunologische fenotypes te identiﬁceren in het ontstoken darm slijmvlies van de vroege IBD? Zowel de aangeboren als de adaptieve immuunreacties zijn sterk afankelijk van migratie van immuun cellen uit hun plaats van oorsprong naar de doelorganen. Het moderne concept van T-cel homing is gebaseerd op de hypothese dat alleen geactiveerde en efector memory T (TEM) cellen de niet-lymfoïde weefsels kunnen bereiken, terwijl naïeve (TN) en central memory T (TCM) cellen worden verondersteld uitsluitend te circule- ren tussen secundaire lymfoïde organen via lymfevaten en bloedstroom11. Het meeste onderzoek in IBD heef zich tot nu toe gericht op homing van geactiveerde efector memory en regulatoire T cellen naar de ontstoken darm. Wat naïeve (TN) en central memory

T (TCM) interessant maakt is dat zij aan het begin staan van de inflammatoire cascade en de potentie hebben zich te diferentiëren zowel in de richting van proinflammatoire als regulatoire T cellen. Om de hierboven genoemde onderzoeksvraag te kunnen beantwoorden hebben we een prospectieve studie uitgevoerd in nieuw gediagnosticeerde, onbehandelde IBD pa- tiënten en gezonde controles naar de verschillende T cel subpopulaties in het ontstoken darm slijmvlies, zoals beschreven in hoofdstuk 6. Verschillende subgroepen van patiën- ten werden geïdentificeerd op basis van het rijpingsprofiel van de T-cel subpopulaties.

De meest ‘naïeve’ subgroep bevatte vooral TN cellen, de meest ‘rijpe’ groep voorname- lijk TEM cellen en de tussenliggende groep vooral TCM cellen. In dit pilot onderzoek werd de naïeve groep geassocieerd met bovenste tractus digestivus betrokkenheid (+ L4) en perianale ziekte bij de eerste presentatie in Crohn-patiënten. Bovendien werden hogere percentages regulatoire (CD4+ CD25+ Foxp3+) T cellen en een lagere percentages mucosale (CD103+) T cellen gevonden in IBD patiënten vergeleken met gezonde controles. In hoofdstuk 7 wordt een uitgebreid overzicht verstrekt over de migratie paden van

TN en TCM, inclusief de recente aanwijzingen dat TN routinematig toegang kunnen hebben tot perifere weefsels en in hoge aantallen aanwezig kunnen zijn in het ontstoken darm- weefsel van IBD patiënten. De homing werkwijze van TN richting perifere niet-lymfoïde weefsels is niet volledig opgehelderd, maar het is aannemelijk dat TN zich aan bepaalde types vasculaire endotheel kan binden gezien de hoge niveaus van L-selectine (CD62L) op het oppervlak12. De ligand van CD62L, perifere LN addressin (PNAd) is hoofdzakelijk aanwezig op hoge endotheliale venules (HEV’s), die normaliter in perifere lymflieren aanwezig zijn. Nieuwvorming van ectopische lymfoïde organen, zogenaamde tertiaire lymfoïde organen (TLO’s) met HEV’s is aangetoond gedurende chronische ontsteking, zoals in reumatoïde artritis. De HEV’s binnen deze TLO’s kunnen de homing van TN en 13 TCM naar ontstoken weefsels faciliteren . Gegevens over de aanwezigheid van TN en TCM in de ontstoken darm van IBD-patiënten zijn schaars en beperkt tot patiënten met lang- durige ziekte en gebruik van immunosuppressiva14.

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Op zoek naar een verklaring voor de toegenomen instroom van TN en TCM in het ontstoken slijmvlies van een subgroep van patiënten, hebben we de aanwezigheid van HEV- achtige vaten en TLO in de initiële biopten van deze patiënten onderzocht, vóór initiatie van medicijngebruik, zoals beschreven in hoofdstuk 8. Extra-folliculaire PNAd+ HEV- achtige vaten waren bijna uitsluitend aanwezig in de ontstoken dikke darm en de dunne darm van de patiënten in vergelijking met gezonde controles. Bovendien vertoonden de patiënten grote variatie in het aantal extra-folliculaire PNAd+ HEV-achtige vaten en werden ingedeeld in twee groepen: een ‘HEVhigh’ en ‘HEVlow’ groep. De hoge aantallen high HEV-achtige vaten in de ‘HEV ’ correleerde goed met verhoogde percentages TN en low TCM, terwijl de zeer lage aantallen HEV-achtige vaten in de ‘HEV ’ groep werd geas-

socieerd met hogere percentages TEM cellen in de mucosa. Deze gegevens tonen een duidelijk verband tussen de nieuwe formatie van extra-folliculaire HEV-achtige vaten in

ontstoken darmslijmvlies en verhoogde lokale instroom van TN en TCM-cellen. De rol van naïeve T cellen in het ontstaan van darm ontsteking werd aangetoond in diermodellen wanneer toediening van naïeve T cellen (CD4+ CD45RB+) in T-cel-deﬁciënte muizen een IBD-achtig syndroom15,16 veroorzaakte. Bij mensen is de aanwezigheid van naïeve T cellen in de darm mucosa eerder alleen onderzocht bij patiënten onder immu- nosuppressieve behandeling. Een relatief groot aantal naïeve T cellen (CD4+ CD45RA+), geassocieerd meet een grote hoeveelheid TNFalfa en een lage hoeveelheid IFNgamma, werd waargenomen in de intestinale mucosa van zowel Crohn als Colitis ulcerosa pati- enten14. Bovendien werd onlangs gemeld dat in IBD patiënten het aantal mucosale CD4+ CD45RA+ T cellen daalde tijdens behandeling met corticosteroïden of TNF blokkers, terwijl efector memory T cellen (CD4+ CD45RO+) onveranderd bleven17. Weinig is bekend over het homing mechanisme van de naïeve T cellen in de perifere weefsels, maar een relatie met lymfoïde neogenese in ontstoken weefsel is voorgesteld18. Verhoogde ex- pressie van PNAd HEV-achtige vaten werd beschreven in ontstoken darmmucosa van lang bestaand IBD in vergelijking met niet-ontstoken darm mucosa van andere IBD pati- enten19. Daarnaast zijn verhoogde aantallen lymfollikels waargenomen in chirurgische resectie specimen van Crohn- patiënten20. Tot nu toe richtte het meeste onderzoek zich op verschillen tussen IBD patiënten en gezonde controles, terwijl verschillen tussen de verscheidene klinische IBD feno types minder aandacht hebben gekregen. Op basis van het in dit proefschrif beschreven onderzoek zijn we ervan overtuigd dat de IBD-heterogeniteit in klinische presentatie en ziektebeloop te wijten kan zijn aan verschillende immunologische mechanismen. Een dergelijk mechanisme kan ook nieuwvorming van TLO’s tijdens chronische ontsteking zijn, hetgeen leidt tot verschillende ‘T cel migratie’ fenotypes. Verschillen in T-celmigratiepaden naar de ontstoken darm kunnen de reactie op de nieuwe anti-integrine therapieën sterk beïnvloeden. Het is mogelijk dat ontstekings- reacties bij patiënten met een ‘HEVhigh’ fenotype minder afankelijk zijn van de homing

160 samenvatting en algemene discussie

+ van alfa4beta7 TEM cellen uit de periferie, en een toegenomen rekrutering, activering en diferentiatie van TN en TCM in het ontstoken slijmvlies vertonen. Deze gegevens sug- gereren dat patiënten met een ‘HEVlow’ fenotype meer zouden kunnen proﬁteren van anti-integrine alfa4beta7 therapie dan ‘HEVhigh’ patiënten.

Conclusies en toekomstperspectieven

Gezien de resultaten van onze studies vermelden we dat een volledige primaire diagnostische work-up van IBD patiënten geïndividualiseerd zou moeten worden op basis van het klinische fenotype bij de presentatie, met inbegrip van symptomen en bevindingen bij ileocolonoscopie en histologie. Naast de reeds bekende klinische fenotypen werden verschillende T cel migratie fenotypen geïdentiﬁceerd in nieuw gediagnosticeerde onbehandelde patiënten, gebaseerd op verschillen in TLO vorming en maturatiestatus van verschillende T cel subpopulaties in de ontstoken mucosa. Toekomstig onderzoek zou zich moeten gaan richten op aanvullende experimenten met betrekking tot mucosale immunologische markers in verschillende IBD fenotypes in de vroege fase van de ziekte. De heterogeniteit van IBD en complexiteit van de pathogenese maakt het haast onmogelijk om de ziekte in alle gevallen onder controle te krijgen, door het moduleren van afzonderlijke componenten op sporadische tijdstippen, tijdens het verloop van de ziekte of de behandeling van alle patiënten, op dezelfde wijze als op dit moment gaande is in de behandeling van IBD. Ons streven is om ons onderzoek te vervolgen met verdere opheldering van de heterogeniteit van verschillende IBD fenotypes met betrekking tot geïntegreerde klinische en immunologische fenomenen.

Toekomstig onderzoek moet zich richten op de volgende punten: − Onderzoek naar nieuwe mucosale en serologische immunologische mechanismen in de vroege fase van IBD, waardoor een betere karakterisering van de verschillende immunologische fenotypes mogelijk wordt gemaakt. − Onderzoek naar de veranderingen van deze immunologische mechanismen onder invloed van de behandeling en in de rustige fase van de ziekte tijdens lange termijn follow-up waardoor voorspellende factoren voor ziektebeloop geïdentiﬁceerd kunnen worden. − Uitvoering van prospectieve gerandomiseerde trials met verschillende immunosuppressiva en biologische geneesmiddelen in speciﬁeke, welomschreven subgroepen van klinische en immunologische subtypes, die de weg zal openen voor gepersona- liseerde geneeskunde in IBD.

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References

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162 part IV Appendices

163

list of abbreviations

− IBD Inﬂammatory bowel disease − CD Crohn’s disease − UC Ulcerative colitis − IBDU IBD unclassiﬁed − IC indeterminate colitis − FEG focally enhanced gastritis − MRE Magnetic resonance enterography − CEUS contrast-enhanced ultrasound − SESCD Simple Endoscopy Score for CD − HBI Harvey Bradshaw Index − GALT gut associated lymphoid tissue − PP Peyer’s patches − SLOs secondary lymphoid organs − PNAd peripheral lymph node addressin − MAdCAM-1 mucosal vascular addressin cell adhesion molecule − TCR T cell receptor − TRECs TCR excision circles − RTE recent thymic emigrants

− TCM central memory T lymphocytes

− TEM efector memory T lymphocytes

− TEMRA efector memory T cells re-expressing CD45RA

− TN naive T lymphocytes

− TMN mature naive T lymphocytes − FoxP3 forkhead box P3 − HEVs high endothelial venules − TLOs tertiary lymphoid organs − IQR interquartile range − SD standard deviation

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list of publications

Cinacalcet for hyperparathyroidism in pregnancy and puerperium. C.S. Horjus Talabur Horje, I Groot, D Telting, P Setten, A van Sorge, CS Kovacs, ARMM Hermus, H de Boer. J of Pediatric Endocrinology & Metabolism 2009; 22:741-749. Acute diarree. C.S. Horjus, M. Spanier. Het Voeding Formularium 2010; onder redactie van J.J. van Binsbergen, JA van Dommelen, JM Geleijnse, JR van der Laan. Microalbuminuria is not a valuable marker for relapse in Crohn’s disease. CS Horjus Talabur Horje, MGH van Oijen, AH Davids, AHJ Naber, DJ de Jong. Digestion 2011; 83: 41-45. Klinische les: buikpijn door een microvasculaire oorzaak. C.A.D. Slegers, G.C. Bloems- ma, C.S. Horjus, H. Visser, R.J. Robijn, E. J. Spillenaar Bilgen. Ned TvG 2012; 156: 469-473. Naive T cells in the gut: how to really find them? C.S. Horjus Talabur Horje, MJM Groenen, PJ Wahab, E van Lochem; Scand J of Gastroenterology 2014; Apr, 49 (4): 513. Integrin αEβ7 in Inflammatory Bowel Disease: Friend or Foe? C. Smids, Carmen S. Horjus Talabur Horje, EG van Lochem. Gastroenterology 2016; 151(1): 213-4. The complexity of alpha E beta 7 blockade in Inflammatory Bowel Diseases. C Smids, Carmen S. Horjus Talabur Horje, F van Wijk, E G. van Lochem. J Crohns Colitis.2016 Sep 22. pii:jjw163 Epub ahead of print. Naive T cells in the gut of newly diagnosed, untreated adult patients with inflammatory bowel disease. Carmen S. Horjus Talabur Horje MD, S Middendorp, E van Koolwijk, L Roovers, M JM Groenen, P J Wahab, E van Lochem. Inflamm Bowel Dis, 2014; 20(11): 1902-1909. On naivety of T cells in inflammatory bowel disease: a review. Smids C, Carmen S Horjus Talabur Horje, Wahab PJ, Groenen MJ, Middendorp S, van Lochem EG. Inflamm Bowel Dis, 2015; 21(1): 167-172. Contrast Enhanced Abdominal Ultrasound in the Assessment of Ileal Inflammation in Crohn’s Disease: A Comparison with MR Enterography. Carmen S Horjus Talabur Horje, Bruijnen R, Roovers L, Groenen MJ, Joosten FB, Wahab PJ. PLoS One, 2015; 10(8): e 0136105. Prevalence of Upper Gastrointestinal Lesions at Primary Diagnosis in Adults with In- flammatory Bowel Disease. Carmen S. Horjus Talabur Horje, Jos Meijer, Lian Rovers, Ellen G van Lochem, Marcel JM Groenen, Peter J Wahab. Inflamm Bowel Dis, 2016; 22(8):1896 -1901.

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acknowledgement/dankwoord

Het gereedkomen van dit proefschrif zie ik niet als een afsluiting maar als inspiratie om wetenschappelijk nieuwsgierig te blijven en het onderzoekswerk voort te zetten. Dit proefschrif is het resultaat van vele jaren werk en het was er niet geweest zonder de hulp van verschillende mensen.

Allereerst wil ik alle patiënten bedanken die volledig belangeloos aan onze studies hebben deel genomen, in het vertrouwen dat wetenschappelijk onderzoek de belangrijkste weg is naar een betere behandeling en zelfs genezing van ziektes.

Graag dank ik degenen die mij op weg naar dit resultaat hebben geholpen. Beste peter, jij bent de allereerste geweest die mij heef aangespoord om wetenschappelijk onderzoek te doen in het Rijnstate ziekenhuis. Jouw ambitie en gedreven- heid om een onderzoekslijn op te richten op het gebied van inﬂammatoire darmziekten hebben hiertoe de eerste aanzet gegeven. In de jaren daarna heb ik steeds jouw betrokkenheid ervaren. Jij hield de grote lijnen altijd scherp in de gaten, terwijl je ook oog had voor het praktische schrijfwerk. Beste marcel, samen met Peter heb jij vanaf het begin, de begeleiding van mijn proef schrif voor je rekening genomen. Je was altijd bereid mee te denken en deed heel actief mee met inclusie van patiënten, een belangrijk deel van het onderzoekswerk. Jouw vriendelijke houding gaven vaak een noodzakelijke dosis ontspanning in stressvolle onderzoekssituaties. Lieve ellen, zonder jouw immunologische kennis en bezieling had ik nooit zover kunnen komen. Vanaf het begin durfde jij het aan om samen nieuwe vragen te stellen, zonder te weten of de uitkomst bevredigend zou zijn. Dit zie ik als een hele belangrijke wetenschappelijke habitus, in een onderzoekscultuur die zo vaak anders georiënteerd is. De samenwerking tussen de MDL en de immunologie is in dit ziektebeeld noodzakelijk voor werkelijke vooruitgang. Ik hoop dat we deze bundeling van krachten nog vele jaren kunnen continueren. In het bijzonder wil ik ad van bodegraven danken voor zijn herhaaldelijke hulp bij het schrijven en herschrijven van artikelen. Beste Ad, van een afstand en zonder eigen belang, hebben jouw kritische en verfrissende suggesties en opmerkingen de wetenschappelijke kwaliteit van mijn artikelen aanzienlijk versterkt. Het was voor mij ook erg leerzaam en plezierig om met iemand met zulk een wetenschappelijk inzicht en gevoel voor humor als jij, van gedachten te kunnen wisselen, waarvoor bedankt!

169 acknowledgement/dankwoord

prof. dr. drenth, geachte Joost, zonder jou was deze promotie überhaupt niet mogelijk geweest. Je staat erom bekend hoe snel en scherpzinnig je op manuscripten reageert en van het nodige commentaar voorziet. Als ik jouw bijdrage aan mijn onderzoek bondig onder woorden moet brengen, zou ik willen zeggen: Jij was voor mijn proefschrif de ‘sine qua non’.

Het onderzoek vond plaats in het Rijnstate ziekenhuis op de afdeling MDL in nauwe samenwerking met afdelingen Immunologie, Radiologie en Pathologie. Aldaar ben ik dus ook dank verschuldigd aan velen. Te beginnen op de afdeling Immunologie wil ik elly van koolwijk en alle collega’s in het laboratorium die hebben meegedaan met het analyseren van biopten en bloed monsters, hartelijk danken.

Zo ook wil ik frank joosten, rutger bruijnen, marieke wielemans en alle andere radiologie medewerkers danken voor hun inzet bij het verrichten van contrast versterkte echograﬁeën. Beste Rutger, ook al een tijdje uit Rijnstate vertrokken, wil ik jou nog danken voor je inzet in het beoordelen en reviseren van MRI’s in het kader van onze studie. Mijn dank gaat ook uit naar jos meijer en alle pathologie laboranten die tijd en ener- gie hebben gestopt in het reviseren en/of afwerken van biopten. Daarnaast ben ik dank verschuldigd aan lian roovers voor haar geduldige hulp met de soms wat taaie materie van de statistiek. Op de MDL afdeling in het Rijnstate, waar ik nu ook met veel plezier aan het werk ben, wil ik iedereen danken die de afgelopen jaren hebben meegeholpen met inclusie van patiënten, plannen van onderzoeken, afnemen van biopten en zo voort. Ik zou graag een ieder bij naam willen noemen, maar dan zou ik bijna zeker iemand kunnen vergeten. Ik wil dus alle endoscopie medewerkers, verpleegkundigen, spreekuur assistenten, sec- retaressen en collega’s artsen van harte dank zeggen.

carolijn, jou wil ik bedanken voor jouw enthousiasme en de voortvarendheid waarmee je aan de slag bent gegaan en het onderzoek hebt opgepakt en voortgezet. Onderzoek doen in een perifere kliniek heef naast vele voordelen het nadeel van het ontbreken van collega’s jonge onderzoekers, met wie je naast professionele zaken ook een stuk gezelligheid en ontspanning kan delen. Dat laatste heb ik zelf gemist en ben daarom blij dat we de afgelopen drie jaar samen hebben kunnen optrekken in de wondere wereld van de wetenschap.

In de afgelopen jaren heb ik ook mogen samenwerken met collega’s immunologen uit het Wilhelmina Ziekenhuis en UMC Utrecht. In het bijzonder wil ik sabine middendorp danken voor haar inzet en samenwerking. Ik hoop dat we de komende jaren immunologische en gastroenterologische kennis verder kunnen combineren in dit onderzoeksveld.

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Natuurlijk wil ik de leden van de manuscriptcommissie: prof.dr mihai netea, prof. dr. mathias prokop en prof dr. gijs van den brink, bedanken voor hun bijdrage aan deze promotie. Graag bedank ik ook bas oldenburg, jos meijer, frank joosten en prof. dr. peter siersema voor hun bereidheid plaats te nemen in de oppositie.

Mijn paranimfen veronica en ebelien: verschillende achtergronden, zelfs twee verschillende werelden, een mooie illustratie van wie ik ben. Geboren, getogen en afgestudeerd in Roemenië, was Veronica jaren lang niet alleen mijn studiegenote maar ook kamergenote en dierbare vriendin. Geneeskunde studeren in postcommunistisch Roemenië was achteraf een bijzondere ervaring, ook al hebben we het destijds niet altijd zo ervaren. Naast een degelijke, soms pittige opleiding hebben we doorzettingsvermogen geleerd en blijven geloven in onze dromen. Lieve Veronica, de herinneringen aan samen hard studeren voor zware tentamens, veel lezen, muziek luisteren en af en toe naar de ﬁlm zijn maar een klein deel van onze huidige vriendschap. Wat heerlijk dat je naast mij staat ! Draga Vero, amintirile din anii de studentie cand invatam din greu pentru examene, citeam, ascultam muzica sau mai mergeam la un ﬁlm impreuna, sunt doar o mica parte din pretenia noastra. Ce bucurie ca esti alaturi de mine!

In Nederland integreren en aan het werk gaan in een ziekenhuis was voor mij een ontzettend leerzame ervaring, waarbij je als nieuwkomer in het begin soms onzeker voelt en steun kan gebruiken. Ebelien heb ik in de afgelopen jaren leren kennen als een vrolijke, enthousiaste en vriendelijke collega, altijd bereid om te helpen waar nodig. Lieve Ebelien, jouw opgewekte gemoed en vriendelijke glimlach hebben me vaak de moeilijke momenten helpen relativeren. Dank voor je vriendschap. Wat geweldig dat je naast mij staat !

Tot slot. jelle, julia en michael, mijn lieverds, een simpel dankwoord is nooit genoeg voor jullie geduld, begrip en steun afgelopen jaren. Een geweldige dikke knufel voor alles!

“Dankbaarheid is de herinnering van het hart” jean baptiste massieu, 1743-1818

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about the author

carmen horjus talabur horje was born in Oradea, Ro- mania, on the 13th of May in 1977. Following secondary school, Carmen studied medicine at the Juliu Hatieganu University in Cluj-Napoca between 1995 and 2001. Afer her medical degree she had been working for few months in a hospital in Oradea and moved to Holland in 2002. During her ﬁrst years in Holland she passed the language examinations and several medical exams in order to receive the Dutch BIG registration and be able to work as a medical doctor. Between 2005 and 2008 she worked as a resident on the Internal Medicine Department in Rijnstate hospital, Arnhem. In 2009 she started working on the research that led to this dissertation and between 2011 and 2014 continued her residency in Gastroentero- logy and Hepatology in Rijnstate hospital and the Radboud University hospital in Nijme- gen. Carmen currently works as gastroenterologist in the Rijnstate hospital and lives in Arnhem together with her family.

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