Mucosal and Systemic Immune Modulation by Trichuris Trichiura in a Self&#X2010;Infected Individual

R.Williams This article isprotected Allrights bycopyright. reserved. doi: 10.1111/pim.12394 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen 5 4 3 2 1 Dige Anders a self-infected individual Mucosal andmodulation immune systemic by Article type : Original Paper 07-Oct-2016 : Date Accepted :03-Oct-2016 Date Revised :16-Mar-2016 Date Received Aarhus University Hospital and Gastroenterology of Hepatology Department MD PhD Dige, Anders Corresponding author Trichiura, trichiuris,helminths, immunology Keywords from modulation Immune title Running Copenhagen, 1870 Frederiksberg C, Denmark Denmark Accepted for Section Pathology, Lillebaelt Hospital, Vejle,7100 Denmark FacultySciences, Veterinary Biology, andMedical University of of of Health Disease Department AarhusC, Denmark 8000 Aarhus Hospital, University Rheumatology, of Department Article Biomedicine, Aarhus of Department AarhusC, Hospital, Aarhus 8000 University andGastroenterology, Hepatology of Department 4 , Jørgen Agnholt 1 , Tue Kruse Rasmussen Trichuris trichiura trichiura Trichuris 1 , Jens FDahlerup , Jens 2,3 University, 8000 Aarhus C, Denmark C, Aarhus 8000 University, , Peter Nejsum 1 , ChristianLHvas on and undergone full peer review buthasnot review undergone fullpeer on and Version of Record. PleaseVersion cite thisarticle as 4 , Rikke Hagemann-Madsen , Rikke 1 Trichuris trichiura 5 , Andrew , Andrew in andsystemic humanimmunity. regional observed incirculating T cells. Taken together, T and ashift response was thephase, cell at towards Th1-response expense the a Th2-dominated of anti- eosinophils Plasma and infection. reaction. We observed several indicators of associated chain cytokines Treg- quantitative factors, polymerase and transcription by measured trichiura T. Campylobacter after its clearance following mebendazole treatment. Unexpectedly, the volunteer experienced a during at baseline, obtained were biopsies Colonic mucosal immunity. inappropriate immunological au towards response inappropriate This article isprotected Allrights bycopyright. reserved. inrecent years attention increased The potentialfor helminthic th Introduction with self-infection investigated In how we immunity. study, this human helminths how modulate of little know in we but recent hasyears, attention diseases gained of immune-mediated therapy Helminthic Abstract None Disclosures (AD) [email protected] E-mail: Denmark DK-8000 Aarhus C Noerrebrogade 44 pro-inflammatory T-helper (Th)1-type responses and induce a modified Th2-type responses andinduce response a Th2-type T-helper (Th)1-type modified pro-inflammatory andregulatory T macrophages, cells infections have helminthic indicated diseases Studies andallergic conditions. during autoimmune beneficial ahelminthiceffects host infectionresponses, the to could mediate immunomodulatory immunological immunological clearance anti-inflammatory induction of the escape through Accepted Article Trichuris (T.) trichiura (T.) Trichuris colonisation induced colonisation increased equally expressions of (h)1-, T-helper and Th2-, Th17-, colitis following in an adult man without intestinal erapy in organ-specific immune-mediated diseases hasgained immune-mediated inorgan-specific erapy T. trichiura T. 1–4 1,5,6 . Autoimmune and dise allergic . Autoimmune Trichuris . Furthermore, helminths have been reported to suppress to been reported have helminths . Furthermore, clearance, served andthis as apositive infectious control. modulation of systemic immunity during the immunity duringthe of systemic modulation induce regulatory dendritic cells, regulatory cells, dendritic regulatory regulatory induce our findingsour corroborate that helminths modulate antibodies rose markedly during the inoculation duringthe inoculation antibodies rose markedly to-antigens or allergen that have usedanimal inflammatorymodels of disease affected mucosal and systemic ases are associated with an T. trichiura T. s. Because helminths infection, and infection, T. trichiura T. 1,5,7,8 .

(i.e. 5,000 eggs per gram eggs per 5,000 (i.e. according World to of faeces Organization Health a positive a positive for control mucosal inflammation. served colonoscopy Therefore, as this and colonoscopy. the to follow-up infection prior helminth the immunomodulatory gaininsight to aimed the into we study, single-individual Inthis acquired a unexpectedly cell andcirculating phenotypic T production characteristics. cytokine Theassociated volunteer eggs. Weinvestigated both and regional systemic effects of development of new treatment strategies. the to lead potentially andcould immunity iswarranted human helminths modulate how of from understanding disease-specific infection Anincreased pathology. helminth the associated with of pro-inflammatory cytokine producing T T cells cytokine producing pro-inflammatory of was associated with adecrease in the numbers of responses also inhumanoccurs helminth infections the adaptive studies,itof Th-cell that befurther anticipated modulation may findings inanimal Resultsfrom clinical studies thathave inve (IBD) disease andallergicinflammatory conditions, suchas allergic rhinitis unresponsive that shown parasite-specificbeen T chronic cells infected from individualshelminthic are This article isprotected Allrights bycopyright. reserved. to grams. faecal Based production of the daily 80% 250 acalculation assuming that on volunteer´s a aimed induce period.to moderate study We the throughout He kg psoriasis. weight of 71 and body aconstant was 1.92 m tall untreated maintained mild with intestinal but was diseases with a38 included prior old of history male years volunteer The no anddesign subject Study Methods the hookworm (T.) Trichuris suis helminthic immunological effects by infections of induced the in Ourunderstanding isprimarily based studies individuals of infected areas humans chronically inendemic on Few clinical have the immunological Few by studies induced investigated changes producing T patients cells withcompared non-infected cytokine numbers increased of circulating cells T and regulatory numbers pro-inflammatory reduced of had patients MS helminth-infected inautoimmune study demonstrated One diseases. helminths studies areunderway studies Accepted trichiura T. numbersinvestigators mu observed increased of the Article T. trichiura T. infection 15,16 9,10 Necator americanusNecator possibly as a result of infection-induced regulatory FOXP3+ T T FOXP3+ ascells regulatory aresult of possibly infection-induced , celiacdisease , whereas asthma patients and patients with celiac disease have been infected with 21 ina self-infected volunteer with 2 . However, these studies did not enable the discrimination changes didnot discrimination of enable the studies these . However, . In IBD and MS, the parasite most frequently used is the pig is used the whipworm, and frequently parasite the . InIBD MS, most Campylobacter 17 , andmultiple sclerosis (MS) 2 . infectious colitis after mebendazole-induced clearance clearance colitisof infectious after mebendazole-induced 20 . In a single self-infected ulcerativecolitis. Ina patient, self-infected single effects by changes investigating inmucosal T-cell- regulatory Tcells andanincrease inthe numbers 1,2,4,6,8 cosal IL-4 andIL-22 producing Tcells following out intestinalout disease using living stigatedeffects the of . 13 18 , asthma , have been published, and additional published, andadditional been , have T. trichiura T. 19 . Indeed, anti-parasitic treatment 14 , inflammatory bowel , inflammatory infection in the volunteer 22 ). We estimated). the helminthic therapy in 11,12 . Based on the. Basedon T. trichiura T. 2 . Ithas

described concentrated McMaster method with an analytical sensitivity of 20 eggs per gram (epg) of faeces as dissecting microscope. Eggs in the faeces were identified, and the numbers were estimated using the Intestinal colonisation with adult region, which has been previously reported previously hasbeen region, which reaction-restriction fragment length polymorphism (PCR-RFLP) of the Internal Transcribed Spacer 2 This article isprotected Allrights bycopyright. reserved. of andestimation Identification sampling. the times at of blood were monitored haemoglobin) protei (i.e., C-reactive parameters Biochemical week. second period, blood every andfaecal the samples study During anemia. were obtained loss and diarrhea, of weight for the development week every clinically was monitored volunteer sigmoid colon and the transverse The the colon. colon, caecum/ascending the ileum, terminal this infection. Biopsies at all ileo-colonoscopies we time point thestool cultures were positivefor trichiura with prior self-infection to ileo-colonoscopies underwent volunteer rate. The establishment higher this second eggingestion to mimic infectio“natural trichiura andslightdecreaseinitial eggdose ineosinoph after the lacking 9 based on we, the Because weeks fecal excretion infection. egg in amoderate result that adose would of 600 eggs estimated we of 1:1 hatch and asex ratio eggs would ingested Accepted Switzerland). instructions (Bühlmann Schönenbuch, Laboratories, manufacturer’s Faecal samples were analysed for faecal calprote Faecal calprotectin Israel) days priorYoqneam, tothe2 ileo-colonoscopies. The small intestine was visualised by ca video enteroscopy capsule Video 18S region (rDNA) followed by aBLAS Article eggs, during the established infection, following clearance the infection, eggs, the duringthe of established he voluntarily ingested an small dosing ingested 3 additional Repeated of doses eggs. usedhe voluntarily was in 23 . Eight expelled worms (see below) were identified as T. trichiura T. trichiura T. T search (GenBank) (datanot shown). 24 eggs eggs was verified by identificationvisual ofeggsusinga and by sequence analysis base pairs(bp) of 435 sequence of the andby psule enteroscopy (PillCam SB, Given Imaging, n, total and differential leukocyte counts, and n, counts, leukocyte total and differential Campylobacter jejuni Campylobacter ils, doubted that the volunteer was infected with ctin using acalprotectin ELISA according to the re obtained systematically from locations: 4 the n” aswe expected that this would result in a T. trichiura T. , andfollowing clearance the of by polymerase chain bypolymerase T. trichiura T. , at which T. T. cytometry was performed within 24 hours usingaFACS (BD 24 Biosciences), analyser wasperformed Canto within and10 cytometry This article isprotected Allrights bycopyright. reserved. IL-22, IL-17A, IL-10, levels ofTGF- IL-4, the to expression was performed quantify RT-PCR cytokines mucosal for (RT-PCR) PCR transcriptase Reverse The gatingfor IL-17A, IL-22, single appearances. cell without used events exclude to was andforward-scatter-amplitude height recorded. forward-scatter- The inthewere combination forward-side lymphogate of scatter events IC7821P) and anti-IFN- cat.no. 51-7179-42), anti-IL-4 PE (eBiosciences, cat.no. 12-7049-42), anti-IL22 PE (R&D, cat. no. 557871) prior tofixation, permeabilisation andstai anti-CD8-P 345766), cat.no. (BD, the PBMCssurface-stainedbrief, anti-C were with serum. Staining intracellularserum. for cytokines in AB human heat-inactivated pooled 20% (PBS)with phosphate-buffered-saline twice with washed and thawed Uppsala, Sweden) atSciences, PBMCs Prior the andstored were -140°C. staining, to (GE cells Bio- Healthcare blood (PBMCs) usingFicoll-Hypaque mononuclear isolated were Peripheral T cells circulating in expression and FOXP3 Peripheral blood mononuclear cell isolation and flow cytometric analysis of intracellular cytokines with a runinduplicate samples maximum individual were the all For experiments, ACTB). (GAPDH and genes housekeeping of control means geometric the to relative andexpressed method inter-replicateacceptablecycl 1 differenceof at 60°C. minute at minutes 10 48°C, at minutes 15 parameters: cycle (Life Technologies, cat. no. 4392938) on an MX3000 RQ-PCR machine (Stratagene) with the following ﬂ Hs01076122, Hs02758991, and Hs01060665, respectively) labelled with the FAM-BHQ system as a Hs00961622, Hs00174383, Hs01574154, Hs00932747, Hs00989291, Hs00231122, Hs01085834, β TGF- IL-22, forIL-17A, IL-4, sets and IL-10, primer probe until Predesigned atuse. -80°C stored were Scientific), andthe RNAwas ng/ diluted (Thermo less aconcentration to than 50 Spectrophotometer RNA determined andpurity of the 2000 using aNanoDrop were concentration Austin, Texas, United States, cat.no. AM1906) usinga kit (Ambion, DNA-free free treated inRNase samples were DNase The and diluted water. and Messenger by usingchloroform washing RNAwas pelleted isopropanol. isolated inmethanol homoge were In brief,biopsies the volunteer. the FoxP3, andRORCGATA3, in from (m) RNApurified biopsies messenger intestinal from the obtained previously described previously as FITC (BD, APC-Cy7 FOXP3 intracellular priorand anti-CD4 staining to cat.no. 557871) for surface-stained with anti-CD3 PerCP (BD, cat.no Accepted Article usingthe RT-PCR TaqMan RNA-to-CT was used. Kit performed 1-Step were uorescence/quencher , IFN- γ , GATA3, FoxP3, RORC, GAPDH and ACTB (Life Technologies, cat. no. Hs00174122, 26 . After staining, the cells were fixed in 250 µl PBS with 1% formaldehyde. Flow µl PBS with formaldehyde. 1% fixed in250 the cells staining, were . After γ APC (eBiosciences, cat. no. 17-7319-82). Inparallel, the PBMCs were γ -interferon and IL-4 positive events was based on isotype controls. The The isotype controls. based on positive was andIL-4 events -interferon E-Cy7 (BD, cat.no. 557746), and the PBMCs was performed PBMCsthe was aspreviously described e. The fold change was calculated using the according tothe manufacturer’s instructions. The . 345766), anti-CD25 PE-Cy7 (BD, cat.no. 557741), nised in TRIzol (Life Technologies), and mRNA and was mRNA Technologies), (Life nised inTRIzol D45RO FITC (BD, cat.no. 555492), anti-CD3 PerCP PerCP anti-CD3 555492), cat.no. (BD, FITC D45RO ning withanti-IL17A Alexa-647 (eBiosciences, 95°C and 40 cycles of 15 seconds at and1 at seconds 95°C 15 cycles and40 of 95°C anti-CD4 APC-Cy7 (BD, cat.no. μ L. The samplesL. The β ∆ , IFN- Ct 25 γ 5 ,

. In the study, he signed he informed consent. study, adeclaration of the then paraffin embedded and cut in 3 µm and sections, from sections serial Each in3µm thin with block. cut 3 paraffinembedded each then formalin; hours were in fixed biopsies for neutral buffered were 24 All biopsies 10% approximately biopsies,Forceps per two bowel segment, were placed on Millipore paperandroutinely prepared. Microscopic examination of intestinal biopsies Diva 5.1 software (BD Biosciences). experimentally infected pigs, essentially pigs, infected as described essentially experimentally by produced was Anti- ofanti- Measurement droplets. mucin eosinophil infiltration. Ileac biopsies were also stai for of andeosin with evaluation stained 3serial that sections contained was slide haematoxylin This article isprotected Allrights bycopyright. reserved. InspectorateAnimal (Licence number 2015 T. suis wasstudy registered at www.Clinicaltrials.gov (ClinicalTrials NCT02399683). no. Infection pigs of for aformal to didnot need to notification Ethics legislation, according the the Danish The Committee. Ethics that Health confirmed Research the on Regionstudy, Committees Denmark Central The cells as recommended previously expressing used the CD4+CD3+ T cells with the lowest CD25 expression (lowest quartile) as “non-FOXP3” gating ofFOXP3 positive events was based on a bi conjugate (Sigma-Aldrich) anti-human IgA-HRP goat seraandthen detection antibodies: of addition the before in PBS, BSA 1% blocked with were buffer (pH9.6). Plates incarbonate/bicarbonate of µg/mL ESproducts 5 measured by the BCA assay (Pierce). ELISA plates (Nunc Maxisorb) were coated overnight at 4°C with The volunteer is The volunteer a researcher within statement Ethical control (pooled naïve serum). whichgavedilution an OD atvalue least 3 sta nm with a Multiskan FC plate reader (ThermoFisher). Titres were calculated as the inverse of the Accepted H 0.2M with stopped substrate TMB andthe reaction with developed were Plates Tween 20. 0.02% containing PBS with incubations between 5 washed times were andplates steps 37°C IgG-HRP conjugate for All 1hour (AbdSerotec). at were incubation anti-mouse Article Trichuris material according approvedmaterial of and conducted guidelines the was Danish Experimental the to antibodies was measured usingELISA. wasmeasured antibodies invitro Trichuris culture of adult worms collected from the andcolon caecum of or anti-humanor mouse IgG antibodies in serum in serum antibodies parasitology with main focus on − 15 27 ndard deviations above the mean of the negative negative the above the ndard deviations of mean . Allflow cytometry data were analysed usingFACS − 0201 ological control as previously described, which which described, controlological aspreviously ned with Alcian Blue-PAS for the visualisation for AlcianBlue-PAS ned the visualisation of with 1 Trichuris suis Trichuris (Sigma Aldrich; clone (Sigma 8c/6-39) followed goat by − 28 00760). , and protein content of of ES content was products , andprotein

excretory/secretory (ES) material material (ES) excretory/secretory T. trichiura T. 2 SO 4 . Plates were read at 450 and T. suis . Before One week prior to the scheduled follow-up prior follow-up the to week scheduled One inflammation inallinvestigated colonic regions and ileum. signs Examination acute the revealed biopsies and terminal intestinal and chronic of of andsmall ulcers colon inthe redness,entire A revealed oedema discomfort. colonoscopy abdominal infection. during symptoms the intestinal experience didnot volunteer 2).The (Figure infection before the No (Figure3). single crypts of disruption slight with colonized in superficially the lamina propria. The infiltra localised predominantly that were by inflammation,eosinophils dominated and chronic were but Circulating eosinophils began to increase 4 weeks following the first inoculation. They the increase first began 4weeksThey following inoculation. to eosinophils Circulating all revealeda lightly to moderate duringthe ilealobtained andcolonic biopsies of the examination Microscopic treatment (Figure 2). after andnormalised stool the samples inthe mebendazole apparent began became decrease to eggs when they the but increase to duringcolonisation, continued Capsule enteroscopy the intestineof small was norm (Figure period 1B). study for the rest of the level just both detection above the remained infection but after the dropped the for at established and rose the time infection markedly IgAandIgG Serum period. study the of mebendazole treatment, the faecal samples were ne for 5 consecutive days was used, which was an effective treatment. Two weeks following the This article isprotected Allrights bycopyright. reserved. Clinical course of self-infection with Results Accepted Articledrug efficacy hasbeen reported for low Because post-treatment. eggs to rapidly at week one 300 decreased numbers the egg which after received mebendazole, volunteer the and colon, colon the transverse caecum/ascending the to (Figure Following1A). later presence duringwhich adult acolonoscopy the restricted worms of was egg ofepg and count 3weeks 100 faecal rapidly epg to was countthis observed, increased 7,620 in week eggs and 100 10, in week eggs 200 9, week trichiura 600 with inoculation first the following 9 duringweeks faecal observed microscopy eggs were no Colonisation with eggs, the volunteer ingested an additional 650 eggs in the following 4 weeks (350 eggs in

T.trichiura T.trichiura T. trichiura T. 1 anti- infection. Faecal calprotectin Trichuris , i.e., colon, the withcaecum/ascending and transverse anda cryptitis following self-infection was monitored by faecal by Because eggwas following self-infection monitored counts. antibody levels were below detection level before the the before level detection below were levels antibody ly inflamed mucosa. Th inflamed mucosa. ly T. trichiura T. T. trichiura tion was much more pronounced in the regions eosinophils was observed in the biopsies obtained 29

, a dose regimen of 100 mg mebendazole twice daily daily twice mebendazole mg 100 of regimen dose a , a after .Four 12) weeks second inoculation, this levels remainedlevels normal during the the terminal ileum, butnoeosinophil infiltration. gative and remained negative for the remainder for remainder the negative and remained gative al with no visible worm e inflammatory cells represented both acute ileo-colonoscopy, the volunteer developed ileo-colonoscopy, thevolunteer T. trichiura T. infection. Antibody levels levels infection. Antibody s in any investigation. T. trichiura T. T. trichiura T. T. trichiura T. T. trichiura trichiura T. infection infection

This article isprotected Allrights bycopyright. reserved. ileum increased IL-21, IFN- IL-17A, observed anddescending the we (i.e., expression of colon), Theexpression of investigatedmost Tcell subset-representativein T-betwasonly modest (F increase genes wasdown further during increased andT-bet IL-21 the expression of been resident, although the expression remained increased compared with the baseline. The regulated duringthe respective (Figure and their cytokines 4). factors transcription in increases with increased corresponding subset-representative the similarly transverse colon (Figure 4). The Th1, Th2, Th17, and Treg subset-representative genes were all colonisedregions with cell subset-representative genes (Figure 4). The increased expression was restricted to the colonic determined that T cell different subsets, i.e., Th-1-, the Th2-, Th17- a was associated with asubset-specific T cell response by examining representative genes from the cell responses to the usedWe PCR quantitative (qPCR) analysis intest of T. trichiura normal. ileum were colon andterminal later, 4 the weeks colonoscopy faecal calprotectin levels normalised sponta observed a marked increase in the faecal calprotectin to 830 mg/kg (Figure 2). The symptoms and Faecal cultures proved positive for the patient recovered fromthe investigated T cellsubset-representative genes retu ininfection theregions in which investigatedremaining T cell subset-repre and TGF- the 30% increase (from 2.8% to 3.7%) in the numbers of IL-22 producing CD4+CD45RO+ T cells during producing IL-4 cells circulating T inthe a increase frequency CD4+CD45RO+ of than andmore fold activated memory T cells, CD45RO was used to enrich thesefor cells. We observed amore thantwo- T cells was analysed (Figure Because 5). the prod main response was also reflected insystemic immunity, th investigateTo whether the T. trichiura Accepted T CD4+CD45RO+ cells (range IL-17A-producing frequencies inthe noclearof changes were There %). (from7.1 to infection 33 with 10.6% % T decreased during the cells CD4+CD45RO+ producing Article T. trichiura T. β during the duringthe induces a regional mucosal cytokine response response cytokine mucosal aregional induces modulates systemic immunity immunity systemic modulates infection compared with the baseline T. trichiura T. Campylobacter T. trichiura T. Campylobacter T. trichiura T. T. trichiura T. induced a pronounced increase in the expression of all investigated increaseT of inthe all investigated apronounced expression induced Campylobacter infection. We aimed to elucidate whether the We elucidate to aimed the infection. whether T. trichiura T. during the colonoscopy, i.e., the caecum/ascending and i.e., caecum/ascending the colonoscopy, duringthe Campylobacter Campylobacter infection in infection the colonic regions inwhich the igure 4). In the colonic regions where infection compared with baseline levels. The expression of the colonisation-induced of the modulation mucosal cellT sentative genes was unchanged during the duringthe unchanged was genes sentative had not been resident (Figure (Figure hadnot the resident 4). The levels of been neously duringthefollowing week. At theileo- colitis (Figure 4). inal biopsiesmucosal to investigate the mucosal T species. During this nd Treg cells, inthe qPCR analysis (Table We1). rned to baseline at the 4-week follow-up after e intracellular cytokine pr levels (Figure Thelevels of number (Figure IFN- 5). uction of the selected cytokines isidentified in Campylobacter Campylobacter colitis, the although T. trichiura oduction incirculating oduction T. trichiura T. T. trichiura T. wasabsent Campylobacter colitis, we γ infection - had γ , IL-10

helminths to the disease incidences in which links decreased exposure the to autoimmune increase hypothesis, andclasses of helminths other to exposure of Indeed, the absence helminths. to previous exposure limited very countries with no or industrialised helminthic infectionshelminthic are endemic primarily in chronic infection described infections have parts ofduring helminthic the where world of studies immunological changes activity. indisease Previous changes coincidental immunity or of to helminth-induced reflected modulation immunologicalclarify whether possible observations the colitis, not it ulcerative was active with ina the because study patient was conducted However, ingestion. Because the precise pre-patency period of period precise pre-patency Because the ingestion. Colonisation with calprotectin, which isa sensitive Based on the changes in the mucosaltrichiura expression of Tcell representative genes, we inthe by regions pronounced was granulocytes, which colonised most byeosinophil dominated This increased T cell activation was paralleled a by mild to moderate inflammatory response determined that infection with from theoriginated inoculation. primary dose) grown second (the weeks maturity andbelieveto within thathave 4 the colonisation could worms at 9. unlikely We that was findit performed week fall, asecond to inoculation began This article isprotected Allrights bycopyright. reserved. ulcerative patientcolitis single who wasself-infected with during individual in healthy anintestinally production This studydescribes the succe Discussion Campylobacter 4-, IL-22-, and 0.2-0.6%) (Figure 5) or CD4+FOXP3+ Tregs (range 3.8-4.2%) (data not shown). The frequencies of IL- specific T cell subset because we observed a similarl transverse colon. Interestingly, the mucosal T cell response to was restricted to colonic regions where the Accepted The present study is the first to investig Article production. and T inthe CD4+CD45RO+ circulating change cell of anti-Trichuris antibodies cytokine formation specific cytokines anda of systemic modulation immunity demonstrated the by eosinophilia, included amarked regional increase inmucosal ge provided a positive up colonoscopy, is inthat unexpected unique Our study the the mucosa of monitoring concomitant with disease, . The increased activation of mucosal Tcells was not mirrored anincrease by infaecal 30 . γ colitis (Figure5). -interferon-producing CD4+CD45RO+ T cells decreased during and following the the duringandfollowing T decreased cells CD4+CD45RO+ -interferon-producing ssful self-infection ssful self-infection with T. trichiura T. T. trichiura T. marker intestinal of inflammation 2 . These observations may not be in . These be patients to not applicable observations may in vivo was associated with increa control for intestinalinflammation. The findings main Campylobacter was demonstrated 13 weeks after the initial egg T. trichiura T. microorganisms forms the basis for the hygiene hygiene the for basis the forms microorganisms ne expression of allinvestigated Tcell-subtype y increasedy expression profile of allinvestigated l and systemicT cell-specificresponses.l and immune T. trichiura T. T. trichiura trichiura T. T. trichiura T. ate changes inintest was resident, i.e., the caecum/ascending and i.e., caecum/ascending resident, the was T. trichiura T. colitis, which was present at the follow- T. trichiura T. isunknown infection. Asimilar studyfrom a in a volunteer without intestinal without a volunteer in 32 . Theincreased Tcell activation has been published has been sed mucosal Tcell activation. was not limited to a inal T cell cytokine 31 and eosinophil levels andeosinophil levels 21 . T. infection is of course speculative and should be confirmed in larger studies. However, taking the the taking However, inlarger studies. be confirmed is andshould course speculative of infection human human suis diarrhea andthat this is more severe Campylobacter that shown have Studies eradicated. Although we consider this an unexp This study has Thisstudy clear limitations, the prominentmost of whichis that we only examined one study subject. To what extent the more specific immunological changes we observed superinfection. bacterial in the included individual reflects the general response of human immunity during immunity general response of a human reflects the individual included in the Based on these findings, it has been speculated speculated findings,it Based these that has been on reported inchronically helm during circulatingTregs FOXP3+ numbers of the diseases are associated with theabnormal activation related with diseases autoimmune the treating principle of supports This article isprotected Allrights bycopyright. reserved. cell regulatory response T Th2-like or a polarised more have expected would specific subtype genes.T-cell animal Based in findingsfrom andstudies on patients, models MS one complicated Campylobacter T cells producing and IL-22 was observed colitis patient who was self-infected with and IL-22 productions. However, also However, a IFN- observed we productions. decreased and IL-22 T incirculating production cells. cytokine altered Asystemic response towards trichiura eosinophils could therefore represent asensitive tool to monitor patients in future trials with the clearance of increased Following established. numbers infection circulating asthe was where eosinophil phase changes. changes caused described the have could helminth-induced epithelial cells, although it did not an increase infaecal ca Furthermore, our study indicates that Trichuris Trichuris cells. The increased IL-4 and IL-4 IFN- increased The decreased cells. Campylobacter Campylobacter Accepted Articleself-infection inUC in circulatingTh2 cells from the Th2 response on the expense of the Th1 response in 33,34 . Furthermore, an. Furthermore, Campylobacter . Another indication of systemic immunomodulation by immunomodulation systemic indication of . Another infections to humans to investigate patients’ humans to infections investigate to studies that be inhumans. infections future applying Wemay associated recommend Campylobacter/Trichuris Campylobacter/Trichuris The volunteer developed a developed volunteer The in the colon microflora, but also that co-infected animals more frequently develop develop animals frequently also but in co-infected colonmore microflora, that the has been reported in humans. This individual presented with a very severe and with a severe presented inhumans. reported very has This individual been should be considered eradicated before individuals are infected with with areinfected beforeshould considered individuals eradicated be T. trichiura T. 21 . Our finding of decreased circulating cells. Ourfinding following decreased Th1 of infections lprotectin ina helminth-infected indivi T. in vitro

suis , theplasma eosinophils normalis T. trichiura T. inthic infected MS patients infections inpigs infections associated are with of anincreased abundancy study reported that secretory products from thatproducts secretory reported study 36 increase the cellsincrease the . To our knowledge, only one case of concomitant in animals only infected with either any sign intestinalof inflamma colitis infection has report infection also reported on been inasingle-patient 21 T. trichiura T. Campylobacter . In these studies, however, both studies, disease-related and however, . Inthese γ 37 T. trichiura T. production incirculating T cells suggest aselection of . Our observation highlights that highlights . Ourobservation ected complication it notmay becoincidential. T. trichiura The infection was associated with increased IL-4 susceptibility to , an intestinal T cell response dominated IL-4 by Trichuris Th1of cells. We didnotobserve anincrease in Campylobacter was suggested already during the inoculation inoculation duringthe was suggested already 19 T. trichiura T. . colitis justafter infection, which infection, has previously been ed. Changes in the circulating infections may modulate the the course of modulate infections may duals, should leadduals, should to the suspicion of T. trichiura T. infected individuals.Anincrease γ Campylobacter T. trichiura T. tion, i.e., clinical symptoms or production in circulating T T incirculating production present andany status, Campylobacter T.trichiura was our finding of of finding our was 5,8,19,20 T. trichiura T. T. suis T. Campylobacter because these . In an ulcerative . Inanulcerative hadbeen damaged damaged infection T. trichiura T. Trichuris T. trichiura trichiura T. colonisation or T. 35 and . T. .

and regional mucosal cytokine a represents cytokine that response all activation regional mucosal of general Th-subsets. diseases. treatment autoimmune by play inthe induced future a role helminths of may modulation Immune both human immunity. helminths andsystemic regional modulate that findings support our cells. together, responseTaken incirculating of mononuclear aTh1-like at the expense response dominated a Th2-like of induction via the immunity systemic infection the modulated Furthermore, This article isprotected Allrights bycopyright. reserved. References studies inanimal include infection responses,demonstrated been cell non-T have which may helminthic T andcirculating cells. during CD4+ mucosal The immunity andsystemic modulation of mucosal bya modulated is that immunity demonstrate in individual, althoughone observed immunity, only systemic changes our and consideration, designinto findingsregarding of mucosal study longitudinal 9. Grogan JL, Kremsner PG, Deelder AM et al.. Antigen-specific proliferation andinterferon- proliferation AM PG, al..Antigen-specific et Deelder JL, Kremsner Grogan 9. Taylor MD, dervan Werf N, Maizels RM. T cells 8. Nat Immunol Rev helminths. to 2011; inimmunity anddialogue RM. JE,Maizels Allen Diversity 7. parasites: defining byhelminth RM. Immunomodulation JP, HJ,Hewitson Maizels McSorley 6. parasites: cellular by Immune RM, and regulation M. helminth Maizels Yazdanbakhsh 5. barrier function. Helminthictherapy: mucosal BroadhurstP. MJ,Loke improving MJ, Wolff 4. 183-185. 491: 2012; Nature returns. worm The JV.Autoimmunity: Weinstock 3. knowns. known to unknowns translating the unknown therapies: AR,Fallon PG. Khan Helminth 2. Elliott DE, Weinstock JV.Helminth-host immu 1. Accepted Article that demonstrates study present the Inconclusion, the response of a specific T cellsubtype. epithelial cells. However, this does not alter our conc expressionassociated with the 1,7 . Because we performed qPCRanalysis we performed forcep. Because on the biopsies, increased cytokine mechanisms and mediators. Int J Parasitol 2013; 43: 301-310. 301-310. 43: 2013; JParasitol Int mediators. and mechanisms molecular mechanisms. Nat Rev Immunol 2003; 3: 733-744. Trends Parasitol 2012; 28: 187-194. ParasitolIntJ 2013; 43: 293-299. 83-96. 1247: Sci 2012; Acad AnnNY diseases. immune-mediated haematobium infection. J Infect Dis 1998; 177: 1433-1437. down-regulated production duringSchistosoma are andinterleukin-5 gamma regulated. Trends Immunol 2012; 33: 181-189. 11: 375-388. T. trichiura T.

infection. Ourinvestigation focused on T. trichiura T. infection may also reflect changes in other immune and infection in immune also reflect changes may other nological interactions: prevention and control of inhelminth infection: lusion that these changes were not restricted to T. trichiura T. adaptive immunity in the form of the regulators andthe infection inducesa infection This article isprotected Allrights bycopyright. reserved. Accepted al..Geneti et A,Hansen TV S,Al-Jubury Nissen 24. diagnosis Nations: of andAgriculture Organization the andcontrol Epidemiology, United Food 23. Health World Organization Soil-transmitted2012 helminthiases: eliminating soil-transmitted 22. T V al..IL-22+ cells areassociatedCD4+ with JM, therapeutic et Leung Kashyap MJ, Broadhurst 21. J,Farez impact MF. parasite Correale The of 20. para between Association J,Farez M. Correale 19. Fleming JO, Isaak A, Lee JE et al.. Probioti 18. Daveson AJ,Jones DM, Gaze Set al..Effect 17. RW, Elliott54: inCrohn's disease. DE,Urban al..Trichuris suistherapy Summers 2005; Gut JFet Article 16. RW, Summers Elliott DE, Urban JFetal..Trichuris suistherapy for active ulcerative a colitis: 15. a randomized infection: hookworm al..Experimental et JR, AJ, K Venn Feary Mortimer 14. al.. J,Ronborg P,Arnved Set Tric Bager 13. N, NauschN,Midzi Mduluza 12. have expanded filarialinfections al..At Set homeostasis B, S, Konate Dembele Metenou 11. CL, S,Kumaraswami control King Mahanty parasite-specific Vet al..Cytokine of anergy in 10. lymphocyte subset. J Clin Invest 1993; 92: 1667-1673. 2 T induction a type filariasis. helper of regulatory lymphatic Preferential human of helmith parasites helmith of swine.of 2011-2020. plan strategic helminthiasis as apublichealth problem 60ra88. trichuris trichiurainfection in anulcerative colitis patient. SciTransl Med 2010; 2: 6-11. 233: 2011; Neuroimmunol sclerosis. Ann Neurol 2007; 61: 97-108. multiple sclerosis: aphase 1 study. Mult Scler 2011; 17: 743-754. e17366. 6: One2011; trial. PLoS controlled placebo double-blinded randomised disease--a 87-90. 825-832. 128: 2005; Gastroenterology trial. controlled randomized placebo-controlled trial in Clinasthma. ExpAllergy 40:2010; 299-306. 2010; 125: 123-130. placebo-controlled double-blind, randomized, haematobiuminfections. PLoS One 2011;6: e16860. 5375-5382. 184: 2010; JImmunol cells. Th2 classical not but regulatory T adaptive 188: 68-77. and from humans recovered pigsinasympat T et al.. Regulatory and activated T Schistosoma cells andactivated inhuman al.. Regulatory et T huris suis therapy forova allergic rhinitis: a c helminth administration inrelapsing-remitting of hookwormof infection on wheat challenge inceliac infections the on course of multiple sclerosis. J c analysis suisand of Trichuris Trichuris trichiura site infection andimmune responses in multiple in children:progress report 2001-2010and ric setting in Uganda. Vet Parasitol 2012; clinical trial. JAllergy Clin Immunol This article isprotected Allrights bycopyright. reserved. Dige A,Stoy S, Rasmussen TK et al..Incr 25. 33. Mansfield Gauthier DT, SRetLS, al Mansfield Abner 33. Faecal SherwoodRA. gastrointe markers of 32. Cooper DA, Bundy ES. Trichuris andtrichuria 31. the hypothesis: andthe hygiene immune GA. modulation series on helminths, Review Rook 30. low have A,Namwanje H,Nejsum and efficacy Olsen et mebendazole al..Albendazole P 29. KringelH,Roepstorff A.Trichuris suisexcr 28. LawJP, Hirschkorn DF 27. 1206-1214. 46: 2011; JGastroenterol Scand al.. et B Deleuran CL, Hvas A, Dige 26. 7 ShinJL,GardinerGW, Deitel Wet al.. 37. infections. helminth parasitic and Malnutrition EA. Ottesen MC, Latham LS, Stephenson 36. AbnerSR,HillDE, Turner JR etal..Response 35. porcine microbiota colon ofthe disruption burden-dependent al..Worm S,Li et RW, Wu Li W 34. Accepted Article 985. 3-11. 126: 2009; Immunology hypothesis. the hygiene implications of broader 443-446. 103: 2009; Hyg Med Trop in inKabale Trichuristrichiura District,children against school-age Uganda.Trans R Soc from single-inoculated pigs. Parasite Immunol 2007; 29: 327-330. regulatory T cells. Cytometry A 2009; 75: 1040-1050. fixation/permeabilization buffer combinat versus active Crohn's disease. J Crohns Colitis 2013; 7: 248-255. Parasitology 2000; 121 Suppl: S23-S38. S23-S38. Suppl: 121 2000; Parasitology 738-745. J conditions. 88: 2002; Parasitol invitro under invasion jejuni Campylobacter influence on andthe products excretory-secretory e35470. 7: 2012; One PLoS infection. suis Trichuris by 70-80. 68: Hyg 2003; Med JTrop Am in naive colon Trichuris jejuni suisandCampylobacter immunologically the swine. of of Campylobacter jejuni? A clinical correlate of anexperimental observation. Can J Gastroenterol 2004;175-177. 18: , Owen RE et al.. The importance of Foxp3 antibody and Does whipwormDoes pathogenicity increase the of eased levels ofcirculatingTh17 cells inquiescent .. Enhancement of disease and pathology by synergy by andpathology of disease .. Enhancement etory/secretory antigen-specific antibodies inserum stinal inflammation. JClinPathol 2012; 65: 981- of intestinal Trichurissuis epithelial cellsto of sis in humans. Adv Parasitol 1989; 28: 107-173. 107-173. sis inhumans. 28: Adv Parasitol 1989; ions inidentifying CD4+CD25+Foxp3+ Trichuris Trichuris (A). of anti- treatment levels after No 2 detected eggs The weeks mebendazole were mebendazole. received after rapidly the volunteer dropped duringcolonoscopy. Eggnumbers demonstrated was egg excretion was observed from week 13 and increased until week 16 where The volunteer ingested Figure 2. The course of the remained above the detection limit (B). *: titer < detection limit. markedly after infection. Both IgAandIgG This article isprotected Allrights bycopyright. reserved. andanti- excretion Egg 1. Figure (RT-PCR). Table 1. Table andfigure legends Th1 Table 1 Tables but rose rapidly during the duringthe rapidly accidental rose but treatment. Faecal calprotectin levels following mebendazole normalisation by adrop and eventually inoculation after followed increased Accepted Article Treg Th17 Th2 T-cell specific subtype reverse genes polymerase analysed by chainreactiontranscriptase IgA andIgG 1 were below thedetection limit (titer 50) before the infection and rose T. trichiura Tbet, IFN- IL-10, FoxP3, TGF- RORC, IL-17A, IL-21, IL-22 GATA3, IL-4 T. trichiura Trichuris trichiura eggs twice (week 0 and repeated ingestion at week 9-13). Faecal γ (closed circles) remained normal during

Campylobacter self-infection. 1 levels dropped after mebendazole treatment but treatment dropped after mebendazole levels β

antibodies during the the during antibodies colitis. Levels of plasma eosinophils (open circles) (open eosinophils plasma of Levels T. trichiura T. trichiura T. T. trichiura T. self-infection. infection, presence

This article isprotected Allrights bycopyright. reserved. 4. Figure destruction of singlecrypts was observed (B). was pronouncedmost inflammation colon, a inthe where caecum/ascending slight andtransverse localisedeosinophil granulocytes predominantly both acute andrepresenting inflammation. chronic The cells dominated infiltratory by were duringtheobtained macroscopically signs inflammati of trigger and first-infection post didnot 16 at (redweek duringcolonoscopy arrows) demonstrated 3. Figure cells T -producing IL-17A CD4+CD45RO+ was (C). observed of frequency the duringandafter cells dropped T CD4+CD45RO+ of interferon- -producing andIL-22 CD4+CD45RO+ IL-4- cells of circulating anddecreasedfrequencies T frequency isotype (B). controls on gated based were events positive and (in example interleukin-4 Ausingflow the cytometry and Brefeldin T hours Ionomycin stimulation in cells 4 PMA, following with was evaluated CD4+CD4RO+ production scatter forward appearance andside (A).Cytokine gated upon their plots. were Lymphocytes flow the higher during was (B).genes specific cell blue)) of regional increasedT expression (light The subtype colon (dark (ileum blue) anddescending intestine the regions of non-colonised inthe observed were regions colonised (caecum/ascending (red) the (A) andtransverse colon (yellow)) nochanges while infection there was a marked increase expression of Accepted by production cytokine Tcells CD4+ ofcirculating Modulation 5. Figure Article T. trichiura trichiura T. Regional Regionally increased expression of T cell subtype specific genes. genes. specific subtype of T cell expression increased Regionally γ -producing -producing CD4+CD45RO+ Tcells. Freque infection than duringthefollowing T. trichiura T.trichiura colonisation in the caecum/ascending and transverese colon was andtransverese in caecum/ascending colonisation the infection revealed slightly to on (A). Histological examination of ileac and colonic biopsies superficially inthe lamina propriae. The Campylobacter Campylobacter all investigated T cell subtype specific genes in ncies of IL-4 and IL-22-producing andIL-22-producing IL-4 ncies of Trichuris trichiura trichiura Trichuris moderate inflammatory changes moderate

enteritis. colitis. No changes inthe γ -interferon). Cytokine- -interferon). T. trichiura During the induced increased . Representative T. trichiura T.

This article isprotected Allrights bycopyright. reserved. Accepted Article