With Whipple's Disease -Specific Th1 Response in Patients Whipplei Tropheryma Reduced Peripheral and Mucosal

Reduced Peripheral and Mucosal Tropheryma whipplei -Specific Th1 Response in Patients with Whipple's Disease

This information is current as Verena Moos, Désirée Kunkel, Thomas Marth, Gerhard E. of September 27, 2021. Feurle, Bernard LaScola, Ralf Ignatius, Martin Zeitz and Thomas Schneider J Immunol 2006; 177:2015-2022; ; doi: 10.4049/jimmunol.177.3.2015

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Reduced Peripheral and Mucosal Tropheryma whipplei-Speciﬁc Th1 Response in Patients with Whipple’s Disease1

Verena Moos,2*De´sire´e Kunkel,* Thomas Marth,† Gerhard E. Feurle,‡ Bernard LaScola,§ Ralf Ignatius,¶ Martin Zeitz,* and Thomas Schneider*

Whipple’s disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple’s disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple’s disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-MarseilleT has enabled the first analysis of T. whipplei-specific reactivity of CD4؉ T cells. A robust T. whipplei-specific CD4؉ Th1 reactivity and activation

(expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 tria- Downloaded from thletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple’s disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple’s disease patients and may contribute to the fatal natural course of the disease. The Journal of Immunology, 2006, 177: 2015–2022. http://www.jimmunol.org/ hipple’s disease, first described by George Hoyt concentrations of IL-12p40, a reduced production of IL-12 in Whipple in 1907 (1), is an infectious disease caused monocytes (15, 16), and the presence of M2/alternatively activated W by the actinomycete Tropheryma whipplei (2). The macrophages that favor the development of Th2 responses in the most frequent manifestations of Whipple’s disease are weight loss, intestine (17) may explain these impaired Th1 cell functions. De- diarrhea, polyarthralgia, fever, lymphadenopathy, and cardiac and spite reduced Th1 activity, general immunocompetence seems to CNS symptoms (3). The course of Whipple’s disease is fatal, un- be unaffected, as no systemic opportunistic infections have been less treatment with antibiotics is initiated (4). reported in Whipple’s disease patients. Previous investigations fo- T. whipplei is assumed to be a microorganism present in the cused on unspecific reactivity of lymphocytes and thus could not environmental soil and water (5). An oral route of acquisition was contribute to clarify this apparent discrepancy. Therefore, the in- by guest on September 27, 2021 proposed (6), and in recent studies, T. whipplei DNA was detected vestigation of T. whipplei-specific immune responses, made pos- in the saliva (7), marginal and subgingival plaque (8), and feces (9) sible through the recently established cultivation of T. whipplei, of healthy subjects in which T. whipplei-specific IgG Abs were appears particularly important. Consequently, we used T. whipplei identified in over 70% of the cases (10). Whipple’s disease is very lysate to investigate specific reactivity of peripheral and duodenal rare despite the almost ubiquitous occurrence of T. whipplei in the CD4ϩ T cells in patients with Whipple’s disease and control environment, suggesting that host factors are necessary to permit subjects. an infection. In patients with Whipple’s disease, cutaneous responses to recall Materials and Methods Ags and peripheral T cell proliferation are reduced after stimula- Patients and control subjects tion with PHA, Con A, and Abs to CD2. These functions improve ϩ somewhat during treatment, but remain impaired even in long- We studied CD4 T cell reactivity in 32 patients with different stages of Whipple’s disease (13) (Table I, 24 males (M), 8 females (F), mean age, standing remission (11–14). Apparently, the Th1 reactivity in the 57.2; range, 41–84 years), compared with control groups without evidence periphery and the intestinal mucosa is impaired (14). Low serum of disease: I, 16 young subjects (8 M, 8 F, mean age, 30.3; range, 21–39); II, 27 elderly subjects, age-matched to Whipple’s disease patients (16 M, 11 F, mean age, 54.9; range, 41–88); and III, 11 active triathletes, age- *Medizinische Klinik I, Charite´-Universita¨tsmedizin Berlin, Campus Benjamin matched to young subjects (11 M, mean age, 32.7; range, 25–43). Triath- Franklin, Berlin, Germany; †St. Josefs Krankenhaus, Zell/Mosel, Germany; letes practice swimming in the river Neckar in Heidelberg where T. whip- ‡ § Deutsches Rotes Kreuz Krankenhaus, Neuwied, Germany; Unite´ de Rickettsies, plei was detected in sewage plants (5) and thus are supposed to have Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 6020, Faculte´de ¶ enhanced contact to environmental T. whipplei. As disease-control, 17 patients Medicine, Marseille, France; and Abteilung fu¨r Medizinische Mikrobiologie und 3 Infektions-Immunologie, Charite´-Universita¨tsmedizin Berlin, Campus Benjamin with active tuberculosis (TB) were selected for group IV (10 M, 7 F, mean Franklin, Berlin, Germany age, 48. 9; range, 23–85; 9 with pulmonary, 6 with lymph node, and 1 with bone and ovarian TB, respectively). The diagnosis was based on the detection Received for publication February 14, 2006. Accepted for publication May 16, 2006. of Mycobacterium tuberculosis by Ziehl-Neelsen staining and cultivation. TB The costs of publication of this article were defrayed in part by the payment of page patients were selected for positive tuberculin skin test, and tuberculin-specific charges. This article must therefore be hereby marked advertisement in accordance Th1 reactivity (IFN-␥ expression after stimulation; see below). with 18 U.S.C. Section 1734 solely to indicate this fact. Duodenal biopsies were obtained of 15 Whipple’s disease patients (11 1 This work was supported by European Union Contract No. QLG1-CT-2002-01049, M, 4 F, mean age, 57.6; range, 41–74) (Table I) and in 11 age-matched Deutsche Forschungs Gemeinschaft KFO 104. 2 Address correspondence and reprint requests to Dr. Verena Moos, Charite´, Campus Benjamin Franklin, Medizinische Klinik I, Hindenburgdamm 30, 12203 Berlin, Ger- 3 Abbreviations used in this paper: TB, tuberculosis; SEB, Staphylococcus entero- many. E-mail address: [email protected] toxin B.

Table I. Patient characteristics

Age Samples Investigated Sustained Remission Year of Disease Statusa No. Sex (years) (Sampling Points) History and Treatment Since Diagnosis

I. Active 1 M 52 Blood ϩ DL (4) 2004 disease 2 M 50 Blood (1) 2004 3 F 66 Blood (1) 2004 4 M 63 Blood (1) Study patients of the third arm of the SIMW, 2005 newly diagnosed and untreated at time point of ﬁrst sampling, followed by treatment with ceftriaxone for 2 wk i.v. and TMP-SMZ orally for 3 mo. 5 F 42 Blood (1) 2005 6 M 64 Blood (1) 2005 7 M 59 Blood ϩ DL (2) 2005 8 M 51 Blood (1) 2005 9 M 60 Blood (1) 2005 10 M 47 Blood (1) 2004 11 M 65 Blood (1) 2004 12 M 60 Blood (1) Initial therapy with doxycycline orally for 6 2004 wk, untreated relapse at time point of Downloaded from sampling. 13 M 35 Blood (1) 2 wk ceftriaxone/streptomycin i.v., treated 2004 with TMP-SMZ orally at time point of sampling. 14 M 46 Blood (1) Initial therapy with TMP-SMZ orally for 12 2002 mo, relapse treated with ceftriaxone at

time point of sampling. http://www.jimmunol.org/ 15 M 46 Blood ϩ DL (3) Unknown initial therapy, relapse 2004; 2 wk 2001 ceftriaxone, 3 mo TMP-SMZ orally, 2 days ceftriaxone ϩ doxycycline i.v., 3 wk penicillin i.v., 12 mo TMP-SMZ orally. 16 M 41 Blood (3) 2 wk meropenem i.v., 12 mo TMP-SMZ 2003 orally. II. Remission 17 F 48 Blood ϩ DL (3) 2 wk meropenem i.v., 12 mo TMP-SMZ 2003 treated orally. 18 M 73 Blood ϩ DL (2) 2 wk ceftriaxone i.v., 12 mo TMP-SMZ 2003 orally. 19 M 66 Blood ϩ DL (1) 2 wk ceftriaxone i.v., 12 mo TMP-SMZ 2003 by guest on September 27, 2021 orally. 20 M 50 Blood ϩ DL (1) 1 mo penicillin/streptomycin i.v., 4 years 2000 TMP-SMZ orally. 21 F 55 Blood (1) 33 mo doxycycline, 7 mo TMP-SMZ orally. 2001 III. Sustained 22 M 54 Blood ϩ DL (1) 2 wk meropenem i.v., 12 mo TMP-SMZ 2003 2002 remission orally. 23 M 47 Blood ϩ DL (2) 2 wk meropenem i.v., 12 mo TMP-SMZ 2003 2002 orally. 24 M 84 Blood (1) 2 wk ceftriaxone i.v., 12 mo TMP-SMZ 1984 1983 orally. 25 M 74 Blood ϩ DL (1) Multiple relapses: 1986 TMP-SMZ orally, 1997 1985 1989 ciproﬂoxacin, 1990 doxycycline, 1995 rifampicin ϩ azithromycin, 10/1995 ceftriaxone i.v. followed by chloramphenicol, 11/1995-4/1997 TMP- SMZ orally ϩ IFN-␥. 26 M 63 Blood ϩ DL (2) 12 mo TMP-SMZ orally. 1999 1998 27 F 72 Blood ϩ DL (1) 18 mo doxycycline, 2 years TMP-SMZ 1994 1991 orally. 28 M 64 Blood (1) 1996 1995 29 M 69 Blood (1) 2 wk meropenem i.v., 12 mo TMP-SMZ 2002 2001 orally. 30 F 54 Blood ϩ DL (1) 2003 2002 31 F 43 Blood (1) 2 wk meropenem i.v., 12 mo TMP-SMZ 2004 2003 orally. 32 F 65 Blood ϩ DL (1) 2 wk ceftriaxone i.v., 12 mo TMP-SMZ 2004 2003 orally.

a Classiﬁed at ﬁrst sampling according to Marth et al. (Ref. 13). M, male; F, female; DL, duodenal lymphocytes; TMP-SMZ: trimetoprime-sulfamethoxazole.

subjects during follow-up of gastric ulcer disease (Table II 8 M, 3 F, mean Cell culture age 67.3; range 43–88; no duodenal involvement, Helicobacter pylori- negative at sampling). Permission for the study was obtained from the Cell cultures were grown in RPMI 1640 with Glutamax (Invitrogen Life clinical ethics committee of the Charite´and all subjects gave their written Technologies) supplemented with penicillin/streptomycin (100 U/100 ␮g/ consent to participate. ml; Biochrom), and 2-ME (50 ␮M; Invitrogen Life Technologies) (culture The Journal of Immunology 2017

Table II. Characteristics of control subjects for DL stimulationa

Age No. Sex (years) Samples Investigated History and Treatment Weeks since Ulcer Disease

1 M 69 Blood ϩ DL 12 2 M 71 Blood ϩ DL 14 3 F 72 DL Helicobacter pylorii (HP)-negative 23 gastric ulcer disease without antibiotic treatment. 4 M 60 Blood ϩ DL 8 5 M 44 DL 19 6 M 83 DL 22 7 F 77 Blood ϩ DL 14

8 M 43 Blood ϩ DL Successfully treated HP-positive 16 gastric ulcer (pantoprazole, amoxicillin, clarithromycin) 9 F 48 DL 17 10 M 88 Blood ϩ DL 13 11 M 85 DL 25

a M, male; F, female; DL, duodenal lymphocytes. Downloaded from

medium) at 37°C and 5% CO2. FCS (Sigma-Aldrich) or autologous serum stimulations. Values after stimulation below background level were deﬁned was added as indicated. as 0.

Isolation of PBMC and duodenal lymphocytes T. whipplei-specific stimulations http://www.jimmunol.org/ Blood was collected in sodium-heparinized tubes (Vacutainer; BD Bio- T. whipplei-specific stimulations were established in 10 healthy subjects sciences), and PBMC were separated by Ficoll-Hypaque-gradient with heat-inactivated, sonicated T. whipplei Twist-MarseilleT cultivated in (Pharmacia). confluent MRC5 fibroblasts (ATCC number CCL-171; American Type Duodenal lymphocytes were isolated from six to eight biopsies as de- Culture Collection) (10, 22). The concentration in the preparation was scribed (18). In short, minced biopsies were digested for3honashaker in ϳ109 T. whipplei/ml (determined by quantitative PCR of 16S rDNA (22)) culture medium supplemented with 10% FCS, HEPES (25 mM; Invitrogen and 104 MRC5/ml (determined from a confluent monolayer of MRC5). Life Technologies), amphotericin (250 ␮g/ml; PAA Laboratories), trypsin Lysate was titrated and a dilution of 1/100 (107 bacteria/ml and 102 MRC5/ inhibitor (100 ␮g/ml; Sigma-Aldrich), DNase (100 ␮g/ml; Roche), and ml) induced highest cytokine production in PBMC (data not shown). Ly- collagenase type CLS III (300 ␮g/ml; Biochrom). The suspension was sate of uninfected MRC5 (102 cells/ml; provided by Dr. H. W. Mittru¨cker, filtered through a mesh, followed by a cell strainer (70 ␮m; BD Bio- Max-Planck Institute for Infection Biology, Berlin, Germany) was used as 7 sciences), and washed twice in PBS containing 0.5% BSA (Sigma- negative control and lysate of cell-free grown T. whipplei (10 bacteria/ml) by guest on September 27, 2021 Aldrich). Viability of all cell preparations was Ͼ90%. (23) to exclude host factors of infected MRC5.

CFSE labeling Generation of short-term T cell lines 7 PBMC were washed twice in PBS, suspended at 1 ϫ 10 cells/ml in PBS CD4ϩ T cell lines were generated as described previously (24). Briefly, 106 containing CFSE (0.5 ␮M/ml; Molecular Probes), incubated for 3 min at PBMC/ml in culture medium (5% autologous serum) were stimulated with room temperature, and washed two times with culture medium (10% FCS). T. whipplei lysate (107 bacteria/ml). Subsequently, IL-2 (20 U/ml; Proleu- kin S; Chiron-Behring) was added on days 2, 5, and 9. On day 14, cell lines Stimulation of PBMC, duodenal lymphocytes, and whole blood and autologous CFDA-labeled PBMC (1:4, 106 cells/ml) were restimulated Ag-specific effector T cells were determined through short-term (6–12 h) as described for PBMC for 6 h. stimulation (19). Brefeldin A (10 ␮g/ml; Sigma-Aldrich) was added for the last3htoassess cytokine production. All preparations were washed and Blocking of endotoxins with polymyxin B resuspended after fixation in PBS/0.5%BSA/0.02%NaN3 (PBA), and Polymyxin B was added at 2 ␮g/ml to stimulations of PBMC of healthy stored at 4°C. subjects with LPS (0.1 mg/ml; Sigma-Aldrich) and T. whipplei lysate (107 Duodenal lymphocytes and PBMC were stimulated in culture medium bacteria/ml). (5% FCS) at 106 cells/ml in the presence of anti-CD28 (1 ␮g/ml, clone CD28.2, low endotoxin, no NaN3, BD Biosciences) for 12 h, and fixed with Flow cytometric analysis 4% formalin in PBS. For stimulation of duodenal lymphocytes, autologous CFDA-labeled PBMC were added at a ratio of 1:2. Ag-specific CD4ϩ T cells were analyzed by four-color FACS analysis (19). Fresh heparinized blood (500 ␮l) was stimulated in 15-ml polypro- Cell preparations were washed in PBA, blocked at room temperature 10 pylene tubes (Eppendorf) in the presence of anti-CD28 and anti-CD49d min in 50 ␮l of PBA containing 2% Beriglobin (Behring), then the Abs ␮ ␮ (20) at 2 g/ml (clone 9F10, low endotoxin, no NaN3; BD Biosciences) for were added in 50 l of PBA in dilutions determined before use (data not 6 h. At the end of incubation, 50 ␮l of 20 mM EDTA (pH 7.5) was added, shown) for 15 min at room temperature. Cells were washed and resus- incubated for 10 min at room temperature and mixed vigorously. Nine pended in PBA for analysis. Intracellular staining (IFN-␥, IL-2, IL-10, volumes of FACS-lysing solution (BD Biosciences) were added for 15 min TNF-␣) was performed in the presence of 0.5% saponin (Sigma-Aldrich). at room temperature for lysis of erythrocytes and fixation. Data were acquired on a FACSCalibur (BD Biosciences), and collected and Tuberculin (6 U/ml; Chiron Behring), lysate of CMV (6 ␮g/ml purified analyzed with CellQuest software (BD Biosciences). CMV grown on human fibroblasts; Biodesign), tetanus toxoid (10 ␮g/ml; Gates were set on lymphocytes on a sideward-/forward-scatter dot blot, Aventis), lysates of heat-inactivated Giardia lamblia trophozoites (5 ϫ and on CD4ϩ cells. At least 50,000 CD4ϩ peripheral and 10,000 CD4ϩ 104/ml; Seramun Diagnostica), and actinomycetes closely related to T. duodenal lymphocytes were analyzed. The following Abs were used: anti-CD3 whipplei (21) (Cellulomonas hominis, and Cellulosimicrobium cellulans (UCHT1), anti-CD4 (SK3), anti-CD154 (CD40L, TRAP1), anti-IFN-␥ (synonym: Cellulomonas cellulans), each at 107 bacteria/ml) were used as (B27), anti-TNF-␣ (Mab11), anti-IL-2 (MQ1–17H12), and anti-IL-10 control Ags. (JES3-19F1) from BD Biosciences; anti-CD4 (MT310), anti-CD25 (ACT- Staphylococcus enterotoxin B (SEB) (2 ␮g/ml; Sigma-Aldrich) served 1), anti-CD69 (FN50) from DakoCytomation; and anti-CD69 (TP1.55.3), as positive control. Negative controls contained no supplements (for SEB), and anti-HLA-DR (Immu357) from Caltag Laboratories. Mouse IgG1, and anti-CD28, or anti-CD28 and anti-CD49d (for Ag-specific stimula- mouse IgG2b, and rat IgG2 (all BD Biosciences) served as isotype tions), respectively, and were subtracted from the values obtained after controls. 2018 Ag-SPECIFIC REACTIONS IN WHIPPLE’S DISEASE

Statistical analysis stimulated with proteinase K-digested T. whipplei lysate. The stim- Data were analyzed by means of the Kruskal-Wallis test. Mann-Whitney’s ulation with digested lysate caused loss of Th1 responses (Fig. 1c). comparison test was used for post hoc analysis. Values of p Ͻ0.05 were ϩ considered significant. T. whipplei-specific CD4 T cell reactivity in Whipple’s disease patients compared with healthy and disease control subjects Results Peripheral blood. T. whipplei-specific T cell responses in whole Establishing a T. whipplei-specific stimulation blood of 32 mainly middle aged patients with different stages of Optimal Ag dose. Lysate from T. whipplei-infected MRC5 fibro- Whipple’s disease (Table I) were compared with those of three blasts, uninfected MRC5, and SEB were used to stimulate PBMC groups of healthy subjects (young subjects, age-matched subjects, in fresh whole blood of healthy subjects. The stimulation revealed and active triathletes), and to those of patients with TB. Patients a dose-dependent increase in the percentage of IFN-␥ϩcells of with Whipple’s disease revealed a significantly reduced percentage ϩ CD4ϩ T cells, and virtually all IFN-␥ϩ cells expressed CD69. A of T. whipplei-specific IFN-␥-producing CD4 T cells compared maximal cytokine response was achieved with 107 bacteria/ml, with the four control groups (Fig. 2a). The frequency of T. whip- ϩ ϩ ϩ subsequently used for additional experiments (Fig. 1a). plei-specific CD69 IFN-␥ cells of CD4 lymphocytes was high- Short-term T cell line. T. whipplei-specific, short-term T cell est in the group of triathletes, followed by the age-matched sub- lines were established from three healthy subjects to confirm Ag jects and the young subjects. TB patients revealed the lowest T. specificity of the reaction. The frequency of T. whipplei-specific whipplei-specific reactivity of the control groups. The reactivity of CD69ϩIFN-␥ϩ cells of CD4ϩ lymphocytes increased after TB patients was still significantly higher than in Whipple’s disease Downloaded from re-stimulation with 107 bacteria/ml at day 14 (Fig. 1b). The patients but significantly lower than in triathletes and age-matched reactivity to MRC5 (data not shown) and SEB remained un- controls (Fig. 2a). ϩ ␥ϩ ϩ changed (Fig. 1b). The individual frequencies of CD69 IFN- cells of CD4 T cells did not differ using different lysates of cell-free grown T. Specificity of T. whipplei-specific stimulation. Polymyxin B was whipplei (107 bacteria/ml) to stimulate PBMC of 10 patients with added to stimulations of healthy subjects to preclude that remnants Whipple’s disease and 10 healthy subjects (data not shown). of endotoxins in the T. whipplei lysate induce the reactivity. Poly- http://www.jimmunol.org/ myxin B reduced the frequency of CD69ϩIFN-␥ϩ cells of CD4ϩ lymphocytes reacting against LPS to 64 Ϯ 8.3% whereas the T. whipplei-specific Th1 reactivity was not affected (Fig. 1c). To show that proteins or peptides contained in the lysate induce the observed Th1 reactivity, PBMC of 10 healthy subjects were by guest on September 27, 2021

FIGURE 1. Establishing a T. whipplei-specific stimulation in healthy FIGURE 2. T. whipplei-specific stimulation (107 bacteria/ml) of whole subjects. a and b, One representative example of 10 (a) respective 3 (b) blood and duodenal lymphocytes. Individual percentages of experiments. a, Stimulation of whole blood with SEB, lysate of MRC5, and CD69ϩIFN-␥ϩ cells of CD4ϩ lymphocytes and median are shown. a, lysate of T. whipplei-infected MRC5 (106 or 107 bacteria/ml). b, Stimula- Whole blood. b, Duodenal lymphocytes. c, T. whipplei-specific reactivity tion of a T. whipplei-specific short-term T cell line with SEB and T. whip- in different stages of Whipple’s disease (WD). d, Time course of T. whip- 7 plei lysate (10 bacteria/ml). c, Stimulation of PBMC in the presence of plei-specific reactivity of single patients. Cyoung, young healthy subjects; ϭ polymyxin B (PB)(n 5), and with proteinase K-digested (Pkdig) T. whip- Tri, healthy triathletes; Cage-matched, age-matched healthy subjects; WD, plei lysate (n ϭ 10). The percentage of CD69ϩIFN-␥ϩ cells of CD4ϩ WD patients; TB, TB patients; (I3II), status changed from active disease 3 lymphocytes stimulated without PB and with undigested T. whipplei lysate (I) to remission under treatment (II); (II III), status changed from remis- was set as 100%. Median and SD are shown. sion under treatment (II) to sustained remission (III). The Journal of Immunology 2019

Duodenal mucosa. The percentage of T. whipplei-specific CD4ϩ stimulus and stimulation with apathogenic actinomycetes was per- duodenal lymphocytes expressing IFN-␥ was significantly lower in formed to exclude cross-reactivity being responsible for the ob- patients with Whipple’s disease compared with age-matched sub- served reactivity to T. whipplei. The reactivity (percent of CD154 jects (Table II) (Fig. 2b). (CD40L)ϩIFN-␥ϩcells of CD4ϩlymphocytes) and the proportion Subgroup analysis of patients with Whipple’s disease. Because of individuals reacting to G. lamblia trophozoites, and the actino- the proliferative capacity of T cells seems to recover after treat- mycetes C. hominis and C. cellulans closest related to T. whipplei ment, we considered the development of T. whipplei-specific re- were similar in patients and healthy subjects (Fig. 3b). activity in the course of Whipple’s disease. The percentages of T. CD4؉ T cell reactivity to SEB. The percentage of peripheral whipplei-specific Th1 cells did not differ between patients with CD69ϩIFN-␥ϩ cells of CD4ϩ T cells of patients with Whipple’s active Whipple’s disease, remission under treatment, and sustained disease reacting to the superantigen SEB was similar compared remission (Fig. 2c). In addition, the individual reactivity of patients with the four control groups (Fig. 4a). In addition to intracellular tested at two to three different time points representing distinct expression of IFN-␥, the percentage of CD4ϩ T cells expressing disease activities had no major variations (Fig. 2d). IL-2, IL-10, and TNF-␣ was determined in 14 Whipple’s disease

ϩ patients and 9 age-matched subjects after SEB-stimulation. No sig- CD4 T cell reactivity to other bacterial and viral Ags niﬁcant differences in the percentage of CD4ϩ T cells expressing :CD4؉ T cell reactivity to tetanus toxoid, tuberculin, actinomy- IL-2 (Whipple’s disease: 5,03 Ϯ 3,94, age-matched subjects ϩ ϩ cetes, G. lamblia, and CMV. Common Ags were used to inves- 4.09 Ϯ 3.27% IL-2 cells of CD4 T cells), and TNF-␣ (Whip- tigate the general Ag-speciﬁc reactivity of Whipple’s disease pa- ple’s disease: 8.19 Ϯ 4.94, age-matched subjects: 8.29 Ϯ 5.83%

ϩ ϩ Downloaded from tients. A similar proportion of patients, age-matched subjects, and TNF-␣ cells of CD4 T cells) were detected. IL-10 was detected ϩ TB patients showed Th1 reactivity to CMV and tetanus toxoid only in a low percentage of CD4 cells compared with IL-2 and (Fig. 3a). Additionally, the frequencies of IFN-␥ϩ CMV- and tet- TNF-␣. However, IL-10-expression was similar in Whipple’s dis- anus toxoid-specific CD4ϩ T cells in these three groups were sim- ease patients and control subjects (Whipple’s disease: 0.29 Ϯ 0.22, ϩ ϩ ilar (Fig. 3a). age-matched subjects: 0.42 Ϯ 0.27% IL-10 cells of CD4 T Obviously, all of the TB patients revealed reactivity to tubercu- cells). lin whereas only 36% of patients with Whipple’s disease, and 44% SEB stimulation of duodenal lymphocytes resulted in a signif- http://www.jimmunol.org/ ϩ of age-matched subjects showed a reaction. As expected, TB pa- icantly higher percentage of IFN-␥-expressing CD69 cells of ϩ tients revealed a significantly higher frequency of tuberculin-spe- CD4 lymphocytes in patients with Whipple’s disease than in age- cific CD69ϩIFN-␥ϩ cells of CD4ϩ T cells compared with Whip- matched subjects (Fig. 4b). ple’s disease patients as well as age-matched subjects (Fig. 3).

Because giardiasis might be associated with Whipple’s disease ϩ (25), lysates of G. lamblia trophozoites were used as additional Activation status of CD4 PBMC after T. whipplei-speciﬁc and SEB stimulation The expression of the activation markers CD69 and CD154 was by guest on September 27, 2021 investigated to exclude that repressed IFN-␥ expression causes the reduced reactivity to T. whipplei. The percentage of peripheral CD4ϩ T cells of patients with Whipple’s disease expressing CD69 and CD154 was reduced compared with all control groups after stimulation with T. whipplei lysates (Fig. 5, a and b). As for IFN-␥ expression, the percentage of CD69ϩ cells of CD4ϩ lymphocytes of TB patients was signiﬁcantly reduced compared with the two groups of healthy subjects (Fig. 5a). In contrast, stimulation with SEB resulted in similar percentages of CD4ϩ T cells expressing CD69 and CD154 in Whipple’s disease patients and control subjects (Fig. 5, c and d).

FIGURE 3. Stimulation of whole blood. a, CMV-lysate, tuberculin, and tetanus toxoid. b, C. hominis, C. cellulans (107 bacteria/ml), and G. lamblia FIGURE 4. SEB stimulation of whole blood and duodenal lympho- trophozoites (5 ϫ 104/ml). Median and SD of persons with an Ag-speciﬁc cytes. Individual percentages of CD69ϩIFN-␥ϩ cells of CD4ϩ lympho- reactivity of Ͼ0.03% CD69ϩIFN-␥ϩ cells respective 0.02% cytes and median are shown. a, Whole blood. b, Duodenal lymphocytes. ϩ ␥ϩ ϩ CD154 (CD40L)IFN- cells of CD4 lymphocytes are shown. Cyoung, young subjects; Tri, triathletes; Cage-matched, age-matched subjects; [%]reacting, Percent of persons with reactivity. WD, WD patients; TB, TB patients. 2020 Ag-SPECIFIC REACTIONS IN WHIPPLE’S DISEASE

patients neither CD69ϩ nor CD25ϩ of CD4ϩ T cells could be stimulated to produce additional IFN-␥.

Activation status of freshly isolated PBMC and duodenal lymphocytes Patients with Whipple’s disease showed a signiﬁcantly increased percentage of activated (CD4ϩCD25ϩ, CD3ϩHLA-DRϩ) duodenal and peripheral lymphocytes compared with healthy age- matched subjects (Fig. 7).

Discussion In the present study, we found that the Th1 response of peripheral and mucosal effector CD4ϩ T cells of healthy individuals was consistent and dose dependent when stimulated with a lysate of T. whipplei whereas this T. whipplei-specific Th1 response was significantly reduced in patients with Whipple’s disease. The response in uninfected individuals increased after restimulation and was characterized by the expression of CD154, indicating the specificity of the reaction (26). Proteinase K digestion of the T. whip- Downloaded from plei lysate abolished stimulative capacity, pointing at proteins as stimulus. Cross-reactivity with apathogenic actinomycetes was not observed as the frequency of T. whipplei-specific Th1 cells (percent of IFN-␥ϩ cells of CD4ϩ T cells) of control subjects was much higher and the reactivity to C. cellulans and C. hominis was

similar in control subjects and in Whipple’s disease patients. In http://www.jimmunol.org/ addition, neither endotoxin nor possible host factors from infected MRC5 were responsible for the T. whipplei-specific reactivity. In healthy subjects, T. whipplei-specific peripheral Th1 cells ϩ FIGURE 5. Activation of CD4 lymphocytes after T. whipplei-specific were found in a frequency similarly as described for other Ags and SEB stimulation of whole blood. Individual percentages and median (27). This consistent T. whipplei-specific cellular immunity in are shown. a and b, Stimulation with T. whipplei and the percentage of (a) CD69ϩ and (b) CD154ϩ cells of CD4ϩ lymphocytes, respectively. c and d, SEB stimulation and the percentage of (c) CD69ϩ and (d) CD154ϩ cells of ϩ CD4 lymphocytes. Cyoung, young subjects; Cage-matched, age-matched sub- by guest on September 27, 2021 jects; WD, WD patients; TB, TB patients.

Influence of IL-2 addition to T. whipplei-specific stimulation To overcome a possible anergy of T cells, whole blood from healthy subjects and Whipple’s disease patients was stimulated with T. whipplei lysate, either alone or in the presence of a high dose of exogenous IL-2 (50 U/ml). The addition of IL-2 did not significantly enhance the percentage of CD69ϩ of CD4ϩ T cells expressing IFN␥ (Fig. 6). In healthy subjects, only CD25ϩ of CD4ϩ T cells could be significantly stimulated by IL-2, while in

FIGURE 6. Addition of exogenous IL-2 to T. whipplei-specific stimu- FIGURE 7. Expression of CD25 and MHC II (HLADR) on freshly iso- lations. Whole blood of healthy subjects (n ϭ 4) and WD patients (n ϭ 9) lated duodenal and peripheral lymphocytes. Individual percentages and was stimulated with T. whipplei with or without IL-2 (50 U/ml). Median median are shown. a, The percent of CD25ϩ cells of CD4ϩCD3ϩ lym- and SD of the percentage of IFN-␥ϩ cells of CD4ϩ lymphocytes are phocytes. b, The percentage of HLADRϩ cells of CD3ϩ lymphocytes. shown. Cage-matched, age-matched subjects; WD, WD patients. The Journal of Immunology 2021 healthy individuals is probably induced by regular contact of the We found enhanced cell activation of peripheral and duode- enteric immune system with T. whipplei. Indeed, the presence of T. nal T cells of Whipple’s disease patients independent of disease whipplei-specific IgG Abs in the serum and T. whipplei DNA in status as described (13, 31). Ongoing infection might be sim- the gastrointestinal tract of some healthy individuals (7, 9, 10) ulated by remnants of T. whipplei that are persistent in affected support this hypothesis. Additionally, according to our results, ac- tissues (3) even after successful treatment. Correspondingly, tive triathletes, exposed to open watercourses, revealed the highest suppression of T cell reactivity often occurring during persis- ϩ percentages of T. whipplei-specific CD4 T cells followed by the tent infection (32) might be responsible for the impaired reac- age matched, and finally the young subjects. TB patients showed tivity of Whipple’s disease patients to mitogens (11–15). reduced T. whipplei-specific responses compared with healthy sub- Hence, some of the proposed mechanisms for T. whipplei-spe- jects. Therefore, attributes that predispose for mycobacterial infec- cific unresponsiveness can be excluded. Cytokines like IL-10 tion or the infection per se seem to impair reactivity to T. (33) and TGF-␤ (34) have been shown to act suppressively. whipplei too. However, in this study, Whipple’s disease patients did not show In addition, the mucosal T cell response to T. whipplei was enhanced percentage of IL-10-producing CD4ϩ PBMC after studied. In accordance with the results from the peripheral blood, stimulation with SEB and serum levels of TGF-␤ have been T. whipplei-specific Th1 cells in duodenal lymphocytes of healthy shown to be similar compared with healthy subjects (16). Con- subjects (cured gastric ulcer patients) were more frequent than in sequently, neither TGF-␤ nor IL-10 seem to be suppressive fac- patients with Whipple’s disease. The activation status of the tors in Whipple’s disease. Exogenous IL-2 did not result in an freshly isolated duodenal lymphocytes of these control subjects increase in T. whipplei-specific Th1 reactivity of Whipple’s dis- and the reactivity to SEB was significantly lower than in Whip- Downloaded from ple’s disease patients. Though, we cannot exclude definitively that ease patients. In addition, IL-2 induced enhanced expression of ␥ former gastric ulcer disease influences Ag-specific reactivity to T. IFN- only in preactivated T cells expressing CD25 of healthy whipplei in the duodenum. But because we see no signs of general subjects (35). Thus, T cell anergy or activity of regulatory T enhanced activation in the duodenal lymphocytes of those control cells does not seem to explain the specific unresponsiveness we subjects, we thus assume that the T. whipplei-specific reactivity is observed in Whipple’s disease.

not enhanced by previous gastric ulceration. The reduced response Macrophages seem to be of central importance in the devel- http://www.jimmunol.org/ of patients with Whipple’s disease to T. whipplei was consistent, opment of the disease (36). Recently, it has been shown that and independent of disease activity and treatment. After T. whip- intestinal macrophages of Whipple’s disease patients display in plei-specific stimulation, the frequency of CD4ϩ cells expressing vivo the phenotype of M2/alternatively activated macrophages CD69, an early marker of activation (19), and CD154, a marker for (17) that favor the development of Th2 responses and inhibit the assessment of Ag-specific T cell responses (26), was reduced protective Th1 polarization (37). This fact explains the dereg- in Whipple’s disease patients compared with control subjects. ulated Th1/Th2 response in Whipple’s disease we have previ- Thus, insufficient T cell activation, and not only repressed IFN-␥ ously described (14). In addition, untreated Whipple’s disease expression, accounts for reduced Th1 reactivity. patients reveal elevated serum levels of IL-16 which favors T. Impaired Th1 reactivity of PBMC (secretion of IFN-␥) and pro- whipplei replication in vitro (38). One hypothesis about the role by guest on September 27, 2021 liferation to various mitogens in vitro has been described in Whip- of IL-16 in vivo is that it contributes to a general recruitment of ple’s disease (11–15). Reduced serum IgG2 (15), the association of CD4ϩ T cells in an inflammatory process resulting in cells re- T. whipplei-specific IgM with Whipple’s disease (10), and eradi- sponsive to cytokine stimulation, but refractory to Ag-specific cation of the causative organism through adjunctive IFN-␥ therapy stimulation (39) because it interferes with cell-mediated im- in one patient (28) indicate the pathogenetic relevance of poor Th1 mune response (40) and induces tolerogenic dendritic cells (41). responses in vivo. However, SEB that was used in this study as However, IL-16 alone was not sufficient to account for T. whip- unspecific stimulus seems to have similar efficiency in PBMC of plei replication (38). Indeed, recent own studies refer to dimin- Whipple’s disease patients and healthy subjects, just as described ished monocyte functions and displacements of circulating den- before for anti-CD2 respective anti-CD3 and anti-CD28 (15). dritic cell phenotypes in Whipple’s disease (V. Moos, D. Moreover, stimulation with SEB resulted in higher reactivity in Kunkel, R. Ignatuis, M. Zeitz, and T. Schneider, manuscript in CD4ϩ duodenal lymphocytes of Whipple’s disease patients than of preparation). Thus, a general Th1-suppressive and tolerogenic healthy subjects. In addition, Ag-specific responses to tetanus tox- intestinal milieu may lead to reduced degradation of invading T. oid, CMV, tuberculin, G. lamblia, and actinomycetes were com- whipplei, and disturbed processing and presentation of T. whip- parable in PBMC of the study groups. Thus, the Th1 unrespon- plei Ags. Hereditary genetic predisposition (42) and specific siveness of Whipple’s disease patients seems to be limited to only host factors (43) have been shown to influence the spread of selected stimuli. Consequently, we hypothesize that a T. whipplei- intracellular bacteria and the deletion of a single IFN-␥-induc- specific immune defect contributes to the very rare susceptibility to the ubiquitously present bacteria. However, we cannot exclude that ible gene induced exclusive susceptibility to Toxoplasma gondii the actinomycete itself plays a role in the induction of this defect. in mice (44). In the case of infection with T. whipplei, the This hypothesis is supported by two major aspects: 1) CD4 im- invading pathogen might induce specific susceptibility in the munodeficiencies (for example AIDS) do not seem to correlate Th1 system of predisposed human hosts that facilitates bacterial with a more frequent incidence of Whipple’s disease, and 2) pa- invasion and impairs immunological clearance. Although we tients with Whipple’s disease do not suffer more often from op- cannot predict the detailed mechanism yet, we established a portunistic infections known to be associated with impaired CD4ϩ basis for further investigations with this study. T cell activity like toxoplasmosis, Pneumocystis carinii, or non- In summary, we have detected a reduced T. whipplei-specific tuberculosis mycobacterial infections (29, 30). Only giardiasis was Th1 response of Whipple’s disease patients while peripheral and found recently to be associated with Whipple’s disease (25). How- mucosal T cells of healthy subjects and TB patients reveal a sig- ever, the similar G. lamblia-specific Th1 reactivity in healthy sub- nificant reactivity. A proposed T. whipplei-specific defect might jects and Whipple’s disease patients casts a common predisposing explain why the ubiquitous bacillus causes symptomatic infection immune defect into doubt. only in certain hosts. 2022 Ag-SPECIFIC REACTIONS IN WHIPPLE’S DISEASE

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