ORIGINAL ARTICLE
B Cells Exposed to Enterobacterial Components Suppress Development of Experimental Colitis Esben Gjerl ff Wedebye Schmidt, MSc,* Hjalte List Larsen, BSc,* Nanna Ny Kristensen, MSc, PhD,* Steen Seier Poulsen, MD, PhD,† Mogens Helweg Claesson, MDSc,* and Anders Elm Pedersen, MD, PhD*
Background: B cells positively contribute to immunity by antigen presentation to CD4þ T cells, cytokine production, and differentiation into antibody secreting plasma cells. Accumulating evidence implies that B cells also possess immunoregulatory functions closely linked to their capability of IL-10 secretion.
Methods: Colitis development was followed in CD4þCD25 T cell transplanted SCID mice co-transferred with B cells exposed to an enterobacterial extract (ebx-B cells). B and T cell cytokine expression was measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA).
Results: We demonstrate that splenic B cells exposed to ebx produce large amounts of IL-10 in vitro and express CD1d and CD5 previously
known to be associated with regulatory B cells. In SCID mice transplanted with colitogenic CD4þCD25 T cells, co-transfer of ebx-B cells significantly suppressed development of colitis. Suppression was dependent on B cell-derived IL-10, as co-transfer of IL-10 knockout ebx-B cells failed to suppress colitis. Ebx-B cell-mediated suppression of colitis was associated with a decrease in interferon gamma (IFN-c)-producing TH1 cells and increased frequencies of Foxp3-expressing T cells.
Conclusions: These data demonstrate that splenic B cells exposed to enterobacterial components acquire immunosuppressive functions by which they can suppress development of experimental T cell-mediated colitis in an IL-10-dependent way. (Inflamm Bowel Dis 2012;18:284–293) Key Words: Regulatory B cells, colitis, IL-10, enterobacteria
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he existence of regulatory B cells (Bregs) was proposed more than 30 years ago1,2 and their immunosuppressive potential has now been proven in experimental animal models.3,4 In humans, the data on Bregs are limited and almost exclusively based on observational studies, showing exacerbation of disease following B cell-depleting therapy in patients suffering from various autoimmune disorders.5–8 B cell-mediated suppression of intestinal inflammation has been demonstrated in different murine models of colitis. B cells play a protective role in the TCRa/ model Received for publication April 6, 2011; Accepted April 12, 2011. From the *Department of International Health, Immunology and Microbiology, Panum Institute, University of Copenhagen, Denmark, † Department of Biomedicine, Panum Institute, University of Copenhagen, Denmark. Supported by the Danish Agency for Science, Technology and Innovation; Colitis Crohn’s Foundation; Aage and Johanne Louis-Hansen Foundation; Aase and Ejnar Danielsen Foundation; Augustinus Foundation; Lundbeck Foundation. Reprints: Esben Gjerl ff Wedebye Schmidt, MSc, or Anders Elm Pedersen, MD, PhD, Department of International Health, Immunology and Microbiology, Panum Institute, Building 18.3.52, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark (e-mail:
[email protected] or
[email protected]). C 2011 Crohn’s & Colitis Foundation of America, Inc. Copyright V DOI 10.1002/ibd.21769 Published online 25 May 2011 in Wiley Online Library (wileyonlinelibrary. com).
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of colitis,9 which requires B cell expression of IL-10 and CD1d.10 B cell suppression of Gai2/ and CD4þCD45RBhigh T cell transfer colitis is independent of CD1d but requires B cell intrinsic IL-10 and regulatory CD8þ T cells.11,12 IL-10 was also found to be a prerequisite for B cell suppression of dextran sulfate sodium (DSS) colitis but not for suppression of spontaneous colitis in nuclear factor of activated T cells c2 (NFATc2) deficient RAG/ mice.13 Bregs have also demonstrated IL-10dependent suppression of experimental autoimmune encephalomyelitis (EAE),14–17 and collagen-induced arthritis (CIA)18 and transfer of lipopolysaccharide (LPS)-activated19 or BCR-stimulated20 B cells was shown to mediate suppression of diabetes in NOD mice. The ability to secrete IL-10 seems to be a general trait for the various described subsets of Bregs.21 Other or subsequent mechanisms like secretion of TGF-b,22 FasLmediated induction of apoptosis,19 suppression of dendritic cell function,23 and regulatory T cells (Tregs) activation16 have been indicated as well. Today, a true marker for Bregs, as Foxp3 for Tregs, is still needed but expression of CD5 and high expression of CD1d have been associated with IL-10-producing B cells and are phenotypic characteristics of Breg subsets.21 Recently, IL-10-competent splenic CD1dhighCD5þCD19high B cells, named B10,24 have shown Inflamm Bowel Dis Volume 18, Number 2, February 2012
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suppressive effects in EAE,25 contact hypersensitivity (CHS),24 murine lupus,26 and colitis.27 In addition, marginal zone (MZ) B cells and transitional-2 marginal zone precursor (T2-MZP) B cells both express CD1d, secrete IL-10 upon activation,28,29 and suppress development of CHS30 and CIA.31 Of importance, stimulation through Toll-like receptors (TLRs) triggers IL-10 production in B cells32 and may, together with BCR ligation, very well be necessary for optimal Breg activation and function. This feature, in particular, could have an important impact on colitis development, as intestinal barrier leakage might allow inflow of different TLR ligands and recognition of enteroantigenic components.33,34 We have previously shown that B cells pulsed with an enterobacterial extract (ebx-B cells) are extremely efficient antigen-presenting cells for enteroantigen-specific CD4þ T cells in vitro.35 We also demonstrated activation of Tregs in vitro by ebx-B cells but not by ebxpulsed DCs.35 Since ebx represents the enteroantigenic repertoire and may contain multiple TLR ligands, we were encouraged to investigate the potential regulatory effect of ebx-B cells in vivo during the development of T cell transfer colitis. Identification of a linkage between enterobacteria and activation of immunosuppressive Bregs could beneficially be targeted in IBD treatment.
MATERIALS AND METHODS Mice Six to eight-week-old female BALB/cA, C.B-17 SCID mice (Taconic, Bomholt, Denmark) and IL-10 knockout mice on a BALB/c background (Jackson Laboratory, Bar Harbor, ME) were housed under controlled microbial environment conditions in the animal facility of the Panum Institute, University of Copenhagen. All animal experiments were carried out in accordance with authorization from the Committee for Animal Surveillance, the Danish Ministry of Justice, Contract no. 2008/561-146.
Preparation of Ebx The ebx was prepared from BALB/cA mice. The fecal content of colon and cecum was dissolved in ice-cold phosphate-buffered saline (PBS) followed by sonication, centrifugation, and sterile filtration. Protein concentration was determined by BCA assay, as described previously.36 The extract was kept at 80C until use.
Purification of Cells
CD4þ T cells were purified from BALB/cA mesenteric lymph nodes (MLN) using CD4 Dynabeads and CD4 DETACHaBEAD (Dynal, Oslo, Norway) according to the manufacturer’s instructions. Next, CD4þCD25þ regulatory T cells were labeled with PE-conjugated anti-CD25 antibody and anti-PE antibody-coated microbeads (Both Miltenyi Bio-
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tec, Bergisch Gladbach, Germany) and depleted by magnetic separation on an autoMACS Separator. Purity of transferred CD4þCD25 T cells was >98%. Splenic B cells were labeled with anti-CD19 microbeads (Miltenyi Biotec) and positively isolated on an autoMACS Separator, yielding a purity >98%.
Colitis Model Colitis was induced in SCID mice by transfer (intraperitoneally [i.p.]) of 0.3 106 purified CD4þCD25 T cells. Every other day mice were weighed and clinical signs of colitis development were followed (weight loss, diarrhea, and rectal prolapse). After euthanization, rectum (distal 2 cm colon) was excised, weighed, and used for histopathological scoring (see below). Purified B cells were incubated with ebx (400 lg/mL) for 24 hours, washed, and injected i.p. simultaneously with T cells.
Histopathology The distal 2 cm of the colon were fixed in 4% paraformaldehyde and embedded in paraffin. The samples were cut longitudinally and stained with periodic acid Schiff and hematoxylin and eosin. Histopathological examination of sections was done in a blinded manner, scoring inflammation from 0 to 5: 0, no signs of inflammation; 1, slight infiltrations in lamina propria by mononuclear cells and some crypt elongation; 2, moderate mononuclear cell infiltration of lamina propria, increased mitotic activity in the epithelial layer and moderate crypt elongation; 3, as in 2 but patchy mononuclear cell infiltration in the submucosal layer, extensive elongation of the crypts with some depletion of goblet cells; 4, as in 3, but extensive crypt destruction, microabscesses, and diffuse mononuclear infiltration of the submucosal layer; 5, as in 4, but including ulcerative lesions of the epithelial layer.
Flow Cytometry Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ) and Mouse Regulatory Staining Kit (eBioscience, San Diego, CA) according to the manufacturers’ description. In brief, after stimulation for 3–4 hours with phorbol myristate acetate (PMA) (50 ng/mL) and ionomycin (750 ng/mL) in the presence of GolgiStop cells were labeled with specific antibodies against cell-surface markers. Then the cells were fixed and permeabilized and stained for intracellular markers. Labeled cells were analyzed on a BD LSR II or FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using FACSDiva or CellQuest software (BD Biosciences). Antibodies used: CD4-APC, IL-17A-PE, IL-4-PE, IFNc-PE-Cy7, CD19-APC, CD1d-FITC, CD5-PerCP, CD80-PE, CD86-PE, and IL-10-PE (BD Biosciences), Foxp3-FITC (eBioscience). Recommended isotype controls were included in all experiments.
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FIGURE 1. B cells exposed to ebx produce IL-10. (A) Splenocytes were stimulated for 5 hours with PMA/ionomycin (PMA/I) alone or in combination with ebx or LPS and IL-10 production was measured in CD19þ B cells by flow cytometry. Frequencies indicate IL-10þ of B cells. One representative of three experiments is shown. (B) IL-10 was measured by ELISA in supernatants from purified B cells stimulated with ebx for 24, 48, and 72 hours. One of two experiments is shown. (C) CD1d and CD5 expression was measured in IL-10-producing (IL-10þ) and IL-10-nonproducing (IL-10) CD19þ B cells. Frequencies indicate CD1dhighCD5þ of B cells. Contour plots show double staining and histograms show expression of each marker. One of three experiments is shown. (D) CD1d and CD5 expression was measured in splenocytes freshly isolated or stimulated for 24 hours with ebx or LPS. One of three experiments is shown. (E) Expression of CD80 and CD86 were measured by flow cytometry on unstimulated B cell subsets, characterized by CD1d and CD5 expression levels. Average of three individual experiments is shown. Error bars ¼ SD.
ELISA Supernatants were collected from cultures of purified B cells stimulated with ebx for 24, 48, and 72 hours. Concentration of IL-10 was detected by sandwich ELISA, using capture antiIL-10 (BD Biosciences, Cat. no. 554422), biotinylated detection anti-IL-10 (BD Biosciences, Cat. no. 554423) and recombinant mouse IL-10 (PeproTech, Rocky Hill, NJ) for standard.
Statistics Two-tailed Student’s t-test was used to compare the means of two data points. Statistic calculations were done with GraphPad Prism software (San Diego, CA).
RESULTS B Cells Exposed to Ebx Produce IL-10 Previously, we demonstrated that enteroantigen-specific CD4þ T cells are efficiently stimulated in vitro by
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splenic B cells pulsed with a colonic ebx,35 giving rise to T cell responses restricted by MHC class II histocompatibility antigens.37 B cells are known to produce IL-10 when exposed to bacterial components like LPS.32 Therefore, we measured the production of IL-10 in splenic B cells exposed to ebx, LPS, or saline for 5 hours in the presence of phorbol myristate acetate (PMA) and ionomycin (PMA/I) (Fig. 1A). Stimulation with ebx was the strongest inducer of IL-10, resulting in 6.7% IL-10-positive B cells, compared to 4.7% induced by LPS. In addition, we measured IL-10 amounts in supernatants from purified B cells stimulated for 24, 48, and 72 hours with different concentrations of ebx (0, 40, and 400 lg/mL) (Fig. 1B). This clearly showed a timeand dose-dependent induction of IL-10 in B cells by ebx. An IL-10 competent splenic B cell subset has previously been identified and phenotypically characterized as CD1dhighCD5þ.24 The phenotype of ebx induced IL-10producing B cells is shown in Figure 1C. Close to 20% of
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the IL-10-positive B cells were CD1dhighCD5þ compared to 0.3% of the IL-10-negative B cells. In addition, all IL10-producing B cells showed a generally higher expression of both CD1d and CD5 (Fig. 1C). Upregulation of CD1d by B cells during intestinal inflammation has been associated with suppression of colitis.10 Consequently, we examined the expression of CD1d and CD5 on B cells after prolonged exposure to ebx (Fig. 1D). After 24 hours of exposure to ebx more than 10% B cells were CD1dhighCD5þ, which is a 5 times increase compared to unexposed B cells. It is noteworthy that prolonged exposure to LPS did not result in a similar upregulation of CD1d and CD5. Expression levels of B7 costimulatory molecules have also been associated with IL-10-producing Bregs.16 To examine if B7expression patterns correlated with Breg phenotypes we measured the expression of CD80 and CD86 on CD1d and CD5-positive B cells (Fig. 1E). Interestingly, the highest frequency of CD80-expressing B cells was within the CD1dhighCD5þ population (30%), whereas less than 5% of the CD1dlowCD5 B cells were CD80-positive. CD86 was mainly expressed on CD1dhigh B cells, although more evenly distributed between the B cell subpopulations.
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idea of the quantitative B cell requirement for suppression, mice received either 107 or 106 ebx-B cells. Mice transplanted with T cells alone developed colitis, as expected; lost body weight, increased rectum weight, and showed intestinal inflammation (Fig. 3A–C). Transfer of 107 wt ebxB cells completely abrogated weight loss and significantly reduced rectum weight and intestinal inflammation, as also shown in Figure 2. Recipients of 107 IL-10 KO ebx-B cells initially increased in body weight but then started to lose weight 4 weeks after cell transfer. Their rectum weights were comparable to that of recipients of T cells alone and their histological colitis score, although decreased, did not significantly differ from that of the colitic control mice. Mice co-transplanted with only 106 ebx-B cells, wt, or IL-10 KO showed an intermediate body weight curve between that of healthy and colitic controls (Fig. 3A). The rectum weight was significantly reduced in mice co-transplanted with 106 wt ebx-B cells but not in recipients of 106 IL-10 KO ebx-B cells (Fig. 3B). Although not significant, recipients of 106 wt ebx-B cells showed a clear tendency of colitis suppression (Fig. 4C). These results indicate that ebx-B cell-mediated suppression of colitis requires IL-10 secreting B cells.
B Cells Exposed to Ebx Suppress Colitis
Adoptive transfer of CD4þCD25 T cells to SCID mice causes the development of colitis characterized by weight loss, loose stools, diarrhea, and colon inflammation. Mice were co-transplanted with 107 purified splenic B cells unexposed or exposed to ebx (ebx-B cells) for 24 hours (Fig. 2). Mice receiving T cells alone significantly lost body weight compared to non-transplanted healthy control mice and diseased mice showed severe diarrhea (Fig. 2A and not shown). Interestingly, transfer of ebx-B cells completely inhibited the T cell induced loss of body weight and these mice did not have diarrhea. Mice co-transferred with B cells not exposed to ebx initially lost weight comparable to the colitic mice but then started to gain weight after 3 weeks. Intestinal inflammation was determined by histopathological examination and weight of rectum, both of which were significantly reduced in mice co-transplanted with ebx-B cells (Fig. 2B–D) as compared with mice transplanted with T cells alone. Co-transfer of B cells not exposed to ebx did not reduce intestinal inflammation as evaluated by the rectum weight and the histopathological scoring.
B Cell-mediated Suppression of Colitis Is IL-10-dependent Since the immunosuppressive capacity of B cells has been closely linked to their IL-10-producing potential,21 CD4þCD25 T cell transplanted SCID mice were co-transplanted with ebx-B cells purified from wildtype (wt) or IL10 knockout (IL-10 KO) mice. In addition, to obtain an
Ebx-B Cell-mediated Suppression of Colitis Is Not Caused by Homeostatic Regulation By use of flow cytometry, we examined if protection from colitis by ebx-B cell co-transfer results from competition for space by ebx-B cell proliferation or was associated with their anatomical distribution. B cells (CD19þ) were not present in the colonic lamina propria of transplanted mice (not shown). However, B cells were readily detected in both spleen and MLN several weeks after cell transfer (Fig. 4A). Recipients of protective ebx-B cells and unexposed B cells showed similar patterns of B cell distribution, although protected mice tended to have relatively more B cells in spleen and MLN. We also measured the distribution of IL-10 KO ebx-B cells, which failed to suppress colitis development, and they showed similar distribution and frequencies as wt ebx-B cells (Fig. 4B). Notably, B cells apparently migrated in large quantities to MLN, i.e., the activation site of colitogenic T cells. CD4þ T cells were also equally present in both spleen and MLN of mice transplanted with ebx-B cells or unexposed B cells (Fig. 4C) and similar distribution was seen when comparing recipients of wt and IL-10 KO ebx-B cells (Fig. 4D). These data show that the colitis-suppressing ebx-B cells do not compromise adoptively transferred T cell expansion in lymphoid tissues. Altogether, these results strongly indicate that the suppressive effect of ebx-B cells on colitis development is not due to in vivo competition for space by homeostatic proliferation of B cells.
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FIGURE 2. B cells exposed to enterobacterial extract suppress colitis development. (A) Relative body weight change of SCID mice untransplanted or transplanted with 0.3 106 CD4þCD25 MLN T cells alone (T alone) or in combination with 107 splenic B cells stimulated for 24 hours with ebx (ebx-B) or 107 freshly isolated untouched B cells (un-B). Error bars ¼ SEM. (B) Weight of rectums (distal two cm of colon) from mice in A. (C) Representative H&E-stained histological sections of colons from mice transplanted with nothing, T cells alone or T cells and ebxB cells. Scale bar ¼ 200 lm. (D) Histopathological scoring of the distal colons of mice in A. One representative of two experiments is shown. Dots represent individual mice. ***P < 0.001. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
T Cell Phenotypes Are Altered During B Cell Regulation of Colitis
Since the pathology of CD4þCD25 T cell transfer colitis is mediated by a mixed TH1/TH17 response,38 we speculated whether colitis suppression by ebx-B cells might be associated with altered differentiation patterns of the transferred CD4þ T cells. Hence, we measured the levels of TH1, TH2, and TH17, based on production of IFN-c, IL4, and IL-17A, respectively, in MLN and spleen of mice reconstituted with CD4þCD25 T cells alone or in combination with ebx-B cells or unexposed B cells. Only very few IL-4-producing cells were detected and no difference between groups was observed (not shown). In contrast,
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both TH1 and TH17 cells were abundantly present in spleen and MLN of all groups of mice, including ebx-B cell recipients with very low scores of colitis. Levels of IFN-c-producing TH1 cells were significantly reduced in both spleen and MLN by co-transfer of ebx-B cells (Fig. 5). Co-transfer of unexposed B cells resulted in a similar but smaller decrease in IFN-c-producing cells, however, only being significant in MLN. In contrast, suppression of colitis was associated with significant upregulation of TH17 responses in spleen and MLN compared to colitic control animals. Recipients of unexposed B cells also showed elevated levels of TH17 cells, being significant in MLN. In addition, we measured the frequencies of T cells producing both
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transfer colitis41 and a subset of these IL-10-producing Tregs does not express Foxp3.42 Therefore, we analyzed IL-10 production in the T cells from mice co-transplanted with ebx-B cells obtained from wt or IL-10 KO animals (Fig. 7). Surprisingly, suppression of colitis by transfer of wt ebx-B cells was associated with a drastic decrease in IL-10-producing CD4þ T cells in both spleen and MLN. However, transfer of IL-10 KO ebx-B cells mediated a similar decline in IL-10-producing T cells. It should be noted that by far the majority of IL-10-producing T cells did not co-express Foxp3 (not shown). Thus, the data suggest that B cells per se suppress the development of IL10 secreting T cells and that Tr1 cells are not involved in ebx-B cell-mediated suppression of colitis.
DISCUSSION
FIGURE 3. B cell secretion of IL-10 is required for optimal suppression of colitis. SCID mice were transplanted with MLN CD4þCD25 T cells alone (T cell) or in combination with 107 or 106 B cells from wt or IL-10 KO mice exposed to ebx for 24 hours. (A) Relative body weight change over time after T and B cell transfer. Error bars ¼ SEM. (B) Weight of rectums (distal 2 cm of colon) 6 weeks after T and B cell transfer. (C) Histopathological scoring of the distal colons. Dots represent individual mice. **P < 0.01, ***P < 0.001.
In the present study we demonstrate that B cells exposed to ebx secrete IL-10 and are capable of suppressing T cell transfer colitis and that suppression is associated with skewed T helper responses and increased Treg levels. Our finding that B cells secrete IL-10 upon exposure to ebx is in line with previous demonstrations of IL-10 production in naı¨ve B cells upon activation through TLRs,15,32 as ebx probably comprises several TLR ligands. Combined stimulation through multiple TLRs synergistically enhances
IL-17A and IFN-c (IL-17AþIFN-cþ double positive), suggested to play a significant role in colitis pathology.39 However, levels of IL-17þIFN-cþ double positive cells were not influenced by B cell transfer (Fig. 5).
Ebx-B Cell Transfer Results in Elevated Numbers of Foxp31 Tregs Tregs have the capacity to reduce or prevent CD4þCD25 T cell transfer colitis development.40 Hence, we studied whether protection from colitis by ebx-B cells was associated with Treg development. Expression of Foxp3, the signature marker of Tregs, in CD4þ T cells from spleen and MLN of mice transplanted with T cells alone or in combination with B cells was studied (Fig. 6). Mice cotransplanted with colitis-suppressive ebx-B cells showed 4 and 5 times the number of Foxp3-positive CD4þ T cells in MLN and spleen, respectively. Nevertheless, levels of Foxp3-positive cells were also increased in mice cotransplanted with unexposed B cells, though not to the same degree. In fact, a similar increase in Foxp3 expression was also observed in recipients of IL-10 KO ebx-B cells (data not shown). As already known, IL-10 itself can induce IL-10-producing regulatory T cells (Tr1 cells), which can suppress
FIGURE 4. Suppression of colitis by ebx-B cells is not due to proliferative competition for space. (A) Frequencies of CD19þ B cells were measured by flow cytometry in spleen and MLN of SCID mice transplanted with T cells in combination with ebx exposed B cells (ebx-B) or untouched B cells (un-B). (B) Frequencies of CD19þ B cells in spleen and MLN of recipients of CD4þCD25 T cells and 107 or 106 ebx-B cells from wt or IL-10 KO mice. (C) Frequencies of CD4þ T cells in spleen and MLN of mice transplanted with T cells and ebx-B cells or untouched un-B cells. (D) Frequencies of CD4þ T cells in spleen and MLN of mice transplanted with T cells and ebxB cells from wt or IL-10 KO mice.
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FIGURE 5. Suppression of colitis by ebx-B cells is associated with altered TH1 and TH17 responses. Frequencies of CD4þ T cells producing IFN-c, IL-17A, or both were measured by flow cytometry in spleen and MLN of SCID mice transplanted with T cells alone or in combination with B cells exposed to ebx (ebx-B) or untouched (un-B). Graphs show average frequency (4–5 mice/group) of cytokine-producing cells in CD4þ population. One of two experiments is shown. Error bars ¼ SD. *P < 0.05, **P < 0.001, ***P < 0.0001.
IL-10 production in B cells,32 which is in accordance with an increased IL-10-inducing ability of ebx compared to TLR4 ligation alone by LPS. We observed elevated expression of CD1d and CD5 in the IL-10-producing splenic B cells exposed to ebx. Expression of these surface molecules has previously been associated with immunosuppressive B cells.10,18,24,31,43–45 In particular, the simultaneous expression of CD1d and CD5 indicates that ebx activates the recently identified splenic Breg subset named B10 cells.24 B10 cells are characterized as IL-10 competent CD1dhighCD5þ B cells and they have demonstrated their suppressive capacity in various CD4þ T cell-mediated disease settings as EAE,25 CHS,24 murine lupus,26 parasitic infection,46 and colitis.27 In fact, B10 cell frequencies increased in mice upon colitis development,27 in line with the increase in CD1dhighCD5þ B cells after prolonged exposure to ebx. The significance of Bregs in human IBD has yet to be determined. Only few studies indirectly suggest the involvement of Bregs in IBD. B cell depleting therapy has caused exacerbation8 and development7 of ulcerative colitis (UC). In addition, levels of B-1a cells (CD5þ) seem to be decreased in both UC and Crohn’s disease (CD) patients.47–49 B cell-mediated suppression of experimental colitis has been demonstrated in different mouse models.9,12,13 The data presented here offer new aspects to the suppressive role of B cells in colitis. Like in the Gai2/ and TCRa/ transfer models, we demonstrated suppression of T cell-mediated colitis. However, suppression of Gai2/ colitis was dependent on regulatory CD8þ T cells,12 which are absent in our model. In fact, B cell-mediated suppression of CD4þCD45RBhigh T cell transfer colitis, which is comparable to our model, also required CD8þ T cells.12 Potentially regulatory cell subsets are present in TCRa/ mice but involvement of such in suppression of TCRa/ colitis has not been addressed. Importantly,
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TCRa/ colitis is mediated by a TH2 response,50 in contrast to our model, and suppression in this model required B cell-expression of IL-12.51 Similar to the Gai2/ and TCRa/ models, B cellderived IL-10 is a prerequisite for suppression in our model. Although IL-10 seems to be important for Breg function in general,21 we observed a tendency of suppression in IL-10 KO transplanted colitic mice. In a spontaneous innate model of colitis (RAG/NFATc2/ double knockout mice) splenic B cells have demonstrated suppressive capability independent of IL-10 but dependent on a native BCR repertoire.13 Similar to the high frequency of CD4þ T cells (5%) constitutively recognizing ebx
FIGURE 6. Increased Foxp3þ Tregs in B cell transplanted mice. Intracellular expression of Foxp3 was measured by flow cytometry in CD4þ T cells from spleen and MLN of SCID mice transplanted with T cells alone or in combination with B cells exposed or unexposed to ebx. Frequencies of Foxp3-positive cells of the CD4þ population are shown. One of two experiments is shown. Dots represent individual mice. **P < 0.001, ***P < 0.0001.
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FIGURE 7. Transfer of B cells results in reduced levels of IL-10-producing CD4þ T cells. SCID mice were transplanted with T cells alone or in combination with 107 or 106 B cells from wt or IL-10 KO mice. Approximately 6 weeks after transfer, frequencies of IL-10-producing CD4þ T cells were measured in spleen and MLN. Graphs show average frequencies (5–8 mice/group) of IL-10-positive cells in CD4þ population. Error bars ¼ SD. Asterisks indicate significant difference from T alone. *P < 0.05, **P < 0.001, ***P < 0.0001.
antigens,37 a high frequency of ebx recognizing B cells might exist. Therefore, we cannot exclude that T cell suppression by ebx-B cells is in part mediated through transcytosis-dependent enteroantigen uptake and presentation by B cells carrying BCRs specific for ebx. In accordance with this idea, we showed previously a superior enteroantigendependent T cell stimulatory capacity mediated by ebx-B cells in vitro.35 In line with this notion, Breg-mediated suppression of EAE14 and CHS24 was shown to require specific antigen, suggesting the presence of B cells recognizing EAE relevant autoantigens. Suppression of colitis by ebx-B cells was associated with a decreased TH1 response, also observed during B cell-mediated suppression of EAE,52 CIA,18 and diabetes.19 However, we also observed an increased frequency of TH17 cells in B cell transplanted mice, which is in contrast to findings in EAE.15 B cells can impede TH1 cell development by suppressing IL-12 secretion by DCs23 and, since IFN-c strongly counteracts TH17 cell differentiation,53,54 suppression mediated by Breg may polarize in favor of TH17 cell development. However, the relevance of this finding needs further analysis. We observed elevated levels of Foxp3-expressing Tregs in mice transplanted with colitis-suppressing ebx-B cells. This is in accordance with similar findings in other studies, demonstrating increased activation of regulatory T cells in B cell-mediated suppression of colitis,12 murine lupus,26 and EAE.16 Furthermore, splenic B cells can induce regulatory CD4þ and CD8þ T cell subsets by MHC-restricted presentation of ocular autoantigen55 and human CD40 stimulated B cells have the capability of inducing56 and expanding57 Tregs in vitro. As 1% of CD4þCD25 T cells express Foxp358—a frequency we also observed in colitic mice transplanted with T cells only—it is uncertain whether the increase in Foxp3þ Tregs observed after ebx-B cell transfer is due to de novo differentiation of Tregs or expansion of transferred Foxp3þ
T cells. However, since ebx-B cell, but not ebx-pulsed DCs, can activate Tregs in vitro,35 our present data suggest that ebx-B-mediated Treg activation also occurs in vivo. Notably, B cells failed to suppress colitis in SCID mice transplanted with CD4þCD45RBhigh T cells,12 which are naı¨ve cells and comprise no Foxp3þ Tregs.58 Because transfer of B cells caused a decrease in IL-10-producing T cells, it appears that the B cell-mediated suppression of colitis demonstrated here did not rely on T cell-derived IL-10. In support, Foxp3þ Tregs can efficiently suppress T cell responses in vitro42 and even transfer colitis59 independent of IL-10. However, further experiments are needed to conclude on the involvement of Tregs in suppression of colitis by ebx-B cells. In conclusion, we demonstrate the presence of IL-10 secretion by B cells exposed to enterobacterial components. Such B cells acquire an immunosuppressive potential capable of suppressing the development of experimental T cell transfer colitis. B cell-induced suppression of colitis is associated with a decreased TH1 response and elevated levels of Foxp3þ Treg.
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