Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis - PubMed (original) (raw)

Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis

Marika C Kullberg et al. J Exp Med. 2002.

Abstract

We have previously demonstrated that interleukin (IL)-10-deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4(+) T cells from IL-10 KO animals and that the cotransfer of CD4(+) T cells from H. hepaticus-infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4(+) cells are contained within the CD45RB(low) fraction and unexpectedly were found in both the CD25(+) and the CD25(-) subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25(+) and CD25(-) CD45RB(low) CD4(+) cells block colitis involves IL-10 and not transforming growth factor (TGF)-beta, as treatment with anti-IL-10R but not anti-TGF-beta monoclonal antibody abrogated their protective effect. In vitro, CD45RB(low) CD4(+) cells from infected WT mice were shown to produce IL-10 and suppress interferon-gamma production by IL-10 KO CD4(+) cells in an H. hepaticus antigen-specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.

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Figures

Figure 1.

Figure 1.

_H. hepaticus_–infected but not naive RAG KO mice develop intestinal inflammation after reconstitution with CD4+ T cells from IL-10 KO mice. (A) Uninfected or _H. hepaticus_–infected RAG KO mice were inoculated intravenously with 3 × 105 CD4+ cells from the MLNs of 11-wk infected IL-10 KO mice, and intestinal pathology was analyzed 8, 15, 22, and 29 d later (solid bars). Naive and infected RAG KO animals receiving no cells were analyzed in parallel (open bars). Bars represent mean cecal histology scores ± SD of three or four mice per group. *, P < 0.05 compared with infected mice receiving no cells. (B–D) Histology of ceca of representative sections from the mice shown in A analyzed 29 d after cell transfer: (B) infected RAG KO without cell transfer; (C) infected RAG KO receiving CD4+ cells from infected IL-10 KO mice; or (D) uninfected RAG KO receiving CD4+ cells from infected IL-10 KO mice. Crypt abscesses (solid arrows), ulcer (open arrow). H&E staining. Bar, 200 μm.

Figure 1.

Figure 1.

_H. hepaticus_–infected but not naive RAG KO mice develop intestinal inflammation after reconstitution with CD4+ T cells from IL-10 KO mice. (A) Uninfected or _H. hepaticus_–infected RAG KO mice were inoculated intravenously with 3 × 105 CD4+ cells from the MLNs of 11-wk infected IL-10 KO mice, and intestinal pathology was analyzed 8, 15, 22, and 29 d later (solid bars). Naive and infected RAG KO animals receiving no cells were analyzed in parallel (open bars). Bars represent mean cecal histology scores ± SD of three or four mice per group. *, P < 0.05 compared with infected mice receiving no cells. (B–D) Histology of ceca of representative sections from the mice shown in A analyzed 29 d after cell transfer: (B) infected RAG KO without cell transfer; (C) infected RAG KO receiving CD4+ cells from infected IL-10 KO mice; or (D) uninfected RAG KO receiving CD4+ cells from infected IL-10 KO mice. Crypt abscesses (solid arrows), ulcer (open arrow). H&E staining. Bar, 200 μm.

Figure 2.

Figure 2.

CD45RBlow CD4+ cells from _H. hepaticus_–infected WT mice protect RAG KO animals from colitis induced by IL-10 KO CD4+ cells plus bacterial infection. (A) _H. hepaticus_–infected RAG KO mice (solid bars) were inoculated with CD4+ cells from infected IL-10 KO mice and CD4+ cells from naive or infected WT mice as indicated (4 × 105 of each population). Intestinal pathology was analyzed 4 wk later. Naive (open bar) and infected RAG KO animals receiving no cells were included as controls. Bars represent mean histology scores ± SD of three mice per group except for groups receiving WT cells alone, in which case, due to limited cell numbers, only two mice per group were used. Similar results were seen in ascending colon (although histology scores were lower) and when disease was induced by the transfer of CD4+ cells from naive IL-10 KO mice (not depicted). (B) Infected RAG KO mice were given either no cells or CD4+ cells from infected IL-10 KO mice and CD4+, CD45RBhi CD4+, or CD45RBlow CD4+ cells from infected WT mice as indicated (4 × 105 of each population). Pathology in the cecum (solid bars) and ascending colon (gray bars) were analyzed 4 wk later. Bars represent mean histology scores ± SD of six or seven mice per group pooled from two separate experiments except for the group receiving IL-10 KO CD4+ cells plus infected WT CD4+ cells (n = 3), which was included in only one of the experiments. Statistical significance was tested for groups receiving IL-10 KO cells. *, P < 0.05 compared with mice receiving IL-10 KO cells alone.

Figure 3.

Figure 3.

Intestinal pathology of _H. hepaticus_–infected RAG KO mice receiving CD4+ cells from IL-10 KO and WT mice. (A–F) Representative cecal sections of the infected RAG KO mice described in Fig. 2 A receiving (A) no cells, (B) infected IL-10 KO CD4+ cells, (C) infected IL-10 KO CD4+ cells plus infected WT CD4+ cells, (D) infected IL-10 KO CD4+ cells plus naive WT CD4+ cells, (E) infected WT CD4+ cells alone, and (F) naive WT CD4+ cells alone. H&E staining. (G and H) Immunohistochemical staining for CD3+ T cells (arrows) in colonic sections of infected RAG KO recipients receiving (G) infected IL-10 KO CD4+ cells alone and (H) infected IL-10 KO CD4+ cells plus infected WT CD45RBlow CD4+ cells. Bar, 200 μm.

Figure 4.

Figure 4.

CD25− CD45RBlow as well as CD25+ CD45RBlow CD4+ cells from infected WT mice protect RAG KO mice against colitis. Infected RAG KO mice were given either no cells or 3 × 105 CD4+ cells from infected IL-10 KO mice either alone or with 3 × 105 CD45RBlow CD4+ cells, 3 × 105, 105, or 3.3 × 104 CD25− CD45RBlow, or CD25+ CD45RBlow CD4+ cells from infected WT mice as indicated. (A) Pathology in the cecum and (B) ascending colon was analyzed 4 wk later. •, an individual mouse; —, the average for each group. Data shown are pooled from two separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with infected mice receiving IL-10 KO cells alone.

Figure 5.

Figure 5.

CD25− CD45RBlow and CD25+ CD45RBlow CD4+ cells from infected WT mice block colitis via an IL-10–dependent but TGF-β–independent mechanism. Infected RAG KO mice were given either no cells or CD4+ cells from infected IL-10 KO mice and CD25− CD45RBlow or CD25+ CD45RBlow CD4+ cells from infected WT mice (3 × 105 of each population) and treated with control mAb, anti–IL-10R (both at 1 mg/wk), or anti–TGF-β as indicated. (A) In the first experiment, mice were given 2 mg of anti–TGF-β per wk for 4 wk, (B) and in the second experiment, they were given 4 mg of anti–TGF-β per wk for 2 wk before analysis. •, the cecal histology score from an individual mouse; —, the average for each group. Similar results, although lower histology scores, were seen for the ascending colon where CD25− cell recipients treated with control or anti–TGF-β mAb displayed indistinguishable scores in B. In A, anti–IL-10R–treated groups had P values of <0.01 and <0.05 compared with control mAb–treated groups, whereas scores for mice given anti–TGF-β were not significantly different from those receiving control mAb. Similar results were seen in B except that it was not technically possible to perform Mann-Whitney U test statistics on recipients of CD25− cells due to identical scores of all mice receiving control mAb.

Figure 6.

Figure 6.

T cell–derived IL-10 mediates the disease-protective effect of WT CD45RBlow CD4+ cells. (A) Infected RAG/IL-10 double deficient mice were given either no cells or infected IL-10 KO CD4+ cells and infected WT CD4+ CD45RBlow cells as indicated (4 × 105 of each population) and then treated with control mAb or anti–IL-10R mAb for 4 wk. Bars represent mean cecal histology scores ± SD of three mice per group. (B) Infected RAG KO mice (solid bars) were inoculated with CD4+ cells from infected IL-10 KO, WT, and IL-4 KO mice as indicated (3 × 105 of each population) and then treated with control mAb or anti–IL-10R mAb for 4 wk. Naive (open bar) and infected RAG KO animals receiving no cells were included as controls. Bars represent mean cecal histology scores ± SD of three mice per group from one representative experiment out of two performed. Statistical significance was tested for groups receiving IL-10 KO cells. *, P < 0.05 compared with mice receiving infected IL-10 KO cells alone.

Figure 7.

Figure 7.

CD45RBlow CD4+ cells from naive WT mice are unable to protect RAG KO recipients from colitis induced by pathogenic T cells plus H. hepaticus infection. (A) Infected RAG KO mice were given either no cells or CD4+ cells from infected IL-10 KO mice and CD45RBlow CD4+ cells from naive or infected WT mice (4 × 105 of each population), and tissues were analyzed 4 wk later. Bars represent mean cecal (solid bars) and colonic (gray bars) histology scores ± SD of three mice per group. (B and C) Uninfected or _H. hepaticus_–infected (Hh-inf.) RAG KO mice were given either no cells or CD45RBhi CD4+ cells from naive WT mice and CD45RBlow CD4+ cells from naive or infected WT mice (3 × 105 of each population). Body weights were measured weekly and tissues were analyzed after 4 and 7.5 wk for infected and uninfected recipients, respectively. (B) Results shown represent mean body weights (expressed as a percentage of the weight 2 d after cell transfer) of four mice per group from the uninfected RAG KO recipients. No difference in body weight was observed between the four groups of infected recipients over the 4-wk time period that these groups were being followed (not depicted). (C) Bars represent mean cecal (solid bars) and colonic (gray bars) histology scores ± SD of three or four mice per group. Statistical significance was tested for groups receiving pathogenic cells. *, P < 0.05 compared with mice receiving infected IL-10 KO CD4+ cells alone (A) or naive WT CD45RBhi CD4+ cells alone (C).

Figure 8.

Figure 8.

CD45RBlow CD4+ cells from infected WT mice inhibit IFN-γ production by IL-10 KO CD4+ cells and produce IL-10 after in vitro stimulation with SHelAg. (A) Responder CD4+ cells from infected IL-10 KO mice were cultured with APC, SHelAg, and indicated numbers of CD45RBlow CD4+ cells from naive (○) or infected (•) WT mice. IFN-γ was measured in 72-h supernatants. (B) CD45RBlow CD4+ cells from naive (□) or infected (▪) WT mice were cultured with IL-10–deficient APC and indicated Ag. IL-10 was measured in 72-h supernatants. No IFN-γ, IL-4, or IL-5 was detected in cultures stimulated with SHelAg plus IL-2. (C) CD45RBlow cells from naive or infected WT mice were cultured with IL-10–deficient APC and SHelAg plus IL-2. After removal of supernatants at 72 h, IL-2 was added for an additional 18 h before cells were stimulated with PMA plus ionomycin and analyzed for the expression of IL-10 and IFN-γ by intracellular staining. The FACS® dot plots shown are gated on CD4+ cells. In the same assay, the cells were negative for intracellular IL-4. The results shown in A–C are representative of two or three experiments performed.

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