Antigen-presenting cell production of IL-10 inhibits T-helper 1 and 17 cell responses and suppresses colitis in mice - PubMed (original) (raw)

Antigen-presenting cell production of IL-10 inhibits T-helper 1 and 17 cell responses and suppresses colitis in mice

Bo Liu et al. Gastroenterology. 2011 Aug.

Abstract

Background & aims: Mice that are deficient in interleukin (IL)-10 develop colitis, mediated by T-helper (Th)1 and Th17 cells, and IL-10-producing regulatory T (Treg) cells suppress colitis, implicating IL-10 in maintaining mucosal homeostasis. We assessed the relative importance of immunoregulatory IL-10 derived from T cells or from antigen presenting cells (APCs) in development of intestinal inflammation.

Methods: CD4(+) cells from germ-free (GF) or specific pathogen-free (SPF) IL-10(-/-) or wild-type mice were injected into IL-10(-/-), Rag2(-/-) mice or Rag2(-/-) mice that express IL-10. After 6-8 weeks, we evaluated inflammation, spontaneous secretion of cytokines from colonic tissue, and mRNA levels of the transcription factor T-bet and the immunoregulatory cytokine transforming growth factor (TGF)-β. CD4(+) T cells were co-cultured with bacterial lysate-pulsed APCs and assayed for cytokine production, FoxP3 expression, and TGF-β-mediated Smad signaling.

Results: CD4(+) cells from GF or SPF IL-10(-/-) or wild-type mice induced more severe colitis and higher levels of inflammatory cytokines in IL-10(-/-), Rag2(-/-) mice than in IL-10-replete, Rag2(-/-) mice. Co-cultures of IL-10(-/-) or wild-type CD4(+) T cells plus bacterial lysate-pulsed APCs from IL-10(-/-) mice contained more interferon (IFN)-γ, IL-12/23p40, and IL-17 than co-cultures of the same T cells plus APCs from wild-type mice. CD11b(+) APCs were required for these effects. Blocking IL-10 receptors increased production of IFN-γ and IL-12/23p40 whereas exogenous IL-10 suppressed these cytokines. IL-10-producing APCs induced TGF-β-mediated, retinoic acid-dependent, differentiation of FoxP3(+) Treg cells, whereas blocking the retinoic acid receptor, in vitro and in vivo, reduced proportions of FoxP3(+) Treg cells.

Conclusions: IL-10 produced by APCs regulates homeostatic T-cell responses to commensal bacteria.

Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Colonic inflammation in IL-10 wt Rag2−/− or IL-10 ko Rag2−/− recipients reconstituted with either SPF or GF IL-10 ko or wt CD4+ cells

(A) Representative H&E stained sections of the distal colons of recipient mice 6-8 wk after transfer of SPF CD4+ cells (20× magnification). (B) Blinded histologic scores in the large intestine and (C) spontaneous secretion of IFN-γ by colonic fragments of recipients of SPF CD4+ cells. (D) Representative H&E stained sections of the distal colons of recipient mice 6-8 wk after transfer of GF CD4+ cells. (E) Blinded histologic scores and (F) IFN-γ secretion by colonic fragments of recipients of GF CD4+ cells. Results show mean ± SEM, pooled from three separate experiments, with (ko→ko, n=14; ko→wt, n=12; wt→ko, n=11; wt→wt, n=11) for recipients of SPF CD4+ cells and (ko→ko, n=7; ko→wt, n=12; wt→ko, n=11; wt→wt, n=14) for recipients of GF CD4+ cells. *p< 0.05, **p<0.01 & ***p<0.001.

Figure 2

Figure 2. Cytokines produced by MLN cells after transfer of SPF or GF IL-10 ko or IL-10 wt CD4+ T cells

Six-eight weeks after transfer of either SPF (A) or GF (B) CD4+ cells, unseparated MLN cells from IL-10 ko Rag2−/− or IL-10 wt Rag2−/− recipients were stimulated with CBL for 72 hours and IFN-γ, IL-17 and IL-12/23p40 secretion were measured. Spontaneous IL-10 by colonic fragments was evaluated. Results show mean ± SEM (number of mice/group same as Figure 1). *p< 0.05, **p<0.01, ***p<0.001.

Figure 3

Figure 3. Time course of histological evaluation of colitis and proinflammatory cytokine production in recipient mice

(A) Histologic score, (B) spontaneous IL-12/23p40 in colonic cultures, and amounts of (C) IFN-γ and (D) IL-12/23p40 produced by unfractionated MLN cells stimulated with CBL (10 μg/ml) after 3 day culture in vitro for mice evaluated 1, 2, 4, 6 and 12 weeks after transfer of SPF IL-10 ko CD4+ T cells to IL-10 ko Rag2−/− or IL-10 wt Rag2−/− recipients. ** p< 0.001 and *p<0.05 or p value shown above the bar vs. IL-10 wt Rag2−/− recipients. Results show mean ± SEM, 6-8 mice per group at each time point.

Figure 4

Figure 4. In vitro cytokine production

Splenic CD4+ T cells from SPF IL-10 ko or IL-10 wt mice were co-cultured with CBL-pulsed APC from IL-10 ko Rag2−/− or IL-10 wt Rag2−/− mice for 72 hours and (A) IFN-γ (B) IL-17, (C) IL-12/23p40 (D) IL-10 concentrations were measured. Representative results (one of three separate experiments) are shown as mean ± SEM of triplicate culture supernatants. ***p<0.001 vs. co-cultures containing IL-10 wt APC. §§§p<0.001 vs co-cultures containing IL-10 ko APC. □p<0.001 vs IL-10 wt APC alone or in co-culture with IL-10 ko CD4+ cells.

Figure 5

Figure 5. CD11b-enriched APC regulate cytokine production

APC from IL-10 ko Rag2−/− or IL-10 wt Rag2−/− mice were separated into CD11b-enriched (CD11b+) or CD11b-depleted (CD11b−) populations by positive or negative magnetic bead sorting, respectively. Co-cultures contain either IL-10 ko or IL-10 wt splenic CD4+ T cells and CBL-pulsed APC. (A, B) IL-17 or (C, D) IFN-γ was measured in supernatants collected 72 hours after co-culture initiation. Representative results (one of three separate experiments) are shown as mean ± SEM of triplicate culture supernatants. *p< 0.05, **p<0.01.

Figure 6

Figure 6. Induction of FoxP3+ Treg cells by IL-10 wt Rag2−/− or IL-10 ko Rag2−/− APC

(A) Intracellular FoxP3 expressed by IL-10 wt CD4+ cells co-cultured with IL-10 wt or IL-10 ko Rag2−/− APC for 4 days in the presence or absence of TGF-β1 plus LE540 or vehicle (DMSO). (B) Intracellular FoxP3 in CD4+ cells from recipients treated in vivo with LE540 or vehicle (DMSO in soybean oil). (C) Expression of Aldh1a1 and Aldh1a2 mRNA relative to β-actin in MLN from IL-10 ko Rag2−/− or IL-10 wt Rag2−/− recipient mice. Representative results (one of two separate experiments) are shown as mean ± SEM of triplicate qPCR. *p< 0.05, **p<0.01. (D) FoxP3 expression by IL-10 wt CD4+ cells co-cultured with IL-10 wt APC in the presence of TGF-β1(1 ng/ml) and anti-IL-10R or isotype control (30 μg/ml). (E) FoxP3 expression by IL-10 wt CD4+ cells co-cultured with IL-10 ko APC in the presence of TGF-β1 (1 ng/ml) with or without recombinant IL-10 (500 pg/ml) for 4 days. Representative results (one of three experiments) are shown for D and E.

Figure 7

Figure 7. Smad3 and P-Smad3 protein and TGF-β1 mRNA expression

APC from IL-10 wt Rag2−/− and IL-10 ko Rag2−/− mice co-cultured with IL-10 wt CD4+ cells were stimulated with TGF-β1 in the presence of CBL (10 μg/ml). Cells were harvested at times shown and Western blot analysis visualized Smad3 and P-Smad3. (A) Representative blots from two experiments are shown. (B) Densitometric analysis of the Western blot shown in (A) p-Smad3/actin left panel, p-Smad3/total Smad3 right panel. (C) TGF-β1 mRNA expression in MLN from IL-10 ko Rag2−/− or IL-10 wt Rag2−/− recipient mice reconstituted with IL-10 ko or IL-10 wt CD4+ cells. Representative results (one of three experiments) are shown as mean ± SEM of triplicate qPCR. (D) TGF-β1 mRNA expression in co-cultured IL-10 wt CD4+ cells with CBL-pulsed APC from either IL-10 ko Rag2−/− or IL-10 wt Rag2−/− mice. Representative results (one of two separate experiments) are shown as mean ± SEM of triplicate qPCR.

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