Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis - PubMed (original) (raw)

. 2009 Nov;10(11):1178-84.

doi: 10.1038/ni.1791. Epub 2009 Sep 27.

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Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis

Masako Murai et al. Nat Immunol. 2009 Nov.

Abstract

Regulatory T cells (T(reg) cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b(+) myeloid cells in recombination-activating gene 1-deficient (Rag1(-/-)) recipient mice was needed to prevent the colitis induced by transferred CD4(+)CD45RB(hi) T cells. In Il10(-/-)Rag1(-/-) mice, T(reg) cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor-deficient (Il10rb(-/-)) T(reg) cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the T(reg) cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on T(reg) cells to maintain Foxp3 expression.

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Figures

Figure 1

Figure 1

IL-10-deficient Treg cells prevent colitis. (a) Body weight of _Rag1_−/− mice given sorted _Il10_−/− or wild-type (WT) CD4+CD25+ Treg cells, together with CD4+CD45RBhi T cells, or of _Rag1_−/− mice given CD4+CD45RBhi T cells alone (control; None), presented relative to initial body weight. Data are pooled from two independent experiments with six mice each (error bars, s.d.). (b) Histology scores of sections of the large intestine at 6 weeks after the cell transfer described in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. NS, not significant. Data are pooled from at least two independent experiments.

Figure 2

Figure 2

_Rag1_−/− host IL-10 is required for Treg cell function. (a) Body weight of _Rag1_−/− or _Il10_−/−_Rag1_−/− hosts given CD4+CD45RBhi T cells plus sorted _Foxp3_gfp Treg cells, presented relative to initial body weight. Data are pooled from two independent experiments with ten mice each (error bars, s.d.). (b) Proximal colon of _Rag1_−/− and _Il10_−/−_Rag1_−/− mice at 6 weeks after the donor cell transfer described in a; sections are stained with hematoxylin and eosin. Original magnification, ×100; scale bars, 100 µm. Data are representative of one of three independent experiments. (c) Histology scores of sections of the large intestine at 6 weeks after the cell transfer described in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (two-tailed Student’s _t_-test). Data are pooled from three independent experiments with a total of nine mice.

Figure 3

Figure 3

Foxp3 is downregulated in _Il10_−/−_Rag1_−/− recipients. (a) Composite ratios of CD45.1+ to CD45.2+ TCRβ+CD4+ cells in the spleen (Spl), PLNs, MLNs and LPL of _Rag1_−/− or _Il10_−/−_Rag1_−/− recipient mice at 6 weeks after injection of 4 × 105 CD4+CD45RBhi T cells derived from C57BL/6 (CD45.1+) mice, plus 1 × 105 _Foxp3_gfp (CD45.2+) Treg cells. (b) Foxp3 expression in the cells in a, gated on TCRβ+CD4+ CD45.2+ cells. Bracketed lines indicate the Foxp3− population. max, maximum. (c) Foxp3− cells in the gated TCRβ+CD4+ CD45.2+ populations in b. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (two-tailed Student’s _t_-test). Data are pooled from three independent experiments with a total of nine mice (a (mean and s.d.) and c) or are representative of one of three independent experiments (b).

Figure 4

Figure 4

Loss of function by Treg cells from _Il10_−/−_Rag1_−/− recipients. (a,b) Flow cytometry of intracellular IFN-γ in CD4+CD45RBhi T cells derived from C57BL/6 (CD45.1+) mice transferred with _Foxp3_gfp (CD45.2+) Treg cells into _Rag1_−/− or _Il10_−/−_Rag1_−/− hosts; plots are gated on the TCRβ+CD4+ CD45.2− progeny of donor CD4+CD45RBhi T cells (a) and the TCRβ+CD4+ CD45.2+ progeny from donor _Foxp3_gfp + Treg cells (b), isolated from spleen (Spl), MLNs and LPL in mice at 6 weeks after donor cell injection and then stimulated with PMA and ionomycin. Numbers in plots indicate percent IFN-γ-producing cells. (c) Suppressive function in vitro of sorted TCRβ+CD4+ CD45.2+ cells from MLNs of _Rag1_−/− or _Il10_−/−_Rag1_−/− recipients of CD45.1+ CD4+CD45RBhi and CD45.2+ CD4+CD25+CD45RBlo Treg cell populations, cultured for 4 d together with CFSE-labeled CD45.1+ naive T cells; after stimulation of cultures, CFSE dilution was assessed by flow cytometry. Data are representative of one of three (a,b) or two (c) independent experiments.

Figure 5

Figure 5

_Il10rb_−/− Treg cells fail to prevent colitis. (a) Body weight of _Rag1_−/− recipients of C57BL/6 (CD45.1+) CD4+CD45RBhi T cells transferred together with wild-type or _Il10rb_−/− (CD45.2+) Treg cells, presented relative to initial body weight. Data are pooled from two independent experiments with a total of ten mice (error bars, s.d.). (b) Proximal colon of recipient mice at 6 weeks after injection of cells as described in a; sections are stained with hematoxylin and eosin. Original magnification, ×100; scale bars, 100 µm. Data are representative of one of three independent experiments. (c) Histology scores of sections of the large intestine at 6 weeks after the cell transfer described in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (two-tailed Student’s _t_-test). Data are pooled from three independent experiments with a total of nine mice. (d) Foxp3 expression by cells isolated from the spleen, PLNs, MLNs and LPL of the recipient mice in a, with gating on TCRβ+CD4+CD45.2+ cells. Bracketed lines indicate the Foxp3− population. Data are representative of one of three independent experiments with a total of nine mice. (e) Foxp3− cells in the TCRβ+CD4+ CD45.2+ T lymphocyte populations described in d. Each symbol represents an individual mouse; small horizontal bars indicate the mean. *P < 0.001 (two-tailed Student’s _t_-test). Data are pooled from three independent experiments with a total of nine mice.

Figure 6

Figure 6

Foxp3 is lost ‘preferentially’ by _Il10rb_−/− Treg cells in mice with colitis. (a) Ratio of CD45.1+ to CD45.2+ CD90.1−CD4+TCRβ+ cells isolated from spleen, PLNs, MLNs and LPL of _Rag1_−/− recipients at 6 weeks after injection of 8 × 105 C57BL/6 (CD90.1+) CD4+CD45RBhi T cells, transferred with 2 × 104 wild-type (CD45.1+) Treg cells and 2 × 104 _Il10rb_−/− (CD45.2+) Treg cells. Data are pooled from two independent experiments with a total of four mice (mean and s.d.). (b) Flow cytometry of Foxp3 expression by cells isolated from a _Rag1_−/− recipient mouse as described in a, with gating on CD90.1−CD4+TCRβ+CD45.2− cells (wild-type Treg cells) or CD90.1−CD4+TCRβ+CD45.2+ cells (_Il10rb_−/− Treg cells). Bracketed lines indicate the Foxp3− population. Data are representative of one of two independent experiments. (c) Foxp3− cells in the CD90.1−CD4+TCRβ+CD45.2− (wild-type Treg) and CD90.1−CD4+TCRβ+CD45.2+ (_Il10rb_−/− Treg) populations isolated from _Rag1_−/− mice as described in a. Each symbol represents an individual mouse; small horizontal bars indicate the mean. Data are pooled from two independent experiments with a total of four mice. (d) Foxp3− cells in CD90.1−CD4+TCRβ+CD45.2− (wild-type Treg) or CD90.1−CD4+TCRβ+CD45.2+ (_Il10rb_−/− Treg) populations isolated from _Rag1_−/− recipients of 4 × 105 C57BL/6 (CD90.1+) CD4+CD45RBhi T cells, transferred together with 1 × 105 wild-type (CD45.1+) and 1 × 105 _Il10rb_−/− (CD45.2+) Treg cells. Each symbol represents an individual mouse; small horizontal bars indicate the mean. * P < 0.01; ** P < 0.001 (two-tailed Student’s _t_-test). Data are pooled from two independent experiments with a total of four mice.

Figure 7

Figure 7

Kinetics of IL-10 expression by Treg cells and host cells. (a) GFP+ cells in IL-10 reporter mice. Numbers adjacent to outlined areas (top row) indicate percent GFP+ cells among gated naive splenocytes (TCRβ+CD4+CD45RBhi) and Treg splenocytes (TCRβ+CD4+CD45RBloCD25+) from _Il10_gfp mice; numbers in top right quadrants (bottom row) indicate percent CD45+GFP+ cells in tissues from _Il10_gfp_Rag1_−/− mice. MFI, mean fluorescence intensity. (b) Flow cytometry of GFP+ cells in tissues 7 d after transfer of a mixture of CD45.1+ CD4+CD45RBhi T cells and CD45.2+ _Il10_gfp Treg cells (ratio, 4:1). Top row, Treg cells gated as CD45.2+ TCRβ+CD4+ cells; bottom row, gated TCRβ−CD4− nonlymphoid cells. Numbers adjacent to outlined areas and in top right quadrants indicate percent GFP+ cells. (c) Real-time PCR analysis of Il10 mRNA in sorted Treg cells (left), CD11b+ cells (middle) and CD11c+ dendritic cells (right) from various sites (keys) before transfer (0) or at 1, 2 and 6 weeks after transfer as in b. Data are from representative one of two independent experiments with six mice (a,b) or are pooled from two independent experiments with six mice (c; mean and s.d.).

Figure 8

Figure 8

IL-10-producing CD11b+ myeloid cells prevent the downregulation of Foxp3. Flow cytometry of Foxp3 expression 3 weeks after the injection of 5 × 106 _Rag1_−/− or _Il10_−/−_Rag1_−/− intestinal CD11c+CD11b+F4/80+ cells (a) or CD11c+CD11bF4/80− cells (b) into _Il10_−/−_Rag1_−/− recipients, transferred intravenously on days 0 and 7 (where ‘day 0’ is the day of T cell transfer) with 4 × 105 CD4+ CD45RBhi (CD45.1+) cells in the presence of 1 × 105 (CD45.2+) Treg cells from _Foxp3_gfp mice. Plots are gated on TCRβ+CD4+CD45.2+ splenocytes. Data are representative of one of two independent experiments with a total of three mice.

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References

    1. Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int. Immunol. 1993;5:1461–1471. - PubMed
    1. Powrie F, et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity. 1994;1:553–562. - PubMed
    1. Powrie F, Correa-Oliveira R, Mauze S, Coffman RL. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 1994;179:589–600. - PMC - PubMed
    1. Mottet C, Uhlig HH, Powrie F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 2003;170:3939–3943. - PubMed
    1. Annacker O, et al. CD25+CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 2001;166:3008–3018. - PubMed

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