A protective function for interleukin 17A in T cell-mediated intestinal inflammation

Abstract

Interleukin 23 (IL-23) and IL-17 have been linked to the pathogenesis of several chronic inflammatory disorders, including inflammatory bowel disease. Yet as an important function for IL-23 is emerging, the function of IL-17 in inflammatory bowel disease remains unclear. Here we demonstrate IL-17A-mediated protection in the CD45RBhi transfer model of colitis. An accelerated wasting disease elicited by T cells deficient in IL-17A correlated with higher expression of genes encoding T helper type 1-type cytokines in colon tissue. IL-17A also modulated T helper type 1 polarization in vitro. Furthermore, T cells deficient in the IL-17 receptor elicited an accelerated, aggressive wasting disease relative to that elicited by wild-type T cells in recipient mice. Our data demonstrate a protective function for IL-17 and identify T cells as not only the source but also a target of IL-17 in vivo.

Figure 1

IL-17A-deficient CD45RB^hi T cells induce an aggressive wasting disease in Rag1^−/− recipient mice. (a) Body weights of Rag1^−/− recipients of intraperitoneally injected purified CD45RB^hi CD4⁺ T cells from Il17a^−/− mice or unmanipulated wild-type (WT) C57BL/6 mice on day 0, presented as percent of original weight. Data are group averages from one experiment (error bars, s.e.m.). (b) Weight loss distributions of each individual mouse (single symbols) and composite statistics of all recipient mice. Means (small horizontal bars) at day 14 (P = 0.4155): recipients of wild-type cells (n = 35), 99.26 (s.d., 4.168); recipients of Il17a>R>−/− cells (n = 32), 99.22 (s.d., 6.057). Means at day 28: recipients of wild-type cells (n = 24), 96.71 (s.d., 7.493); recipients of Il17a^−/− cells (n = 21), 90.87 (s.d., 8.155). *, P ≤ 0.01. Data are representative of three independent experiments. (c) Gross organ morphology of the cecum and ascending colon from the recipient mice in b at day 28. Results are representative of three experiments. (d) Histology of colon tissues from the mice in a. Top, colon tissues at day 14 from an unmanipulated wild-type mouse (i), a recipient of Il17a^−/− cells (ii) or a recipient of wild-type cells (iii). *, edema. Bottom, colon tissues at day 28 from a recipient of Il17a^−/− cells (iv and v (higher magnification of box in iv)), or wild-type cells (vi and vii (higher magnification of box in vi). Arrowheads indicate the presence (iv,v) or absence (vi,vii) of ulcerated epithelium. Scale bars, 500 μm. Results are representative of two experiments. (e) Quantification of pathological changes in the mice in a, assessed as ‘severity scores’. Data are representative of two experiments (error bars, s.e.m.). (f) Quantitative RT-PCR analysis of mRNA transcripts encoding cytokines (vertical axes), measured in ascending colon tissue from the mice in a on day 28. Each dot represents an individual mouse; scores are presented as ‘fold increase’ relative to baseline expression in colon tissue from unmanipulated wild-type littermates after normalization to expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase; change in cycling threshold method). Means for Ifng (P ≤ 0.0001): recipients of wild-type cells (n = 14), 89.79; recipients of Il17a^−/− cells (n = 17), 263.8. Means for Spp1 (P ≤ 0.001): recipients of wild-type cells (n = 13), 4.56; recipients of Il17a^−/− cells (n = 17), 41.25. Means for Il6: recipients of wild-type cells (n = 14), 60.48; recipients of Il17ra^−/− T cells (n = 13), 18.62. *, P ≤ 0.001 (unpaired, two-tailed Student's t-test). Data are representative of three experiments.

Figure 2

IL-17 modulates T_H1 differentiation. (a) Flow cytometry analysis of IL-17R on the cell surface of naive CD4⁺ T cells assessed directly after isolation (Naive) or after 2 d or 4 d of culture in T_H1-polarizing conditions. Light lines, isotype-matched control antibody; dark lines, antibody to IL-17R. Data are representative of two experiments. (b) Immunoblot analysis (IB) of the immunoprecipitation (IP) of IL-17R from freshly isolated naive CD4⁺ T cells or from T cells cultured for 4 d in T_H1-polarizing conditions in vitro with (right) or without (left and middle) amidase treatment. Actin, loading control for protein content in cell lysates. α-IL-17R, antibody to IL-17R. Results are representative of two independent experiments. (c) Real-time PCR analysis of gene expression in T_H0 effector cells or in T_H1 effector cells at day 4 generated in vitro in the presence (+ IL-17) or absence of recombinant IL-17 (20 ng/ml). Data are representative of three experiments. (d) Immunoblot analysis (left) of T_H1 effector cells at day 4 generated in vitro in the presence (+) or absence (−) of recombinant IL-17 (rIL-17) and/or IL-17-neutralizing antibody (α-IL-17). Right, differences in expression of phosphorylated STAT1 (p-STAT1), normalized to actin and presented relative to that of day-4 T_H1 cells cultured alone. Data are representative of three experiments.

Figure 3

IL-17 suppresses the induction of T-bet in maturing T_H1 cells. (a) Real-time PCR analysis of Tbx21 in T_H0 effector cells or in T_H1 effector cells polarized in vitro in the presence (+ IL-17) or absence of 2 ng/ml or 20 ng/ml (in parentheses) of recombinant mouse IL-17 after 48 h or 96 h of culture, normalized to Hprt1 expression and presented as ‘fold increase’ relative to that of T_H0 cells. (b) Immunoblot analysis of T-bet in the 96-hour cultures in a. Actin, loading control. Data are representative of three or more independent experiments (a,b). (c) Proliferation of cells in T_H1 effector cultures at day 4, differentiated in the presence or absence of 2 ng/ml or 20 ng/ml (in parentheses) of recombinant mouse IL-17, assessed by incorporation of [³H]thymidine. Data are representative of two independent experiments. (d) Tbx21 expression in day-4 T_H1 effector cells generated in vitro from wild-type or Il17a^−/− naive CD4⁺ CD45RB^hi T cells, normalized to Hprt1 expression and presented as ‘fold increase’ relative to that of T_H0 cells. Data are representative of three experiments. (e) Enzyme-linked immunosorbent assay of the release of IFN-γ from naive wild-type or Il17a^−/− CD4⁺ CD45RB^hi T cells polarized for 5 d in T_H1-type conditions before overnight restimulation. Data are representative of three experiments. (f) Proliferation of the cells in d, assessed by incorporation of [³H]thymidine. Data are representative of three experiments.

Figure 4

Il17ra^−/− CD45RB^hi donor T cells elicit an accelerated wasting disease in Rag1^−/− recipients. (a) Composite weight-loss curves of recipients of adoptive transfer of wild-type or Il17ra^−/− CD45RB^hi CD4⁺ T cells (number of mice with weight loss, Table 1). P ≤ 0.01, time; P ≤ 0.01, experimental group; P ≤ 0.01, time-group interaction (repeated-measures ANOVA). *, P < 0.05, **, P ≤ 0.01, days 35–60 after transfer of cells (post-hoc t-test); means at day 35: recipients of wild-type T cells (n = 8), 108.1; recipients of Il17ra^−/− T cells (n = 8), 99.59. Data are representative of two independent experiments with similar results (error bars, s.d.). (b) Hematoxylin and eosin–stained sections of Rag1^−/− recipient colons obtained at day 28 after adoptive transfer Il17a^−/− or Il17ra^−/− T cells. Scale bars, 500 μm. Results are representative of two experiments. (c) Histological quantification of edema and inflammation in colons from Rag1^−/− recipient mice, obtained at day 28 after adoptive transfer of Il17a^−/− or Il17ra^−/− T cells, presented as ‘severity scores’. There were no statistically significant differences among the groups for any criteria. Data are representative of two experiments (error bars, s.e.m.).