Anti-inflammatory role of IL-17 in experimental autoimmune uveitis

Abstract

Previous studies have shown that IL-17 is a strong proinflammatory cytokine and that IL-17-producing autoreactive T cells play a major role in the pathogenesis of autoimmune diseases. In a previous study, we showed that injection of experimental autoimmune uveitis-susceptible mice with anti-IL-17 Abs blocked subsequent disease development. To determine whether administration of IL-17 to experimental autoimmune uveitis-susceptible Lewis rats and B10RIII mice injected with disease-inducing peptides enhanced disease susceptibility, we injected the recipient animals with various doses of human rIL-17 (hIL-17). Unexpectedly, the treated animals showed significant amelioration of disease; in addition, both the intensity of the autoreactive response and cytokine production by the autoreactive T cells induced by immunization with uveitogenic peptides were significantly decreased. Our results show that IL-17 has anti-inflammatory activity and that this cytokine can suppress the development of autoimmune disease.

FIGURE 1

Injection of Lewis rats and B10RIII mice with small doses of IL-17 significantly ameliorates the development of EAU. A and B, R16-immunized Lewis rats received two i.p. injections of hIL-17 (3 µg/injection) on days 4 and 7 (A) or days 10 and 13 (disease onset) p.i. (B). Disease severity was observed using a slit lamp microscope and graded using previously described criteria. The mean score at each time point is the average for six animals. C and D, IRBP_161–180-immunized B10RIII mice received three i.p. injections of IL-17 (1 µg/injection) on days 0, 4, and 8. Disease severity was monitored by fundoscopy on days 9, 11, and 13 (C) and pathologic examination was performed on day 13 (D). E, T cells isolated at day 13 p.i. from the draining lymph nodes and spleens of R16-immunized Lewis rats with or without IL-17 treatment on days 4 and 7 were enriched by passage through nylon wool and restimulated in vitro with R16 (10 µg/ml) presented by irradiated syngeneic spleen APCs. After 3 days, activated T cell blasts were separated on a Ficoll gradient and injected (3 × 10⁶ cells, i.v.) into naive Lewis rats. Clinical signs were observed by slit lamp biomicroscopy and scored as described in Materials and Methods. The results shown are the mean ± SD for three animals in a single experiment, which was repeated three times with similar results.

FIGURE 2

hIL-17-treated R16-immunized rats have a decreased T cell response to the immunizing peptide. A, Two groups of R16-immunized rats were treated with or without hIL-17 on days 4 and 7, then, on day 10, enriched splenic T cells were pooled and tested in 96-well plates for their proliferative response in the presence of graded doses of R16 and irradiated APCs from naive rats. B and C, The responder T cells from A from hIL-17-treated (B) or nontreated (C) R16-immunized rats were incubated with irradiated spleen cells from either naive or IL-17-treated rats and their proliferative response to Ag stimulation was measured. The proliferative response is expressed as the mean cpm ± SD of triplicate determinations and the result is representative of those for three separate experiments; the SD was <15%. Values of p < 0.05 were labeled with * and those <0.01 were labeled with **.

FIGURE 3

IL-17 treatment preferentially inhibits the activation of IFN-γ⁺ IRBP-specific T cells. A, Enriched T cells from the spleen and draining lymph nodes of R16-immunized rats with or without IL-17 treatment were incubated with the immunizing peptide for 2 days in the presence of syngeneic APCs and the activated T cells separated by Ficoll gradient centrifugation and continuously cultured in cytokine-free medium for 2, 4, or 6 days, then intracellular expression of IFN-γ and IL-17 was assessed by FACS analysis. B, Enriched T cells from spleen and draining lymph nodes of R16-immunized rats with or without IL-17 treatment were incubated with the immunizing peptide for 2 days in the presence of syngeneic APCs, then IFN-γ and IL-17 production was measured by ELISA. C, Enriched T cells from R16-immunized rats not treated with IL-17 were stimulated for 48 h with R16 and APCs in the presence of graded amounts (0, 3, or 30 ng/ml) of hIL-17, then the activated T cells in each group were intracellularly stained with anti-IFN-γ and anti-IL-17 Abs, followed by FACS analysis. The results shown are representative of those in three experiments.

FIGURE 4

IL-17 has an in vitro inhibitory effect on the activation of IRBP-specific T cells. T cells (4 × 10⁵/well) prepared from the spleens of R16-immunized rats (9 days p.i.) were stimulated in 96-well plates with R16 (10 µg/ml) and APCs for 48 h in the presence of graded doses of recombinant hIL-17 (A), LPS (B), or mouse IFN-γ (C) and T cell proliferation was assessed at 48 h by [³H]thymidine uptake. Values of p < 0.05 were labeled with * and those <0.01 were labeled with **.

FIGURE 5

hIL-17 treatment results in enhanced regulatory T cell activity. A, Rats treated with IL-17 express increased numbers of Foxp3⁺ T cells in the periphery and in the inflamed eye. Lewis rats were immunized with R16 with or without IL-17 treatment (two i.p. injections of 3 µg/injection on days 4 and 7), then splenic T cells and eye-infiltrating cells were prepared on day 10 p.i. and the number of CD4⁺Foxp3⁺ T cells were assessed by dual staining with anti-CD4 and anti-Foxp3 Abs and FACS analysis. B, Splenic T cells from hIL-17-treated rats have increased suppressor activity. Two groups (n = 4) of rats were immunized with a pathogenic dose of R16 (50 µg/rat) and one group was also injected i.p. with 50 × 10⁶ splenic T cells isolated on day 15 from R16-immunized rats treated with IL-17 on days 4 and 7 or from R16-immunized rats without IL-17 treatment (control). Disease was monitored and scored. The results shown are the mean ± SD from one representative experiment of two.

FIGURE 6

Protective effect of rat IL-17 on rat EAU. R16-immunized Lewis rats received two i.p. injections of rIL-17 (3 µg/injection) on days 4 and 7. Disease severity was observed using a slit lamp microscope and graded using previously described criteria. The results shown are the mean ± SD for three animals in a single experiment, which was repeated two times with similar results.

FIGURE 7

hIL-17 induces chemokine production by mouse macrophages. Mouse macrophages were cultured from bone marrow cells by medium containing M-CSF (61, 62). Monolayers of macrophages cultured in 24-well plate were exposed to graded doses of mouse IL-17 (mIL-17) or hIL-17 as indicated. Forty-eight hours later, the culture supernatants were assessed for production of CCL2 using ELISA. Values of p < 0.05 were labeled with * and those <0.01 were labeled with **.