IL-6 promotes NK cell production of IL-17 during toxoplasmosis

Abstract

Previous studies have implicated T cell production of IL-17 in resistance to Toxoplasma gondii as well as the development of immune-mediated pathology during this infection. Analysis of C57BL/6 and C57BL/6 RAG(-/-) mice challenged with T. gondii-identified NK cells as a major innate source of IL-17. The ability of soluble Toxoplasma Ag to stimulate NK cells to produce IL-17 was dependent on the presence of accessory cells and the production of IL-6, IL-23, and TGF-beta. In contrast, these events were inhibited by IL-2, IL-15, and IL-27. Given that IL-6 was one of the most potent enhancers of NK cell production of IL-17, further studies revealed that only a subset of NK cells expressed both chains of the IL-6R, IL-6 upregulated expression of the Th17-associated transcription factor RORgammat, and that IL-6(-/-) mice challenged with T. gondii had a major defect in NK cell production of IL-17. Together, these data indicate that many of the same cytokines that regulate Th17 cells are part of a conserved pathway that also control innate production of IL-17 and identify a major role for IL-6 in the regulation of NK cell responses.

Figure 1. NK cells are an early source of IL-17 during toxoplasmosis

A) Kinetics of IL-17A production in the serum from wild-type C57BL/6 (n=3) and RAG^-/- mice (n=3) infected with 20 cysts from the ME49 strain of T. gondii measured by ELISA. Data shown from a representative experiment of three performances. B) Flow cytometry of IL-17A versus IFN-γ from splenocytes isolated from naive C57BL/6 mice (n=3) or from mice infected for five days with 20 cysts from the ME49 strain of T. gondii (n=3). Plots are gated on NK1.1⁺ CD3^- or CD3⁺ cells. Numbers in quadrants indicate the frequency of cells in each. Representative profiles are from three individual experiments.

Figure 2. Depletion of NK cells decreases IL-17A production during T. gondii infection

A) Serum from wild-type C57BL/6 and RAG^-/- mice (n=4) treated in vivo with α-asialo GM1 to deplete NK cells (50 μg ip 1 day prior to infection and thereafter every 3 days in a volume of 0.2 ml for the duration of the experiment) was used to measure IL-17A by ELISA. Data shown from a representative experiment of five performances. *p<0.01 and **p<0.01. B and C) ELISA of IL-17A in the supernatants of splenocytes isolated at day 5 post-infection from wild-type C57BL/6 mice (n =4) or RAG^-/- mice (n=3) treated with or without a-asialo GM1 stimulated for 20 h in the presence or absence of STAg. Data shown from a representative experiment of three performances. *p<0.01.

Figure 3. Factors that regulate NK cell production of IL-17

A) Levels of IL-17A measured by ELISA in the supernatant from uninfected or infected (day 5 pi) RAG^-/- mice (n=3) cultured with media or STAg for 20 h. B) IL-17A levels measured by ELISA in supernatants from cultured splenocytes isolated from infected RAG^-/- mice (day 5 pi) and NK cells purified from infected RAG^-/- mice, stimulated with or without STAg for 20 h. C) ELISA for IL-17A production was performed using supernatants from infected RAG^-/- mice cultured with media alone or STAg in the presence or absence of α-IL-6, α-IL-12/23p40, α-IL-23p19 or α-TGF-β for 20 h. The data represent means (±SD) from 3 independent experiments. *p<0.01 and **p<0.01. D) Supernatants of splenocytes from uninfected or infected RAG^-/- mice (n= 3) cultured with media or IL-6, IL-23, TGF-β or the combination of IL-6, IL-23 and TGF-β for 20 h. The data represent means (±SD) from 3 independent experiments. E) IL-17A ELISA was performed with the supernatants of purified NK cells from uninfected or infected RAG^-/- mice cultured for 20 h with media alone or in the presence of IL-6, IL-23, TGF-β or all three cytokines together. The data represents two independent experiments. F) Supernatants from cultured splenocytes derived from infected RAG^-/- mice (n=3) cultured with media, IL-6 or IL-6 plus IL-2 (ν), IL-15 (Δ) or IL-27 (O) at different concentrations for 20 h. The data represent means (±SD) from three independent experiments.

Figure 4. Examination of IL-6R expression NK cells during toxoplasmosis

A) Flow cytometry of gp130 and IL-6 receptor alpha on gated NK1.1⁺ CD3^- cells from splenocytes of uninfected and infected RAG^-/- mice (n=5). Representative profiles are shown. Numbers in quadrants indicate the frequency of cells in each. The data represents three independent experiments. B) Frequency of IL-6Rα positive cells from splenocytes of uninfected or infected RAG^-/- mice (n=5). Data from a representative experiment. C) Flow cytometry of IL-6Rα and IL-12Rβ2 on NK1.1⁺CD3^- cells from splenocytes of uninfected or infected RAG^-/- mice. Representative profiles from 5 mice are shown. Numbers in quadrants indicate the frequency of cells in each.

Figure 5. Expression of the transcription factor RORyt and T-bet in NK cells

A) Total RNA was prepared from purified NK cells from splenocytes of uninfected and infected RAG^-/- mice (n=3) cultured with media, IL-6, IL-23 or TGF-β alone for 20 h. The mRNA expression was measure by quantitative real-time PCR. The data is represented as fold induction relative to the house-keeping gene β- actin. Data shown representative of 3 independent experiments. B) Flow cytometry of RORγt and T-bet gated on NK1.1⁺ CD3^- cells from splenocytes of uninfected or 5 days post-infection infected RAG^-/- mice (n=6). Numbers in quadrants indicate the frequency of cells in each. The data are representative of four experiments. C) Flow cytometry of IL-6Rα expression on RORyt⁺ or T-bet⁺ NK1.1⁺ cells (dotted line) or RORγt^- or T-bet^- NK1.1⁺ cells (black line). The shaded histogram represents the isotype control. Representative profiles are shown. The data represents four independent experiments.

Figure 6. IL-6 has a critical role in the production of IL-17 by NK cells

A) Serum from wild-type C57BL/6 or IL-6^-/- mice (n=4) was used to measure the production of IL-17A by ELISA on days 3, 5 and 7 post-infection. Data shown are from one representative experiment. B) Flow cytometry of intracellular IL-17 gated on NK1.1⁺ CD3^- cells or CD3⁺ T cells from splenocytes of day six infected wild-type C57BL/6 mice or IL-6^-/- mice (n=5). Numbers in quadrants indicate the frequency of cells in each. Representative profiles are shown. The data presented are from three independent experiments.