Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22

Abstract

The interleukin (IL) 17 family of cytokines has emerged to be critical for host defense as well as the pathogenesis of autoimmune and autoinflammatory disorders, and serves to link adaptive and innate responses. Recent studies have identified a new subset of T cells that selectively produce IL-17 (Th17 cells; Bettelli, E., T. Korn, and V.K. Kuchroo. 2007. Curr. Opin. Immunol. 19:652-657; Kolls, J.K., and A. Linden. 2004. Immunity. 21:467-476), but the regulation of IL-17 production by innate immune cells is less well understood. We report that in vitro stimulation with IL-23 induced IL-17 production by recombination activating gene (Rag) 2(-/-) splenocytes but not Rag2(-/-) common gamma chain(-/-) splenocytes. We found that a major source of IL-17 was CD4(+)CD3(-)NK1.1(-)CD11b(-)Gr1(-)CD11c(-)B220(-) cells, a phenotype that corresponds to lymphoid tissue inducer-like cells (LTi-like cells), which constitutively expressed the IL-23 receptor, aryl hydrocarbon receptor, and CCR6. In vivo challenge with the yeast cell wall product zymosan rapidly induced IL-17 production in these cells. Genetic deletion of signal transducer and activator of transcription 3 reduced but did not abrogate IL-17 production in LTi-like cells. Thus, it appears that splenic LTi-like cells are a rapid source of IL-17 and IL-22, which might contribute to dynamic organization of secondary lymphoid organ structure or host defense.

Figure 1.

IL-23 and zymosan induce IL-17 production in a population of non–B, non–T cells. (A) WT or Rag2^−/− splenocytes were cultured in the presence of the indicated cytokines for 48 h, and IL-17A in culture supernatants was measured by ELISA. The data are means ± SD from duplicate cultures and are representative of four independent experiments (n = 8). **, P < 0.01. (B) WT, Rag2^−/−, or Rag2^−/−γc^−/− splenocytes were cultured in the presence of IL-23 alone, and IL-17A in culture supernatants was measured by ELISA. The data are means ± SD from duplicate cultures and are representative of three independent experiments (n = 6). *, P < 0.05; **, P < 0.01. (C) Sera and splenocytes were harvested from Rag2^−/− mice after injection of 5 mg zymosan or PBS (Control). (left) IL-17A in sera was measured by ELISA. (right) The relative expression levels (/18S) of IL-17A mRNA were analyzed by quantitative PCR (q-PCR) and are depicted as fold induction relative to cells 1 h after treatment with zymosan. The data are means ± SD of results from two mice per group per time point and are representative of two independent experiments. ND, not detected.

Figure 2.

CD4⁺CD3⁻lineage⁻ cells produce IL-17. Rag2^−/− splenocytes were cultured in the absence (Control) or presence of IL-23 for 24 h. The proportion of IL-17A–producing CD3⁻ cells was evaluated by intracellular staining. (A) NK1.1⁺ versus IL-17A⁺ cells gated on CD3⁻ cells are shown. (B) IL-17A⁺ versus CD3⁺ cells (nongated; left) and IL-17A⁺ versus CD4⁺ cells gated on CD3⁻ cells (right) are shown. Data are representative of three independent experiments.

Figure 3.

Isolated CD4⁺CD3⁻CD11c⁻B220⁻ LTi-like cells produce IL-17 and constitutively express IL-23R, RORγt, AHR, and CCR6. (A) Isolated CD4⁺CD3⁻CD11c⁻B220⁻ LTi-like subsets from Rag2^−/− splenocytes (top) were cultured in the absence (Control) or presence of IL-23 (or with PMA/Iono) for 24 h, and IL-17A in culture supernatants was measured by ELISA (bottom). Data are means ± SD from duplicate cultures and are representative of two independent experiments. (B) Stat3^fl/fl or Stat3^fl/fl; MMTV-Cre splenocytes were cultured in the absence (Control) or presence of IL-23 for 24 h. The proportion of IL-17A–producing LTi-like cells was evaluated by intracellular staining. Data are means ± SD from duplicate cultures and are representative of four independent experiments (n = 8). *, P < 0.05. (C) Total RNA was prepared from LTi-like cells isolated from Rag2^−/− spleens. The relative expression levels (/18S) of the indicated mRNA was analyzed by q-PCR and are depicted as fold induction relative to fresh B cells (or fresh naive T cells for AHR mRNA expression). Data are representative of three independent experiments. M-T, memory T cells; N-T, naive T cells. (D) The expression of CCR6 on LTi-like cells of WT splenocytes was analyzed by FACS. The shaded histogram indicates staining with isotype-matched control antibodies. The continuous line histogram indicates the surface expression level of CCR6 on LTi-like cells. Data are representative of three independent experiments. ND, not detected.

Figure 4.

Zymosan induces IL-17 production in splenic LTi-like cells in vivo. (left) Intracellular staining of IL-17A was performed with in vitro conventional staining (top) or in vivo staining (bottom). For conventional staining, Rag2^−/− mice were challenged with PBS (Control) or 12.5 mg zymosan i.p. Splenocytes were isolated 2 h later and stimulated with PMA/Iono and BFA for 2 h in vitro. The proportion of IL-17A–producing LTi-like cells was evaluated by intracellular staining. For the in vivo staining, Rag2^−/− mice were injected with PBS (Control) or 12.5 mg zymosan i.p. together with 0.25 mg BFA i.v. (reference 22). The proportion of IL-17A–producing LTi-like cells in spleens was directly evaluated by intracellular staining. (right) The mean values (horizontal bars) for IL-17A–producing LTi-like cells were calculated for in vitro staining (top, n = 6) and in vivo staining (bottom, n = 6). Data are representative of three (top) or two (bottom) independent experiments. ***, P < 0.001.

Figure 5.

LTi-like cells produce high levels of IL-22. (A) Sera and splenocytes were harvested from Rag2^−/− mice 3 h after injection of PBS (Control) or 5 mg zymosan. (left) IL-22 in sera was measured by ELISA. (right) The relative expression levels (/18S) of IL-22 mRNA were determined by q-PCR and are depicted as fold induction relative to control cells. The data are means ± SD from two mice and are representative of two independent experiments. (B) WT or Rag2^−/− splenocytes or isolated LTi-like cells were cultured in the presence of IL-23 for 24 h, and IL-22 in culture supernatants was measured by ELISA. The data are means ± SD from duplicate cultures and are representative of two independent experiments.