IL-17A produced by gammadelta T cells plays a critical role in innate immunity against listeria monocytogenes infection in the liver

Abstract

IL-17A is originally identified as a proinflammatory cytokine that induces neutrophils. Although IL-17A production by CD4(+) Th17 T cells is well documented, it is not clear whether IL-17A is produced and participates in the innate immune response against infections. In the present report, we demonstrate that IL-17A is expressed in the liver of mice infected with Listeria monocytogenes from an early stage of infection. IL-17A is important in protective immunity at an early stage of listerial infection in the liver because IL-17A-deficient mice showed aggravation of the protective response. The major IL-17A-producing cells at the early stage were TCR gammadelta T cells expressing TCR Vgamma4 or Vgamma6. Interestingly, TCR gammadelta T cells expressing both IFN-gamma and IL-17A were hardly detected, indicating that the IL-17A-producing TCR gammadelta T cells are distinct from IFN-gamma-producing gammadelta T cells, similar to the distinction between Th17 and Th1 in CD4(+) T cells. All the results suggest that IL-17A is a newly discovered effector molecule produced by TCR gammadelta T cells, which is important in innate immunity in the liver.

FIGURE 1

Expression and function of IL-17A in the liver of L. monocytogenes-infected mice. A, Wild-type (WT) mice were infected via the intraperitoneal route with viable L. monocytogenes. IL-17A expression of liver mononuclear cells was analyzed by real time PCR method. The data are expressed as the increase in expression level compared with that before infection. The result shown is representative of three independent analyses.*, p < 0.05 compared with day 0. WT or IL-17A^−/− mice were infected with viable L. monocytogenes, and bacterial burden in the liver was determined on day 1 and 5 after the infection (B), or liver mononuclear cells (MNC) were collected, and counted (C, left panel). The cells were stained with mAb against leukocyte surface molecules and analyzed by FCM. The ratio of neutrophils (Gr1⁺CD11b⁺) was determined and the absolute number of the cell was calculated (C, right panel). D, WT (open bar) or IL-17A^−/− (filled bar) mice were inoculated with heat-killed L. monocytogenes, liver MNC were collected on day 5 of infection, and neutrophil number was determined.*, p < 0.05 compared with C57BL/6 mice. The analyses were conducted more than three times, and representative data is shown in this figure.

FIGURE 2

Pivotal role of IL-17A in the formation of granulomatous lesions at an early stage of L. monocytogenes infection. Mice were infected with L. monocytogenes and were sacrificed at day 5 of infection for histological examination. A, Sections of the liver of wild-type (WT) (upper panels) or IL-17A^−/− (lower panels) mice were examined by H & E staining (original magnification ×40 for left panels and ×200 for right panels). B, Sections of WT (upper panels) or IL-17A^−/− (lower panels) were examined by confocal laser scanning microscopy with CD11b mAb (green, left panels) and CD3 mAb (red, middle panels) staining (original magnification ×200). Merged image of CD11b and CD3 staining are shown in the right panels. C, Kinetics of serum ALT levels after L. monocytogenes infection of WT (○) or IL-17A^−/− (■) mice is shown to indicate the degree of liver injury after the infection. *, p < 0.05 compared with wild-type mice. The data shown are representative of three independent experiments with more than five mice in each group.

FIGURE 3

Identification of IL-17A producing cells in the liver of L. monocytogenes-infected mice. Wild-type mice were infected with L. monocytogenes, and liver mononuclear cells were isolated. A, The liver mononuclear cells were analyzed on day 5 of the infection by FCM to identify IL-17A producing cells in the lymphocyte subsets, including TCR γδ T cells (TCR Cδ⁺), αβ T cells (TCR Cβ⁺), NK cells, NK T cells (NK1.1⁺), CD4⁺ T cells, and CD8⁺ T cells. B, The liver mononuclear cells were prepared on day 1 and 5 of the infection, and ratio and absolute number of IL-17-producing TCR Cβ⁺ or TCR Cδ⁺ T cells were analyzed. *, p < 0.05. C, The liver mononuclear cells were prepared on day 5 of the infection and sorted into TCR αβ⁺ or TCR γδ⁺ T cells. The cells were cultured in the culture dishes coated with anti-CD3 mAb (10 μg/ml) or not coated with mAb. Culture supernatants were collected 24 h later and IL-17A was detected by ELISA. D, TCR γδ T cells were sorted from the liver mononuclear cells on day 5 after the infection and analyzed by FCM for their expression of IL-17A and IFN-γ. The experiments were repeated more than three times and a representative example is shown.

FIGURE 4

Restricted TCR Vγ repertoire of IL-17A-producing TCR γδ T cells. A, Wild-type (WT) mice were infected with L. monocytogenes, liver mononuclear cells on day 5 were isolated, and TCR γδ T cells were enriched using the MACS system. The TCR Vγ and Vδ repertoire of the γδ T cells was analyzed by PCR and agarose gel electrophoresis. B, IL-17A-producing TCR γδ T cell subpopulations were analyzed in the liver mononuclear cells on day 5 of L. monocytogenes-infected WT mice by FCM using anti-Vγ mAb after analysis gate was set on TCR Cδ⁺ cells (lower panels). The data shown are representative of two or three independent experiments. C, The liver mononuclear cells were collected on day 1 or 5 after the infection and analyzed for Vγ expression and IL-17 production as in B. Three samples consisting of pooled liver mononuclear cells of two to three mice were analyzed and mean and SD was plotted. D, IL-17A production of TCR γδ T cells was also analyzed in the liver mononuclear cells of L. monocytogenes-infected Vγ4/6^−/− mice on day 5 by FCM after analysis gate was set on TCR Cδ⁺ cells. E, Sections of the liver of WT (upper panels) or Vγ4/6^−/− (lower panels) mice were examined by H & E staining (original magnification ×40 for left panels and ×200 for right panels).

FIGURE 5

TCR γδ T cells producing IL-17A contribute to early protection against L. monocytogenes infection. Irradiated IL-17A^−/− mice were transplanted with BM cells from wild-type (WT), IL-17A^−/−, TCRCδ^−/−, and both IL-17A^−/− and TCRCδ^−/− mice (BM chimeras). BM chimeras were infected i.p. with L. monocytogenes 35 days after the transfer. The bacterial burden was determined on day 5 of the infection. *, p ≦ 0.05. The data shown is a representative of two independent experiments.