Interleukin-22 (IL-22) production by pulmonary Natural Killer cells and the potential role of IL-22 during primary influenza virus infection

Abstract

We set out to test the hypothesis that interleukin-22 (IL-22), a cytokine crucial for epithelial cell homeostasis and recovery from tissue injury, would be protective during influenza virus infection. Recent studies have identified phenotypically and functionally unique intestinal NK cells capable of producing the cytokine IL-22. Unlike gut NK cells that produce IL-22, the surface phenotypes of lung NK cells were similar to those of spleen NK cells and were characteristically mature. With mitogen stimulation, both single and double IL-22- and gamma interferon (IFN-gamma)-producing lung NK cells were detected. However, only the IL-22(+) IFN-gamma(-) lung NK subset was observed after stimulation with IL-23. IL-23 receptor (IL-23R) blocking dramatically inhibited IL-22 production, but not IFN-gamma production. Furthermore, we found that NK1.1(+) or CD27(-) lung NK cells were the primary sources of IL-22. After influenza virus infection, lung NK cells were quickly activated to produce both IFN-gamma and IL-22 and had increased cytotoxic potential. The level of IL-22 in the lung tissue declined shortly after infection, gradually returning to the baseline after virus clearance, although the IL-22 gene expression was maintained. Furthermore, depletion of NK cells with or without influenza virus infection reduced the protein level of IL-22 in the lung. Anti-IL-22 neutralization in vivo did not dramatically affect weight loss and survival after virus clearance. Unexpectedly, anti-IL-22-treated mice had reduced virus titers. Our data suggest that during primary respiratory viral infection, IL-22 seems to a play a marginal role for protection, indicating a differential requirement of this cytokine for bacterial and viral infections.

FIG. 1.

Lung NK cells are phenotypically mature. Freshly isolated lung and spleen cells were stained and gated for CD3⁻ NKp46⁺ NK cells and analyzed for the expression of a panel of markers that are absent (A), downregulated (B), or upregulated (C) on gut NK cells compared to on spleen NK cells (24, 31). In each overlay, gray-filled and unfilled histograms represent the expression of individual markers on spleen and lung NK cells, respectively. The numbers on each plot represent the frequency of each molecule from gated NK cells. Data are representative of the results for three independent experiments.

FIG. 2.

Production of IL-22 by lung NK cells in vitro. Lung cells were isolated and stimulated with either PMA-ionomycin (PMA/Iono) or IL-23 in the absence (A) or presence (B) of anti-IL-23R blocking antibody as described in the text. Gated live CD3⁻ NKp46⁺ lung NK cells were analyzed for intracellular IFN-γ and IL-22. Data are representative of the results for at least three independent experiments.

FIG. 3.

IL-22 production by lung NK cell subsets. Lung cells were isolated and stimulated with either PMA-ionomycin or IL-23. Live CD3⁻ NKp46⁺ lung NK cells were gated for analysis of NK1.1 and IFN-γ or IL-22 (A). Alternatively, lung NK cells were analyzed for CD27 expression after PMA-ionomycin stimulation in comparison with isotype control (shaded histogram) (B), and CD27^+/− subsets were further examined for IFN-γ and IL-22 expression (C). Data are representative of the results for two independent experiments.

FIG. 4.

Production of IL-22 by lung NK cells in vivo. Mice were infected with 5 PFU influenza PR8 virus, and lungs were collected at different time points. Lung single-cell suspensions were purified and counted before surface and intracellular IFN-γ and IL-22 staining was performed and analyzed (A). The absolute number of lung NK cells was obtained by multiplying the total number of lung lymphocytes counted with the percentage of NK cells (B). At each time point, three to five mice were used. Data are representative of the results for two independent experiments.

FIG. 5.

Cytotoxic potential of IL-22-producing lung NK cells. Mice were infected with 5 PFU influenza PR8 virus determined by staining and tissues were collected at different time points for analysis. NK cell cytotoxicity was measured with the expression of CD107a in both lung (A) and spleen (B) samples immediately after cell preparation by FACS. IL-22 production by lung NK cells was also determined by staining and analyzed together with CD107a (C). Data are representative of the results for two independent experiments.

FIG. 6.

Lung IL-22 expression after influenza virus infection. Mice were infected with 5 PFU PR8 influenza virus or 3 × 10⁵ 50% egg infective doses (EID₅₀) of X31 virus as described in Material and Methods. (A) Kinetics of IL-22 level in the lung after influenza virus infection. At each time point as indicated in the figure, three mice were used. (B) Lung IL-22 gene expression after influenza virus infection in comparison to noninfection control. Lung total RNA was extracted and RT-PCR performed as described, and the relative amount of IL-22 was obtained after normalization with the HRPT housekeeping gene. Data are averages for three mice at each time point analyzed. (C) IL-22 levels in the lungs of uninfected mice after anti-NK1.1 antibody depletion. Mice were injected with anti-NK1.1 or isotype control antibodies as described in the text, and lungs were collected at different times for IL-22 detection. Data presented are average values for three mice for each group. (D) IL-22 levels in the lungs of PR8-infected mice after anti-NK1.1 antibody depletion. Mice were depleted with anti-NK1.1 antibodies 12 h before infection, and lung samples were collected for IL-22 measurement at the indicated time. Data presented are average values for six mice for each group.

FIG. 7.

Neutralization of IL-22 after influenza virus infection. Mice were infected with 5 PFU PR8 influenza virus and treated with either anti-IL-22 or isotype control antibodies as described in Materials and Methods. (A) Virus titers of lung and trachea samples collected from anti-IL-22- or isotype control-treated animals at day 5 of infection. Data are averages of the results for three mice in each group analyzed. (B) Change in body weight of infected animals with anti-IL-22 or isotype control treatment was calculated as the percentage of initial weight. Data presented are average values for five mice for each group and representative of the results for two independent experiments.