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. 2016 Jul;20(7):1339-51.
doi: 10.1111/jcmm.12821. Epub 2016 Mar 29.

Natural killer cells regulate Th1/Treg and Th17/Treg balance in chlamydial lung infection

Jing Li  1 , Xiaojing Dong  1 , Lei Zhao  2 , Xiao Wang  3 , Yan Wang  3 , Xi Yang  1   4 , Hong Wang  1 , Weiming Zhao  1
Affiliations

Natural killer cells regulate Th1/Treg and Th17/Treg balance in chlamydial lung infection

Jing Li et al. J Cell Mol Med. 2016 Jul.

Abstract

Natural killer (NK) cell is an important component in innate immunity, playing a critical role in bridging innate and adaptive immunity by modulating the function of other immune cells including T cells. In this study, we focused on the role of NK cells in regulating Th1/Treg and Th17/Treg balance during chlamydial lung infection. We found that NK cell-depleted mice showed decreased Th1 and Th17 cells, which was correlated with reduced interferon-γ, interleukin (IL)-12, IL-17 and IL-22 production as well as T-bet and receptor-related orphan receptor gamma t expression compared with mice treated with the isotype control antibody. In contrast, NK cell depletion significantly increased Treg in cell number and related transcription factor (Foxp3) expression. The opposite trends of changes of Th1/Th17 and Treg led to significant reduction in the Th1/Treg and Th17/Treg ratios. The data implicate that NK cells play an important role in host defence against chlamydial lung infection, mainly through maintaining Th1/Treg and Th17/Treg balance.

Keywords: Chlamydia; Th1/Treg; Th17/Treg; immunoregulation; natural killer cells.

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Figures

Figure 1
Figure 1
NK cell‐depleted mice show more severe disease following Chlamydia muridarum lung infection. BALB/c mice received tail‐vein injection (i.v) with 20 μl anti‐asialo GM1 [Cm/NK (‐)] or control normal rabbit IgG (Cm/isotype) antibody in 50 μl PBS 1 day before and 1 day after intranasal infection (i.n) with C. muridarum (1 × 103 IFU), then every 3 or 5 days were injected with 10 μl anti‐asialo GM1 or isotype until the end of the test. The uninfected control mice were administered with isotype control antibody (noninfected/isotype) following the same schedule. (A) The schematic of NK cell deleption method. (B) Flow cytometric images of NK cell (CD3− DX5+) in lung mononuclear cells from mice treated with anti–asialo GM1 or isotype antibody. (C) The bw changes in the noninfected/isotype, Cm/isotype and Cm/NK (‐) groups. (D) Mice were killed at day 3, day 6, day12 and day 22 after infection, and the lung tissues were homogenized and live chlamydial loads were measured as described in Materials and methods. (E) The lung tissue sections were stained with haematoxylin & eosin for histological analysis under light microscopy at ×200 magnification. (F) The score of tissue inflammatory grades were analysed as described in Materials and methods. At least three independent experiments with 4–5 mice in each group were performed, with one representative experiment is shown. Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01 versus isotype control.
Figure 2
Figure 2
Decreased type 1 immune response in NK cell‐depleted mice following Chlamydia muridarum lung infection. BALB/c mice were treated with anti‐asialo GM1 or isotype antibodies and infected with C. muridarumas described in the legend to Figure 1, and killed at day 6 or day 12 after infection. (A–C) ELISA of IFN‐γ protein expression in splenocytes, lung mononuclear cells and mediastinal lymph node cells (LN) cultured with heat‐inactivated C. muridarum EBs (105 IFU/ml) for 72 hrs. (D) ELISA of IFN‐γ production in serum. (E–G) Summary of intracellular cytokine staining data of IFN‐γ‐producing CD4+ T cells in the different organs. At least three independent experiments with 4–5 mice in each group were performed, with one representative experiment is shown. Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 versus isotype control.
Figure 3
Figure 3
Reduced IL‐17 and IL‐22 expression in NK cell‐depleted mice with chlamydial lung infection. Mice were treated as described in the legend to Figure 1, and killed at day 6 or day 12 after infection, IL‐17A (A–C) and IL‐22 protein levels in 72 hrs culture supermants of splenocytes, lung mononuclear cells, mediastinal lymph node cells (D–F) and in serum (G) were determined by ELISA. At least 3 independent experiments with 4–5 mice in each group were performed, with one representative experiment is shown. Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Reduction in IL‐17+ CD4+T cells response in NK cell‐depleted mice during chlamydial lung infection. Splenocytes, lung mononuclear cells and mediastinal lymph node cells from each group were prepared at days 6 or 12 after infection.IL‐17‐producing CD4+ T cells were quantified by intracellular cytokine staining as described in Materials and methods. (A) Representative flow cytometric images of IL‐17‐producing CD4+ T cells. Summary of the percentage (B–D) and absolute number (E–G) of IL‐17‐producing CD4+ T cells. At least three independent experiments with 4–5 mice in each group were performed, and one representative experiment is shown. Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01.
Figure 5
Figure 5
Increased CD4+ CD25+ Foxp3+ regulatory T‐cell responses in NK cell‐depleted mice. Splenocytes, lung mononuclear cells and mediastinal lymph node cells were isolated at day 6 or day 12 after Chlamydia muridarum infection. (A) Representative flow cytometric images of CD4+ CD25+ Foxp3+ T cells in the different organs. Summary of the percentage (B–D) and absolute number (E–G) of CD4+ CD25+ Foxp3+ T cells. At least three independent experiments with 4–5 mice in each group were performed, with one representative experiment is shown. Data are mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
NK‐cell depletion decreases Th1, Th17 transcription factor (T‐bet, RORγT) but increases Treg and Th2 transcription factor (Foxp3,GATA3). Mice were treated and infected as described in the legend to Figure 1, and killed at day 6 or day 12 after infection. Total RNA extracted from the splenocytes and lung mononuclear cells which were cultured with heat‐inactivated Chlamydia muridarum (105 IFU/ml) for 72 hrs, and transcription factors mRNA expression were measured by Q‐PCR using specific primers. Q‐PCR analysis of T‐bet in splenocytes and lung mononuclear cells (A and B), RORγT in lung mononuclear cells (C) and Foxp3,GATA3 in splenocytes and lung mononuclear cells (D–G) of NK cell‐depleted mice, isotype control and noninfected mice. At least three independent experiments with 4–5 mice in each group were performed, with one representative experiment is shown. Data are shown as mean ± S.E.M. *P < 0.05 , **P < 0.01.
Figure 7
Figure 7
NK‐cell depletion increases TGF‐β production but decreases IL‐12p40 and IL‐6 production. ELISA of IL‐12p40 (A and B), TGF‐β (C and D) and IL‐6 (E and F) in lung mononuclear cells and mediastinal lymph node cells which were cultured with UV‐inactivated Chlamydia muridarum EBs for 72 hrs. At least three independent experiments with 4–5 mice in NK cell‐depleted group and isotype control group were performed, with one representative experiment is shown. Data are mean ± SEM. *P < 0.05, **P < 0.01.
Figure 8
Figure 8
NK‐cell depletion reduces Th1/Treg and Th17/Treg ratio. Th1/Treg and Th17/Treg ratio in isotype control and NK cell‐depleted mice in splenocytes (A and D), lung mononuclear cells (B and E) and mediastinal lymph node cells (C and F) were calculated by the numbers of IFN‐γ‐produced CD4+ T cells or IL‐17‐produced CD4+ T cells by the number of CD4+ CD25+ Foxp3+ regulatory T cells, respectively. At least three independent experiments with 4–5 mice in each group were performed, with one representative experiment is shown. Data are mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.
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