Chlamydial lung infection induces transient IL-9 production which is redundant for host defense against primary infection

Abstract

IL-9/Th9 responses are recently found to be important for innate and adaptive immunity particularly in parasitic infections. To date, the study on the role of IL-9 in bacterial infections is limited and the reported data are contradictory. One reported function of IL-9/Th9 is to modulate Th1/Th17 responses. Since our and others' previous work has shown a critical role of Th1 and Th17 cells in host defense against chlamydial lung infection, we here examined the role of IL-9 responses in Chlamydia muridarum (Cm) lung infection, particularly its effect on Th1 and Th17 responses and outcome infection. Our data showed quick but transient IL-9 production in the lung following infection, peaking at day 3 and back to baseline around day 7. CD4+ T cell was the major source of IL-9 production in the lung infection. Blockade of endogenous IL-9 using neutralizing antibody failed to change Interferon-γ (IFN-γ) and IL-17 production by cultured spleen mononuclear cells isolated from Cm infected mice. Similarly, in vivo neutralization of IL-9 failed to show significant effect on T cell (Th1 and Th17) and antibody responses (IgA, IgG1 and IgG2a). Consistently, the neutralization of IL-9 had no significant effect on disease process, including body weight change, bacterial burden and histopathological score. The data suggest that IL-9 production following chlamydial lung infection is redundant for host defense against the intracellular bacteria.

Fig 1. Kinetics of local IL-9, IL-17 and IFN-γ production following lung Cm infection. C57BL/6 Mice were inoculated intranasally with Cm (1×10³ IFUs) and were sacrificed at the indicated days.

The lungs were isolated and the lung homogenates was prepared to detect IL-9(A), IL-17(B) and IFNγ (C) protein levels by ELISA. One representative experiment of two independent experiments (4 mice in each group) is shown. The results are shown as mean ± SD. The levels of IL-9, IL-17 and IFN-γ at various days post-infection were compared to those on day 0 by ANOVA (*p<0.05;**p<0.01).

Fig 2. Production of IL-9 by lung mononuclear cells following Cm infection. Mice were inoculated intranasally with Cm (1×10³ IFUs) and were sacrificed at day 3 post-infection.

The isolated lung mononuclear cells were detected for IL-9 expression by FACS. (A) Representative intracellular IL-9 staining of CD45+ cells and absolute number of IL9 producing CD45+ cells in the lung. (B) Representative intracellular IL-9 staining and absolute number of IL-9 producing CD4+ (CD4+CD3+) T cells, CD8+ (CD8+CD3+) T cells, NK (NK1.1+CD3-) cells and NKT (CD1d tetramer+NK1.1+) cells in the lung. One representative experiment of two independent experiments (four mice in each group in each experiment) is shown. The results are shown as mean ± SD (*, p＜0.05).

Fig 3. Blockade of endogenous IL-9 had no significant effect on IFN-γ and IL-17 production by ex vivo spleens isolated from Cm infected mice.

Spleen cells from mice infected with Cm at day 3 post-infection were cultured in 48-well plates with UV-inactivated-Cm stimulation in the presence of anti-mouse IL-9 mAb or isotype control at various concentrations as indicated. The culture was proceeded for 72 hours and the concentration of IFN-γ and IL-17 in culture supernatants was determined by ELISA. One representative experiment of three independent experiments is shown. The results are shown as mean± SD of each group.

Fig 4. Intranasal delivery of anti-IL-9 mAb successfully neutralized IL-9 in the lung.

C57BL/6 mice were intranasally administered with 10μg of either anti-mouse IL-9 mAb or isotype control antibody at day -1 (one day before), day 0, day 2 following Cm (1×10³ IFUs) lung infection. On day 3 post-infection, mice were sacrificed and lung homogenates were prepared to detect IL-9 protein by ELISA. One representative experiment of two independent experiments (four mice in each group in each experiment) is shown. The results are shown as mean ± SD (**, p＜0.01).

Fig 5. In vivo neutralization of IL-9 failed to alter IFNγ producing by CD4+ and CD8+ T cells following Cm lung infection.

C57BL/6 mice (4 mice per group) were treated with anti-IL-9 mAb or isotype control antibody at days -1, 0, 2, 4 and 6 following intranasal infection with Cm (1×10³ IFUs) and sacrificed at day 7 after infection. (A) Representative data of IFN-γ-producing CD4+ and CD8+ T cells in each group. (B) & (C) Summary of the percentage of IFN-γ+ CD4+ T cells and IFN-γ+ CD8+ T cells in each group (4 mice/group in each experiment). (D) Lung, spleen and draining LN cells were isolated and cultured with UV-inactivated EBs. The concentrations of IFN-γ in the culture supernatants were measured by ELISA. One representative experiment of three independent experiments (4 mice in each group) with similar results is shown. Data are shown as the mean ± SD.

Fig 6. In vivo neutralization of IL-9 failed to alter IL-17/Th17 production following Cm lung infection.

The mice were treated as described in legend to Fig. 5. On day 7 post-infection, lung, spleen and draining LN cells were isolated and analyzed by intracellular cytokine staining or cultured for 72 h following by ELISA testing of IL-17. (A) Representative staining of IL-17 producing CD4 cell in the lung, spleen and draining LNs; the percentages of IL-17-producing CD4+ T cells were showed in the right upper quadrant of the graph. (B)Summary of the percentage of IL-17 producing CD4 T cells in each group. (C) Lung, spleen and draining LN cells were cultured with stimulation of UV-inactivated Cm EBs, and IL-17 concentrations in 72h culture supernatants were determined by ELISA. Three independent experiments with four mice in each group were performed, and one representative experiment is shown. Data show the mean ± SD.

Fig 7. In vivo neutralization of IL-9 failed to alter serum Ab responses following Cm lung infection.

Mice were treated 10 μg anti-IL-9 mAb or isotype control at days -1, 0 and every two day thereafter following intranasal Cm (1×10³ IFUs) infection. Serum samples were collected at days 7 and 14 post-infection and tested for Cm-specific IgA (A), IgG1 (B) and IgG2a (C). Ab titers were transformed to log 10 values. Pooled data for three experiments (12 mice in each group) are presented. Data is shown as mean ± SD.

Fig 8. The neutralization of endogenous IL-9 has significant impact on infection process and histopathology in the lung.

C57BL/6 mice were intranasally administered with 10μg of either anti-mouse IL-9 mAb or isotype control antibody at one day before and the same day of infection following by every 2 days thereafter. Mice were inoculated intranasally with 1×10³ IFUs of Cm EBs. (A) The percentage of body weight changes in the two groups of mice. (B) Live chlamydial organisms (IFUs) in the lung on day 7 and day 14 after infection in each group. (C) The lung tissue sections from different experiment groups were tained with H&E and observed under light microscopy. (D) Semi-quantitative analysis of lung inflammation and damage (pathological score). Slides were examined by a blinded pathologist and the inflammatory grades were analyzed as described in Materials and Methods. One representative experiment of three independent experiments (four mice in each group in each experiment) is shown. The results are shown as mean ± SD.