CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma

Abstract

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8+ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8+ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8+ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8+ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8+ T cells was observed in female recipient mice. Male CD8+ T cells produced higher levels of IFN-γ than female CD8+ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4+ T cells. Interestingly, IFN-γ receptor expression on CD4+ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8+ T cells and the lower expression of IFN-γ receptor on CD4+ T cells in females compared with males.

Fig 1. Sex differences in inflammatory cell infiltration in BAL fluid.

Male and female WT mice (A) and CD8KO mice (B) were sensitized and challenged. The number of inflammatory cells in BAL fluids were counted on day 5 after OVA challenge. Data are shown as the mean ± SEM from at least three independent experiments (n = 12–16). (C and D) The number of CD4⁺ and CD8⁺ T cells in BAL fluids of WT mice (C) and CD8KO mice (D) on day 5 after OVA challenge is shown. Each column represents the mean ± SEM of four to five mice. Open bars, male mice; closed bars, female mice.*, P < 0.05; NS, not significant.

Fig 2. Sex differences in cytokine production in lung.

Male and female WT mice (A) and CD8KO mice (B) were sensitized and challenged. IL-2, IL-4, IL-5, IL-13 and IFN-γ levels in the lung were measured by ELISA one day after OVA challenge. Data are shown as the mean ± SEM from at least three independent experiments (n = 8–11). Open bars, male mice; closed bars, female mice. *, P < 0.05; **, P < 0.01; NS, not significant.

Fig 3. Sex differences in IL-4 production from BLN cells.

BLN cells from WT mice (A) and CD8KO (B) mice were prepared one day after OVA inhalation and cultured with 3 μg/ml of OVA or 1 μg/ml of LPS for 72 h. The concentration of IL-4 in the culture supernatants was measured by ELISA. Data are shown as the mean ± SD of triplicate cultures. Experiment were repeated twice with similar results. Open bars, BLN cells from male mice; closed bars, BLN cells from female mice. **, P < 0.01; NS, not significant.

Fig 4. Sex differences in the inhibitory effect of BLN CD8⁺ T cells.

Male (A) or female (B) CD4⁺ T cells (2 x 10⁵ cells/well) were cultured with CD8⁺ T cells (2 x 10⁵ cells/well) and splenic CD11c⁺ cells (2 x 10⁵ cells/well) in the presence of OVA (50 μg/ml) for 72 h. Concentration of IL-4 in the culture supernatants was measured by ELISA. Data are shown as the mean ± SD of triplicate cultures. Experiment were repeated twice with similar results. (C and D) Sensitized male or female CD8KO mice were adoptively transferred with CD8⁺ T cells from BLN of sensitized and challenged male (C) or female (D) WT mice. Saline was used as control of the transfer. Three days later, recipient CD8KO mice were challenged with OVA aerosol, and then sacrificed on day 5 post-OVA challenge. The numbers of inflammatory cells in BAL fluids were counted. Data are shown as the mean ± SEM from two independent experiments (n = 4–9). Open bars, male mice transferred with saline; closed bars, female mice transferred with saline; hatched bars, male mice transferred with male CD8⁺ T cells; horizontal line bars, female mice transferred with male CD8⁺ T cells; dotted bars, male mice transferred with female CD8⁺ T cells; vertical line bars, female mice transferred with female CD8⁺ T cells. −, mice transferred with saline; +, mice transferred with CD8⁺ T cells; *, P < 0.05; **, P < 0.01; NS, not significant.

Fig 5. Involvement of IFN-γ in sex differences in the inhibitory effect of BLN CD8⁺ T cells.

(A) Intracellular cytokine expression by CD8⁺ T cells was analyzed using a flow cytometer. Data are shown as the mean ± SD of four mice. The expression of Ifng (B), Gata3 and Tbet (C) in CD8⁺ T cells were measured by quantitative real-time RT-PCR. Data are shown as the mean ± SEM of four to eight mice. (D) Male CD4⁺ T cells (2 x 10⁵ cells/well), CD11c⁺ cells (2 x 10⁵ cells/well) and CD8⁺ T cells (0.66 x 10⁵ cells/well) were cultured with 50 μg/ml of OVA in presence of an anti- IFN-γ mAb (10 μg/ml) or control IgG for 72 h. The concentration of IL-4 in the culture supernatants was measured by ELISA. Data are shown as the mean ± SD of triplicate cultures. Experiment were repeated twice with similar results. *, P < 0.05; NS, not significant.

Fig 6. IFN-γ attenuates IL-4 production from BLN male CD4⁺ T cells.

Male or female CD4⁺ T cells (2 x 10⁵ cells/well) and CD11c⁺ cells (2 x 10⁵ cells/well) were cultured with 50 μg/ml of OVA in presence of rIFN-γ (10 ng/ml) or vehicle for 72 h. Data are shown as the mean ± SD of triplicate cultures. Experiments were repeated twice with similar results. Open bars, male CD4⁺ T cells; closed bars, female CD4⁺ T cells. **, P < 0.01; NS, not significant.

Fig 7. Sex differences in IFN-γ receptor expression on CD4⁺ T cells from WT mice.

(A) Representative profiles of IFN-γ receptor expression on CD4⁺ T cells in BLN of male and female mice are shown. Cut-off lines were determined on the basis of an IgG isotype-matched control profile. The percentages of IFN-γ receptor α⁺ population (B) and IFN-γ receptor β⁺ population (C) in CD4⁺ T cells were analyzed in each group. Data are shown as the mean ± SD of three to five mice. Experiment were repeated twice with similar results. (D and E) CD4⁺ T cells obtained from naïve WT mice were cultured with rIFN-γ (10 ng/ml) for 72 h. The percentage of IFN-γ receptor α⁺ population (D) and IFN-γ receptor β⁺ population (E) in CD4⁺ T cells were analyzed before and after the culture. Data are shown as the mean ± SD of triplicate cultures. Experiments were repeated twice with similar results. **, P < 0.01 compared with male mice; #, P < 0.05; ##, P < 0.01 compared to vehicle pretreatments.