Effect of IFNAR1 deficiency on the Th1-related response.

<p>WT and IFNAR1KO mice were infected intratracheally with <i>C</i>. <i>neoformans</i>. (<b><i>A</i></b>) IFN-γ and IL-12p70 production in the lung homogenates was measured on day 3, 7, 14, and 28. Each column represents the mean ± SD of five to six mice. (<b><i>B</i></b>) Expression of iNOS mRNA in the lungs was measured on day 7 after infection. Each column represents the mean ± SD of five mice. Experiments were repeated twice with similar results and the representative data are shown. <i>NS</i>, not significant; *, <i>p < 0</i>.<i>05</i>.</p

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

<div><p>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 <i>cd8α</i>-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.</p></div

Effect of IFNAR1 deficiency on the Th2-related response.

<p>WT and IFNAR1KO mice were infected intratracheally with <i>C</i>. <i>neoformans</i>. (<b><i>A</i></b>) IL-4, IL-5, and IL-13 production in the lung homogenates was measured on day 3, 7, 14, and 28 after infection. Each column represents the mean ± SD of five to six mice. (<b><i>B</i></b>) LN cells obtained on day 7 post-infection were stimulated with indicated doses of <i>C</i>. <i>neoformans</i> or ConA for 48 h, and production of IL-4 was measured. Each column represents the mean ± SD of triplicate cultures. The lung leukocytes (<b><i>C</i></b>) and LN cells (<b><i>D</i>)</b> were prepared on day 7 post-infection. Expression of IL-4 in CD3⁺ CD4⁺ T cells was analyzed using flow cytometry and the number of IL-4⁺ CD3⁺ CD4⁺ T cells was calculated. Each column represents the mean ± SD of five mice. Experiments were repeated twice with similar results and the representative data are shown. <i>NS</i>, not significant; *, <i>p < 0</i>.<i>05</i>.</p

Effect of IFNAR1 deficiency on the Th17-related response.

<p>WT and IFNAR1KO mice were infected intratracheally with <i>C</i>. <i>neoformans</i>. (<b><i>A</i></b>) IL-17A and IL-23p19 production in the lung homogenates was measured on day 3, 7, 14, and 28. Each column represents the mean ± SD of five to six mice. (<b><i>B</i></b>) The lung leukocytes prepared on day 7 post-infection were stained with Diff-Quick and observed under a light microscope. The amount of cells in each leukocyte fraction was counted. Each column represents the mean ± SD of five mice. Experiments were repeated twice with similar results and the representative data are shown. <i>NS</i>, not significant; *, <i>p < 0</i>.<i>05</i>.</p

<i>Cryptococcus neoformans</i> Infection in Mice Lacking Type I Interferon Signaling Leads to Increased Fungal Clearance and IL-4-Dependent Mucin Production in the Lungs

<div><p>Type I interferons (IFNs) are secreted by many cell types upon stimulation via pattern recognition receptors and bind to IFN-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2. Although type I IFNs are well known as anti-viral cytokines, limited information is available on their role during fungal infection. In the present study, we addressed this issue by examining the effect of IFNAR1 defects on the host defense response to <i>Cryptococcus neoformans</i>. In IFNAR1KO mice, the number of live colonies was lower and the host immune response mediated not only by Th1 but also by Th2 and Th17-related cytokines was more accelerated in the infected lungs than in WT mice. In addition, mucin production by bronchoepithelial cells and expression of MUC5AC, a major core protein of mucin in the lungs, were significantly higher in IFNAR1KO mice than in WT mice. This increase in mucin and MUC5AC production was significantly inhibited by treatment with neutralizing anti-IL-4 mAb. In contrast, administration of recombinant IFN-αA/D significantly suppressed the production of IL-4, but not of IFN-γ and IL-17A, in the lungs of WT mice after cryptococcal infection. These results indicate that defects of IFNAR1 led to improved clearance of infection with <i>C</i>. <i>neoformans</i> and enhanced synthesis of IFN-γ and the IL-4-dependent production of mucin. They also suggest that type I IFNs may be involved in the negative regulation of early host defense to this infection.</p></div

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

<p>(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.</p

<i>C</i>. <i>neoformans</i> infection in IFNAR1KO mice.

<p>WT and IFNAR1KO mice were infected intratracheally with <i>C</i>. <i>neoformans</i>. The number of live colonies in the lungs was counted on day 7, 14, and 28 after infection. Each symbol represents each mouse and bars indicate the mean ± SD of five to six mice. Experiments were repeated twice with similar results and the representative data are shown. <i>NS</i>, not significant; *, <i>p < 0</i>.<i>05</i>.</p

Primer sequences for quantitative real-time RT-PCR.

<p>HPRT: hypoxanthine-guanine phosphoribosyltransferase.</p><p>Primer sequences for quantitative real-time RT-PCR.</p

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

<p>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.</p

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

<p>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.</p