Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection

Abstract

Host defense mechanisms against Pneumocystis carinii are not fully understood. Previous work in the murine model has shown that host defense against infection is critically dependent upon host CD4(+) T cells. The recently described Th17 immune response is predominantly a function of effector CD4(+) T cells stimulated by interleukin-23 (IL-23), but whether these cells are required for defense against P. carinii infection is unknown. We tested the hypothesis that P. carinii stimulates the early release of IL-23, leading to increases in IL-17 production and lung effector CD4(+) T-cell population that mediate clearance of infection. In vitro, stimulation of alveolar macrophages with P. carinii induced IL-23, and IL-23p19 mRNA was expressed in lungs of mice infected with this pathogen. To address the role of IL-23 in resistance to P. carinii, IL-23p19-/- and wild-type control C57BL/6 mice were infected and their fungal burdens and cytokine/chemokine responses were compared. IL-23p19-/- mice displayed transient but impaired clearance of infection, which was most apparent 2 weeks after inoculation. In confirmatory studies, the administration of either anti-IL-23p19 or anti-IL-17 neutralizing antibody to wild-type mice infected with P. carinii also caused increases in fungal burdens. IL-17 and the lymphocyte chemokines IP-10, MIG, MIP-1alpha, MIP-1beta, and RANTES were decreased in the lungs of infected IL-23p19-/- mice in comparison to their levels in the lungs of wild-type mice. In IL-23p19-/- mice infected with P. carinii, there were fewer effector CD4(+) T cells in the lung tissue. Collectively, these studies indicate that the IL-23-IL-17 axis participates in host defense against P. carinii.

FIG. 1.

IL-23 expression in alveolar macrophages in response to P. carinii infection in vitro. (A) MH-S cell IL-23p19 mRNA expression at indicated times after exposure. (B) Splenocyte IL-17 production induced by conditioned supernatants from P. carinii-exposed MH-S cells. MH-S cells were infected with P. carinii for 24 h prior to supernatant harvest and transfer onto BALB/c splenocytes. At the indicated times postexposure, splenocyte supernatants were collected for IL-17 assay. The data are expressed as the means ± SEMs and are representative of three separate experiments. n = 3 per group. C, control.

FIG. 2.

IL-23p19 mRNA expression in BAL cells following P. carinii challenge. BALB/c mice were inoculated with P. carinii, and BAL cells were harvested at the indicated times postinoculation for mRNA assay. The data are expressed as the means ± SEMs and are representative of two separate experiments. n = 4 per group.

FIG. 3.

Lung P. carinii burden in wild-type and IL-23p19−/− mice. C57BL/6 and IL-23p19−/− mice were inoculated with P. carinii and sacrificed for P. carinii rRNA assay of the right lung at the indicated time points postinoculation. The data are expressed as the means ± SEMs of total rRNA per right lung and are representative of five separate experiments. n = 4 per group. *, P < 0.05 compared with C57BL/6 mice at the same time point.

FIG. 4.

Lung P. carinii burden in C57BL/6 mice treated with anti-IL-23p19 Ab. C57BL/6 mice were inoculated with P. carinii and treated with either anti-IL-23 Ab or isotype control Ab twice a week. At the indicated time points postinoculation, total RNA was isolated from the right lungs for assay of P. carinii rRNA expression levels by real-time RT-PCR. The data are expressed as the means ± SEMs and are representative of two separate experiments. n = 4 per group. *, P < 0.05 compared with control mice at the same time point.

FIG. 5.

Chemokine expression in wild-type C57BL/6 and IL-23p19−/− mice during P. carinii infection. The mice were inoculated with P. carinii and sacrificed at the indicated time points postinoculation. The expression of chemokines was measured in whole-lung homogenates of the left lung. The data are expressed as the means ± SEMs and are representative of two separate experiments. n = 4 per group. *, P < 0.05 compared with control mice at the same time point.

FIG. 6.

Lung CD4⁺ effector T-cell recruitment in wild-type C57BL/6 and IL-23p19−/− mice in response to P. carinii infection. At the indicated times after P. carinii inoculation, BAL cells were collected and stained with fluorochrome-conjugated Abs specific for murine CD4, CD44, and CD62L. The absolute numbers of lymphocytes bearing surface expression of these molecules were determined by using a fluorescence-activated cell sorter. The data are expressed as the means ± SEMs and are representative of two separate experiments. n = 4 per group. *, P < 0.05 compared with wild-type mice at the same time point.

FIG. 7.

Lung IL-17 production in wild-type and IL-23p19−/− mice in response to P. carinii. C57BL/6 and IL-23p19−/− mice were inoculated with P. carinii, and lungs were harvested at the indicated time points postinoculation. The right lungs were assayed for mRNA content and the left lungs for protein analysis. (Left) Lung IL-17 mRNA expression levels. (Right) Lung IL-17 protein expression levels. The data are expressed as the means ± SEMs and are representative of three separate experiments. n = 4 per group. *, P < 0.05 compared with wild-type mice at the same time point.

FIG. 8.

Lung P. carinii burden in C57BL/6 mice treated with anti-IL-17 Ab. C57BL/6 mice were inoculated with P. carinii and then treated with anti-IL-17 Ab or isotype control Ab twice a week. At the indicated time points postinoculation, total RNA was isolated from the right lung for assay of P. carinii rRNA expression levels by real-time RT-PCR. The data are expressed as the means ± SEMs and are representative of two separate experiments. n = 4 per group. *, P < 0.05 compared with control mice at the same time points.