Vaccinated C57BL/6 mice develop protective and memory T cell responses to Coccidioides posadasii infection in the absence of interleukin-10

Abstract

High concentrations of lung tissue-associated interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, correlate with susceptibility of mice to Coccidioides spp. infection. In this study, we found that macrophages, dendritic cells, neutrophils, and both CD8(+) and CD4(+) T cells recruited to Coccidioides posadasii-infected lungs of nonvaccinated and vaccinated mice contributed to the production of IL-10. The major IL-10-producing leukocytes were CD8(+) T cells, neutrophils, and macrophages in lungs of nonvaccinated mice, while both Foxp3(+) and Foxp3(-) subsets of IL-10(+) CD4(+) T cells were significantly elevated in vaccinated mice. Profiles of the recruited leukocytes in lungs revealed that only CD4(+) T cells were significantly increased in IL-10(-/-) knockout mice compared to their wild-type counterparts. Furthermore, ex vivo recall assays showed that CD4(+) T cells isolated from vaccinated IL-10(-/-) mice compared to vaccinated wild-type mice produced significantly higher amounts of IL-2, gamma interferon (IFN-γ), IL-4, IL-6, and IL-17A in the presence of a coccidioidal antigen, indicating that IL-10 suppresses Th1, Th2, and Th17 immunity to Coccidioides infection. Analysis of absolute numbers of CD44(+) CD62L(-) CD4(+) T effector memory T cells (TEM) and IFN-γ- and IL-17A-producing CD4(+) T cells in the lungs of Coccidioides-infected mice correlated with better fungal clearance in nonvaccinated IL-10(-/-) mice than in nonvaccinated wild-type mice. Our results suggest that IL-10 suppresses CD4(+) T-cell immunity in nonvaccinated mice during Coccidioides infection but does not impede the development of a memory response nor exacerbate immunopathology of vaccinated mice over at least a 4-month period after the last immunization.

FIG 1

IL-10 production in lungs of vaccinated (ΔT) and nonvaccinated (PBS-Ctl.) C57BL/6 mice did not directly correlate with recruitment of CD4⁺ Treg cells. (A and B) IL-10 cytokine levels (A) and fold changes of transcripts (B) in lung homogenates at the indicated DPC following intranasal challenge with approximately 60 to 80 Coccidioides spores were determined for C57BL/6 mice that were challenged at 16 weeks after the final vaccination. Nonvaccinated and noninfected mice (N) of the same age and gender were included for determination of the baseline amounts of IL-10 transcript and protein. (C and D) The absolute number of Foxp3⁺ CD4⁺ CD25⁺ Treg cells (C) in lung homogenates of the mice was determined at the indicated time points. Total RNA was also isolated from duplicated sets of mice and used for determination of the fold changes of Foxp3 expression at 8 and 12 DPC (D). The assays were conducted in two independent experiments. Data are means ± SEM of 4 mice per group. Asterisks indicate P < 0. 05 compared to nonvaccinated mice.

FIG 2

Diverse subsets of lymphocytes and granulocytes produced IL-10 in the lungs during Coccidioides infection. Pulmonary cells were isolated from the nonvaccinated (PBS-Ctl.) or vaccinated (ΔT) C57BL/6 mice at 8 or 12 DPC. (A) Dot plots of gated IL-10-producing cells in the pulmonary leukocyte population (CD45⁺). (B) Percentages of CD4⁺ versus CD8⁺ cells in the gated IL-10⁺ populations. Numbers juxtaposed to the gates give the mean percentage of the subset populations within the gated IL-10⁺ cells. (C) Percentages of CD11c⁺ versus Ly6G⁺ subsets of granulocytes, which represented DCs and PMNs in the gated IL-10⁺ cell population. (D and E) Total numbers of IL-10-producing CD4⁺ T cells in the lungs were further divided into Foxp3⁺ and Foxp3⁻ subpopulations. The numbers of IL-10-producing CD8⁺ T cells, B cells (BC), dendritic cells, neutrophils (PMN), and macrophages (Mϕ) per lung organ were also measured as described in Materials and Methods at 8 and 12 DPC. Asterisks indicate significantly higher numbers of IL-10-producing cell numbers in vaccinated compared to nonvaccinated mice, while daggers indicate higher numbers in the nonvaccinated mice compared to vaccinated mice (P < 0.05). Data are representative of 2 independent experiments with similar results.

FIG 3

Enhanced acquisition of CD4⁺ T cells in lung homogenates of Coccidioides-infected IL-10^−/− mice. Pulmonary cells were isolated from the nonvaccinated or vaccinated Coccidioides-infected WT and IL-10^−/− mice that were sacrificed at 8 DPC (A and C) or 12 DPC (B and D). Percentages of each immune cell subset within the gated CD45⁺ population were determined as described in Materials and Methods. Data are presented as means ± SEM of 4 mice per group. Asterisks indicate significant differences between the vaccinated and nonvaccinated mice of the same strain, while the dagger represents a significant difference between the IL-10^−/− and WT mice (P < 0.05).

FIG 4

CD4⁺ T cells isolated from vaccinated IL-10^−/− mice secreted higher amounts of Th1-, Th2-, and Th17-type cytokines upon stimulation with Coccidioides antigen than did nonvaccinated mice. CD4⁺ T cells were isolated from splenocytes of WT and IL-10^−/− mice immunized with the ΔT vaccine or PBS at 16 weeks postvaccination. Isolated CD4⁺ T cells were cultured in the presence or absence of Coccidioides T27K antigen. Concentrations of representative Th1 cytokines (IL-2 and IFN-γ) (A and B), Th2 cytokines (IL-4 and IL-5) (C and D), and Th17 cytokines (IL-6 and IL-17A) (E and F) in supernatants of antigen-stimulated immune cells were compared. Statistically significant differences in cytokine production between the ΔT-vaccinated and nonvaccinated mice of the same strain are indicated by asterisks (P < 0.05). Daggers indicate a significant difference between WT and IL-10^−/− mice (P < 0.01). The results are representative of 2 separate experiments with 4 mice per group.

FIG 5

Both ΔT-vaccinated WT and IL-10^−/− mice revealed elevated percentages and numbers of T_EM cells as well as IFN-γ-, IL-5-, and IL-17A-expressing CD4⁺ T cells compared to nonvaccinated mice. (A) Percentages of T_EM (CD4⁺ CD44⁺ CD62⁻) and T_CM (CD4⁺ CD44⁺ CD62L⁺) cells within the gated CD4⁺ T-cell population in the lungs of nonvaccinated (PBS-Ctl.) versus vaccinated (ΔT) mice are shown at 8 or 12 DPC. Numbers above the gates indicate the mean percentages of the gated T_EM and T_CM subsets. (B) The cell numbers of gated, specific cytokine-producing CD4⁺ T cells per lung were determined by intracellular cytokine staining. Asterisks indicate significantly higher cell numbers of responsive T-cell subpopulations in lungs of vaccinated compared to nonvaccinated mice, while the daggers indicate higher numbers of selected cytokine-producing cells in IL-10^−/− mice compared to WT counterparts. Error bars indicate standard errors. The results reported are representative of two independent experiments.

FIG 6

Both vaccinated WT and IL-10^−/− mice significantly reduced the fungal burden in their lungs and spleen and showed prolonged survival compared to nonvaccinated mice. The numbers of CFU of C. posadasii detected in dilution plate cultures of lung (A) and spleen (B) homogenates of WT and IL-10^−/− mice that had been vaccinated with the ΔT vaccine (ΔT) or immunized with PBS (Ctl.) as controls are reported at 14 DPC. All mice were challenged by the intranasal route with 80 viable spores isolated from the virulent strain. The horizontal lines indicate the mean CFU determinations. (C) Survival plots are presented for WT and IL-10^−/− mice vaccinated with the ΔT vaccine or treated with PBS as controls. Asterisks indicate a statistically significant difference (P < 0.05) between CFU in the lungs and spleen of the vaccinated versus nonvaccinated mice of the same strain, while the daggers indicate significantly reduced CFU in the IL-10^−/− mice versus the wild-type counterpart. The results are representative of 2 separate experiments.

FIG 7

Comparative histopathology of Coccidioides-infected lungs revealed greater consolidation of inflammatory tissue in both vaccinated WT and IL-10^−/− mice compared to nonvaccinated mice. Tissue sections were prepared from lungs of the nonvaccinated (A to D) and vaccinated (E to H) mice at 14 DPC. The large abscesses of the nonvaccinated WT and IL-10^−/− mice contained concentrated inflammatory cells without apparent organization (A and C). Representative images revealed higher numbers of Coccidioides spherules surrounded by areas of necrosis in the nonvaccinated WT mice (B) compared to the IL-10^−/− mice (D). Inflammatory cells converged to the sites of spherule rupture and in some cases were visible within the spherule matrix of the nonvaccinated mice (arrows in panel B). High numbers of inflammatory cells also surrounded spherules (S) prior to endosporulation in IL-10^−/− mice. In contrast, both vaccinated wild-type and IL-10^−/− mice showed localized recruitment of inflammatory cells surrounded by normal tissue (E to H) representive of a protective response, compared to nonvaccinated mice. Bars, 500 μm (A, C, E, and G) or 100 μm (B, D, E, F, and H).