CD28 exerts protective and detrimental effects in a pulmonary model of paracoccidioidomycosis

Abstract

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28(-/-)) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28(-/-) mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.

FIG. 1.

Early in infection CD28 is protective, while at the chronic phase, CD28 signaling exerts a deleterious effect. CD28^−/− and control WT C57BL/6 mice were i.t. infected with 1 × 10⁶ fungal cells, and severity of infection was analyzed by CFU counts at four postinfection periods. (A) At weeks 2 and 10 after infection, increased fungal burdens in the lungs of CD28-deficient mice were observed. (B) By week 10, an increased dissemination to livers of CD28^−/− mice was also seen. Unexpectedly, at week 16, a marked fungal growth was detected in the lungs, and the number of fungal cells in the livers of WT mice supplanted that observed with the deficient strain. At week 26 postinfection, no differences in the number of pulmonary CFU were noted, but the number of viable yeasts in the livers of WT mice remained higher than that of CD28-deficient mice. The points represent means ± SEM of the numbers of log₁₀ CFU obtained from groups of six to eight mice. The results are representative of 3 experiments (except week 16). *, P < 0.05, and ***, P < 0.001, compared with WT controls.

FIG. 2.

CD28 costimulation results in increased mortality rates of P. brasiliensis-infected mice. Survival times of CD28^−/− and WT control mice after i.t. infection with 1 × 10⁶ P. brasiliensis yeast cells were determined for a period of 57 weeks. Significant differences in mortality rates were detected; the results are representative of two independent experiments; (n = 6). **, P < 0.01.

FIG. 3.

CD28 deficiency determines impaired production of NO and decreased humoral immunity. Levels of nitric oxide (A) and P. brasiliensis-specific antibodies (B) were assayed, respectively, in organ homogenates and sera of CD28^−/− and WT mice i.t. infected with 1 × 10⁶ yeast cells. NO was measured by the Griess reaction, and sera were assayed for total IgG, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 by using an isotype-specific ELISA as detailed in Materials and Methods. The bars depict means ± SE of NO levels or serum titers (6 to 8 mice per group). The results are representative of 3 experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with WT controls.

FIG. 4.

CD28 deficiency causes a reduced production of pro- and anti-inflammatory cytokines in the lungs and livers of P. brasiliensis-infected mice. Levels of cytokines in organ homogenates of CD28^−/− and WT mice were measured after i.t. infection with 1 × 10⁶ yeast cells. Lungs and livers were collected at weeks 2 and 10 after infection and disrupted in 5.0 ml of PBS, and supernatants were analyzed for cytokine content by capture ELISA. (A) At week 2 after infection, CD28-deficient mice presented reduced levels of IL-2, IL-4, and GM-CSF in the lungs and livers. (B) Decreased levels of IL-12, TNF-α, and TGF-β in the lungs and IL-12, TNF-α, IFN-γ, IL-10, and TGF-β in the livers of deficient mice were seen at week 10 postinfection. The bars depict means ± SEM of cytokine levels (6 to 8 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with WT controls.

FIG. 5.

WT mice presented an increased influx of activated macrophages, T cells, and regulatory CD4⁺CD25⁺FoxP3⁺ T cells to the lungs, whereas CD28-deficient mice showed an increased presence of naïve CD8⁺ T cells. Characterization of leukocyte subsets and the activation profile of cells by flow cytometry in the lung-infiltrating leukocytes (LIL) from CD28^−/− and WT mice inoculated i.t. with 1 × 10⁶ P. brasiliensis yeast cells. At week 10 after infection, lung cell suspensions were obtained and stained as described in Materials and Methods. The acquisition and analysis gates were restricted to macrophages or lymphocytes. (A) Macrophages (møs); (B) CD4⁺ T cells; (C) CD8⁺ T cells. (D) To characterize the expansion of regulatory T cells in LIL, surface staining of CD25⁺ and intracellular FoxP3 expression were back-gated on the CD4⁺ T-cell population. (E) The number of apoptotic and necrotic leukocytes was assessed by flow cytometry using FITC-labeled annexin V and propidium iodide. The data represent the mean ± SEM of the results from 6 to 8 mice per group and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with WT controls. N^r, number.

FIG. 6.

At week 16, the numbers and levels of activation of inflammatory cells from lungs and livers of CD28^−/− and WT mice were similar, but increased numbers of regulatory T cells were detected in the latter strain. Characterization of leukocyte subsets and the activation profile of cells by flow cytometry in the lung- and liver-infiltrating leukocytes (LIL) from CD28^−/− and WT mice inoculated i.t. with 1 × 10⁶ P. brasiliensis yeast cells. At week 16 after infection, lung and liver cell suspensions were obtained and stained as described in Materials and Methods. The acquisition and analysis gates were restricted to macrophages or lymphocytes. (A, B, and C) Lung macrophages and CD4⁺ and CD8⁺ T cells, respectively. (D, E, and F) Liver macrophages and CD4⁺ and CD8⁺ T cells, respectively. (G) To characterize the expansion of regulatory T cells in lungs and livers, surface staining of CD25⁺ and intracellular FoxP3 expression were back-gated on the CD4⁺ T-cell population. The data represent the mean ± SEM of the results from 6 to 8 mice per group and are representative of one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with WT controls.

FIG. 7.

In vivo depletion of CD8⁺ T cells abrogated the differences in fungal loads between WT and CD28-deficient mice. Both mouse strains were treated in vivo with H35 anti-CD8 monoclonal antibodies or control immunoglobulin and i.t. infected with 1 × 10⁶ P. brasiliensis yeasts. (A) The number of viable yeasts recovered from lungs was assessed at week 2 postinfection. (B) Lung-infiltrating CD4⁺ and CD8⁺ T cells and CD11b⁺ macrophages were phenotyped at week 2 postinfection. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with controls.

FIG. 8.

Photomicrographs of pulmonary and hepatic lesions of WT and CD28^−/− mice at week 10 postinfection with 1 × 10⁶ P. brasiliensis yeasts. (A and B) WT mice showed well-organized granulomas containing an elevated number of yeasts surrounded by a halo of mononuclear cells containing a few plasmocytes; most of the lung tissue was preserved, with limited signs of inflammatory cell recruitment. (C and D) CD28-deficient mice presented an elevated number of well-defined epithelioid granulomas containing a large number of yeast budding cells; these lesions occupy a large area of lung tissue and were concomitant with an extensive expansion of the bronchoalveolar lymphoid tissue. No hepatic lesions were observed in WT mice (E and F), while CD28-deficient mice showed a large number of viable yeasts surrounded by granulomatous lesions scattered through the liver parenchyma (G and H). For panels A, C, E, and G: H&E, ×100; for panels B, D, F, and H: Groccot stain, ×100.

FIG. 9.

At week 26 after infection, WT mice present large numbers of lesions and P. brasiliensis yeasts in their livers. CD28^−/− and WT mice were i.t. infected with 1 × 10⁶ fungal cells, and histopathological analyses of lungs and livers were performed at week 26 after infection. (A to D) No differences in pulmonary lesions were noted between WT and CD28-deficient mice. (E to H) Increased numbers of yeast cells and more-severe hepatic lesions were detected in WT mice. For panels A, B, E, and F: HE, ×100; for C, D, G, and H, Groccot stain, ×100. (I) Morphometrical analysis confirmed the more extensive areas occupied by the liver lesions of WT mice. (J) Levels of liver cytokines at week 26 postinfection. *, P < 0.05; **, P < 0.01, compared with WT controls.