Early or late IL-10 blockade enhances Th1 and Th17 effector responses and promotes fungal clearance in mice with cryptococcal lung infection

Abstract

The potent immunoregulatory properties of IL-10 can counteract protective immune responses and, thereby, promote persistent infections, as evidenced by studies of cryptococcal lung infection in IL-10-deficient mice. To further investigate how IL-10 impairs fungal clearance, the current study used an established murine model of C57BL/6J mice infected with Cryptococcus neoformans strain 52D. Our results demonstrate that fungal persistence is associated with an early and sustained expression of IL-10 by lung leukocytes. To examine whether IL-10-mediated immune modulation occurs during the early or late phase of infection, assessments of fungal burden and immunophenotyping were performed on mice treated with anti-IL-10R-blocking Ab at 3, 6, and 9 d postinfection (dpi) (early phase) or at 15, 18, and 21 dpi (late phase). We found that both early and late IL-10 blockade significantly improved fungal clearance within the lung compared with isotype control treatment when assessed 35 dpi. Immunophenotyping identified that IL-10 blockade enhanced several critical effector mechanisms, including increased accumulation of CD4(+) T cells and B cells, but not CD8(+) T cells; specific increases in the total numbers of Th1 and Th17 cells; and increased accumulation and activation of CD11b(+) dendritic cells and exudate macrophages. Importantly, IL-10 blockade effectively abrogated dissemination of C. neoformans to the brain. Collectively, this study identifies early and late cellular and molecular mechanisms through which IL-10 impairs fungal clearance and highlights the therapeutic potential of IL-10 blockade in the treatment of fungal lung infections.

Figure 1

Persistent infection of C57BL6/J mice with C. neoformans strain 52D is associated with early and sustained IL-10 expression. (A, B) C57BL/6 mice were infected by the intratracheal route with C. neoformans strain 52D. At days 0 (uninfected) and the indicated days post infection (dpi) lungs were harvested for analysis. (A) Lung CFU analysis. (B) Representative lung sections from uninfected and infected mice (at 14 and 35 dpi; H&E stain, 400X magnification). At day 14 dpi, note the presence of numerous extracellular yeast within alveolar spaces (see inset, black closed arrows). At 35 dpi, note that most yeast are located intracellularly (see inset, black open arrows) within large macrophages amidst scattered foci of small mononuclear cells displaying lymphocyte morphology. (C) IL-10 production by lung leukocytes harvested at the designated time points post infection (as described in Materials and Methods). (A,C) Data represent mean ± SEM of 5–21 mice assayed individually per time point in two separate experiments; *, p<0.05 ANOVA with Dunnett’s post hoc analysis vs. Day 0 (uninfected).

Figure 2

Early or late IL-10R blockade improves lung fungal clearance. (A) Experimental strategy to block IL-10 signaling. Cohorts of mice infected by the intratracheal route received isotype control Ab (open arrowheads) or anti-IL-10 receptor Ab (black arrowheads) (0.5 mg/200 µl PBS i.p.) during either the early (3, 6 and 9 dpi) or late (15, 18 and 21 dpi) phase of cryptococcal lung infection. At 35 dpi, lungs were harvested from all four cohorts for analysis. (B) Lung fungal burden, quantified by CFU assay, in mice treated with either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. Data represent mean ± SEM of 9–10 mice per experimental cohort assayed individually in two separate experiments; * p < 0.05 by unpaired Student t test comparing treatments at the same time points.

Figure 3

Effect of IL-10 blockade on total lung leukocytes during cryptococcal lung infection. C57BL/6 mice were infected by the intratracheal route with C. neoformans strain 52D. Total lung leukocytes (identified at 35 dpi by CD45+ staining using flow cytometric analysis) in mice receiving either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. Data represent mean ± SEM of 9–10 mice per experimental cohort assayed individually in two separate experiments; * p < 0.05 by unpaired Student t test for comparisons between mice receiving either isotype control Ab or anti IL-10R Ab at the same time point.

Figure 4

Early or late IL-10 blockade increases lung Th1 and Th17 cells during cryptococcal lung infection. (A–D) C57BL/6 mice were infected by the intratracheal route with C. neoformans strain 52D. Total numbers of (A) CD4+ T cells; (B) CD8+ T cells; and (C) B cells (identified at 35 dpi by flow cytometric analysis using the gating strategy described in Materials and Methods) in the lungs of mice receiving either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. (D) The percentages (left panels) and total number (right panels) of CD4+ T cells expressing intracellular IFN-γ (top panels) or IL-17A (bottom panels) was evaluated in mice treated with either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. Data represent mean ± SEM of 9–10 mice per experimental cohort assayed individually per time point in two separate experiments; * p < 0.05 by unpaired Student t test for comparisons between mice treated with isotype control Ab treated versus anti IL-10R Ab at the same time point.

Figure 5

Early or late blockade increases lung exudate macrophages during cryptococcal lung infection. C57BL/6 mice were infected by the intratracheal route with C. neoformans strain 52D. Specific myeloid cell subsets including (A) Polymorphonuclear neutrophils; (B) eosinophils; (C) Ly-6C^high monocytes; (D) CD11b+ DC; (E) exudate macrophages; and (F) alveolar macrophages (identified at 35 dpi by flow cytometric analysis using the gating strategy described in Materials and Methods) in the lungs of mice receiving either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. Data represent mean ± SEM of 9–10 mice per experimental cohort assayed individually per time point in two separate experiments; * p < 0.05 by unpaired Student t test for comparisons between mice receiving either isotype control Ab or anti IL-10R Ab at the same time point.

Figure 6

Late IL-10 blockade increases lung DC and macrophage activation during cryptococcal lung infection. (A, B) C57BL/6 mice were infected by the intratracheal route with C. neoformans strain 52D. (A) Representative histograms displaying isotype control staining (shaded gray histogram), or specific cell surface staining for I-A^b, CD80, and CD86 on CD11b+ DC, exudate macrophages, and alveolar macrophages identified at 35 dpi in the lungs of mice receiving isotype control Ab (dashed line), or anti-IL-10R Ab (solid line) at the early time points (left panels) or late time points (right panels). (B) Mean fluorescence intensity of the indicated markers on each specified subset obtained from mice receiving either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. Data represent mean ± SEM of 9–10 mice per experimental cohort assayed individually per time point in two separate experiments; * p < 0.05 by unpaired Student t test for comparisons between mice treated with isotype control Ab treated versus anti IL-10R Ab at the same time point.

Figure 7

IL-10 blockade reduces fungal burden within the central nervous system. (A and B) C57BL/6 mice were infected by the intratracheal route with C. neoformans strain 52D. Brain fungal burden was quantified by CFU assays in mice receiving either isotype control Ab (white bars) or anti-IL-10R blocking Ab (black bars) during the early phase (left bars) or late phase (right bars) of infection. Brain fungal burden was quantified at (A) 72 hours following the last dose of Ab (12 dpi for mice receiving early treatment; 24 dpi for mice receiving late treatment) or (B) at 35 dpi. Numeric data above the bars indicate the number of mice with any detectable fungal growth in brain tissue. Data represent mean ± SEM of 4–10 mice per experimental cohort assayed individually per time point in 1–2 separate experiments; * p < 0.05 by unpaired Student t test for comparisons between mice treated with isotype control Ab treated versus anti IL-10R Ab at the same time points. (C) C57BL/6 mice were infected by the intravenous route with C. neoformans strain 52D 24 hours after receiving a single dose of antibody treatment. Fungal burden in the brain, lung and spleen was quantified using CFU assays 48 hours after infection in mice receiving isotype control Ab (white bars) or anti-IL-10R Ab (black bars). Data represent mean ± SEM of 6 mice per experimental cohort assayed individually.