MyD88-dependent signaling drives host survival and early cytokine production during Histoplasma capsulatum infection

Abstract

The ability of the innate immune system to trigger an adaptive T cell response is critical to resolution of infection with the fungal pathogen Histoplasma capsulatum. However, the signaling pathways and cell types involved in the recognition of and response to this respiratory pathogen remain poorly defined. Here, we show that MyD88, an adaptor protein vital to multiple innate immune pathways, is critically required for the host response to Histoplasma. MyD88-deficient (MyD88-/-) mice are unable to control the fungal burden and are more sensitive to Histoplasma infection than wild-type, Dectin-1-/-, or interleukin 1 receptor-deficient (IL-1R-/-) mice. We found that MyD88 is necessary for the production of key early inflammatory cytokines and the subsequent recruitment of inflammatory monocytes to the lung. In both our in vitro and ex vivo analyses, MyD88 was intrinsically required in dendritic cells and alveolar macrophages for initial cytokine production. Additionally, MyD88-deficient bone marrow-derived dendritic cells fail to efficiently control fungal growth when cocultured with primed splenic T cells. Surprisingly, mice that lack MyD88 only in dendritic cells and alveolar macrophages are competent for early cytokine production and normal survival, indicating the presence of compensatory and redundant MyD88 signaling in other cell types during infection. Ultimately, global MyD88 deficiency prevents proper T cell activation and gamma interferon (IFN-γ) production, which are critical for infection resolution. Collectively, this work reveals a central role for MyD88 in coordinating the innate and adaptive immune responses to infection with this ubiquitous fungal pathogen of humans.

FIG 1

MyD88 is required for host survival after infection with Histoplasma. (A) Gender- and age-matched MyD88^−/−, Dectin-1^−/−, and IL-1R^−/− mice and their respective wild-type counterparts (n = 6 to 10) were infected intranasally with 1.8 × 10⁴ Histoplasma cells and monitored for survival. Differences in survival were determined using a log-rank test. (B and C) Lungs (B) and spleens (C) of infected MyD88^−/− and C57BL/6J mice were harvested, homogenized, and plated for CFU at the indicated time points (n = 5 or 6 mice/time point for each strain; d, days). All results are representative of at least three independent experiments. *, P < 0.05; ***, P < 0.001; N.D., not detectable. P values were determined using ANOVA. Horizontal bars are means of analyzed samples.

FIG 2

MyD88 is essential for the kinetics of normal cytokine production in the lungs of mice infected with Histoplasma. Lungs of infected MyD88^−/− and C57BL/6J mice were harvested, homogenized, and evaluated for cytokine levels at the indicated time points. *, P < 0.05; ***, P < 0.001; P values were determined by ANOVA. All the results are representative of at least three independent experiments. The bars represent means ± standard deviations (SD) for 5 mice.

FIG 3

Inflammatory cell recruitment to the lung requires MyD88 signaling. Infected lungs were dissociated into single-cell suspensions and analyzed via flow cytometry to determine the numbers of specific cell populations. (A) Total lung cell counts. (B) CD45⁺ cell counts. (C) Alveolar macrophage count as defined by CD11c⁺ SiglecF⁺ autofluorescent cells. (D) Dendritic cell count as defined by CD11c⁺ CD11b⁺ MHC class II^high cells. (E) Neutrophil count as defined by CD11c⁻ CD11b⁺ Ly6C⁺ Ly6G⁺ cells. (F) Monocyte count as defined by CD11c⁻ CD11b⁺ Ly6C⁺ Ly6G⁻ cells. The bars represent the means ± SD of 5 or 6 mice. All results are representative of at least three independent experiments. **, P < 0.01; ***, P < 0.001; P values were determined using ANOVA.

FIG 4

MyD88 is necessary for timely recruitment and activation of T cells and NK cells. (A) Analysis of global cytokine production in the lungs of Histoplasma-infected mice for total IFN-γ and IL-17A. (B to E) Analysis of lung cell counts and percent cells expressing CD69 for γδ TCR⁺ cells (B), NK cells (C), CD8⁺ T cells (D), and CD4⁺ T cells (E). CD4⁺ T cells are defined as CD45⁺ CD3ε⁺ CD4⁺ cells. CD8⁺ T cells are defined as CD45⁺ CD3ε⁺ CD8⁺ cells. γδ TCR⁺ cells are defined as CD45⁺ CD3ε⁺ γδ TCR⁺ cells. NK cells are defined as CD45⁺ CD3ε⁻ NK1.1⁺ cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were determined by ANOVA.

FIG 5

MyD88 is required for T cell proliferation and production of IFN-γ by T cells and NK cells. (A) T cell proliferation in the lungs and mediastinal lymph nodes 7 days p.i. as measured by in vivo BrdU incorporation. (B) Ex vivo intracellular cytokine staining on lung cells 7 days p.i. to detect IFN-γ production in γδ TCR⁺ cells, NK cells, and CD4⁺ and CD8⁺ T cells isolated from the lung. The bars represent the means ± SD of 5 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; P values were determined by ANOVA. (C) Representative fluorescence-activated cell sorter (FACS) plots for IFN-γ-producing CD4⁺ gated populations.

FIG 6

MyD88 is required for appropriate cytokine signaling in alveolar macrophages and bone-marrow-derived dendritic cells in vitro. (A and B) BMDCs (A) and alveolar macrophages (AvMs) (B) from WT and MyD88^−/− mice were harvested and infected in vitro with Histoplasma at an MOI of 1. Supernatants from infected cells were collected in triplicate at 48 h and evaluated for cytokine levels. (C) BMDCs infected with wild-type Histoplasma (MOI = 1) were cocultured with purified splenic WT CD4⁺ T cells. The cells were lysed, and CFU were counted at the indicated time points. *, P < 0.05; ***, P < 0.001; P values were determined by ANOVA. The bars represent the means ± SD of 3 or 4 samples. All the results are representative of at least three independent experiments.

FIG 7

Alveolar macrophages and dendritic cells require MyD88 for cytokine gene expression in vivo. Single-cell suspensions were harvested from infected wild-type, MyD88^−/−, and CD11c-MyD88 mice at 3 days p.i. CD11c⁺ cells (alveolar macrophages and lung dendritic cells) were purified from lung cells via magnetic-bead separation and subjected to RNA isolation followed by analysis of cytokine transcription. (A) C57BL/6J (wild-type) and MyD88^−/− (mutant) CD11c⁺ cells. (B) MyD88^fl/fl (wild-type) and CD11c-MyD88 (mutant) CD11c⁺ cells. **, P < 0.01; ***, P < 0.001; P values were determined by ANOVA. The bars represent the means ± SD of 3 samples harvested from 2 or 3 pooled mice. All the results are representative of at least three independent experiments.

FIG 8

Loss of MyD88-dependent signaling in dendritic cells, alveolar macrophages, or neutrophils is not sufficient to impair host survival, control of fungal growth, or global cytokine production in the lungs. MyD88^fl/fl mice carrying the CD11c-Cre (CD11c-MyD88) or LysM-Cre (LysM-MyD88) transgene were infected with 1.6 × 10⁴ Histoplasma cells and monitored for survival (n = 8 to 10 mice/strain) (A) or fungal burden (B) at 7 days p.i. (C) Whole-lung homogenates of infected CD11c-MyD88 mice were analyzed for cytokine production at 5 days p.i. (n = 5 or 6 mice/strain). (D) RNA harvested from whole lungs of infected LysM-MyD88 mice was analyzed via qRT-PCR for transcription of cytokines at 4 days p.i. (n = 3 to 5 mice/strain). (E) MyD88^fl/fl mice or MyD88^fl/fl mice carrying the Vav-Cre transgene (Vav-MyD88) were infected with Histoplasma and monitored for survival (n = 6 to 9 mice/strain). n.s., not significant; P values were determined by ANOVA.