Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1

Abstract

Coccidioides posadasii is a pathogenic fungus that causes endemic and epidemic coccidioidomycosis in the deserts of North, Central, and South America. How the innate immune system responds to the organism is not well understood. Here we show that elicited mouse peritoneal macrophages respond to spherules (the tissue form of the fungus) by producing proinflammatory cytokines as measured by quantitative PCR of cellular transcripts and by enzyme-linked immunosorbent assay (ELISA) assays for secreted protein. We examined the contribution of Toll-like receptors (TLR) and MyD88 in macrophage responses to formalin-killed spherules (FKS) by comparing cytokine responses of elicited macrophages from different knockout mice. FKS were added to elicited mouse peritoneal macrophages from wild-type, TLR2-/-, and MyD88-/- cells, and wild-type cells made more tumor necrosis factor alpha, MIP-2, and interleukin 6 than did the mutant macrophages. In contrast, the C3H/HeJ mice, which have a point mutation in TLR4, and TLR4-/- B6 mice exhibited no defect in cytokine production compared to the control mice. We also investigated the role of the macrophage beta-glucan receptor, Dectin-1. RAW 264.7 macrophages overexpressing Dectin-1 produced more cytokines in respond to FKS, live spherules, and purified beta-glucan than did control RAW cells. Blockage of Dectin-1 with antibodies inhibited cytokine production in elicited mouse peritoneal macrophages. Taken together, these results show that cytokine responses in mouse peritoneal macrophages to C. posadasii spherules are dependent on TLR2, MyD88, and Dectin-1.

FIG. 1.

C. posadasii FKS induced cytokine responses in elicited mouse C57BL/6 peritoneal macrophages. (A) TaqMan RT-PCR analysis of mRNAs for IL-6, IL-12, MIP-2, IL-10, IL-4, interferon gamma (IFN), and TNF-α in elicited mouse peritoneal macrophages 6 h after incubation with 5 × 10⁴ FKS/ml in comparison to results for unstimulated (Uns) cells. The data are normalized to the 18S level in each sample. Bars are means ± standard errors of the means for duplicate assays. (B) The macrophages were stimulated with various doses of FKS for 16 h, and the culture supernatant was assayed for TNF-α and MIP-2 by ELISA. Data are means ± standard errors for duplicate experiments.

FIG. 2.

Role of Toll-like receptor 4 (TLR4) in peritoneal macrophage response to C. posadasii. Production of MIP-2 by peritoneal macrophages of C3H/HeJ and control C3H/OuJ mice (A) and TLR4^−/− and control C57BL/6 mice (B). Peritoneal macrophages were left unstimulated (Uns) or stimulated with various dosages of lipopolysaccharides Re595 (LPS), C. posadasii FKS, formalin-fixed C. albicans, or 10⁷ particles of heat-killed S. aureus/ml (Staph) for 18 h. The supernatants were tested for MIP-2 release by ELISA. Data represent means ± standard errors for a duplicate experiment. Experiments were performed three times with similar results. *, P < 0.05.

FIG. 3.

Production of TNF-α and MIP-2 by peritoneal macrophages of TLR2-deficient mice. Mouse peritoneal macrophages of TLR2^−/− and C57BL/6J WT mice were left unstimulated (Uns) or stimulated with various concentrations of FKS/ml as indicated, 1 ng of LPS/ml, or 10⁷ particles of heat-killed S. aureus/ml (Staph), and TNF-α (A) and MIP-2 (B) concentrations were measured by ELISA after 18 h of stimulation. Bars represent means ± standard errors for a duplicate assay. Two separate experiments were performed with identical results. *, P < 0.05.

FIG. 4.

Production of MIP-2 by peritoneal macrophages of MyD88-deficient mice. Mouse peritoneal macrophages of MyD88^−/− and C57BL/6J WT mice were left unstimulated (Uns) or stimulated with 5 × 10³, 5 × 10⁴, and 5 × 10⁵ FKS/ml, 1 ng of LPS/ml, or 10⁷ particles of heat-killed S. aureus/ml (Staph), and MIP-2 concentrations were measured by ELISA after 18 h of stimulation. Bars represent means ± standard errors for a duplicate assay. Two separate experiments were performed with similar results. *, P < 0.05.

FIG. 5.

Role of Dectin-1 in host response to C. posadasii. (A) Raw 264.7 cells transfected with the mouse Dectin-1 gene cloned in the vector pFBneo (RAW-Dectin) and cells transfected with vector alone (RAW-pFB) were activated with various concentrations of FKS (left panel) as well as left unstimulated (Uns) or stimulated with 10⁶ particles of heat-killed S. aureus/ml (Staph) or 10⁶ particles of zymosan/ml (right panel). After 18 h of incubation, TNF-α levels in the supernatants were determined by ELISA. Data represent means ± standard errors for a duplicate assay. Four separate experiments were performed with identical results. (B) Raw cells transfected with the mouse Dectin-1 gene cloned in the vector pFBneo (RAW-Dectin) or with vector alone (RAW-pFB) were left unstimulated (Uns) or stimulated with 1.6 × 10⁴ of live C. posadasii spherules/ml (Live) or 1.6 × 10⁴ C. posadasii FKS/ml. TNF-α and MIP-2 levels in the culture supernatants were measured by ELISA after 18 h of stimulation. Bars represent means ± standard errors for a duplicate assay. Two separate experiments were performed with identical results. (C) The effects of anti-Dectin-1 MAb on FKS activation of macrophages. Mouse peritoneal macrophages were left unstimulated (Uns) or stimulated with 5 × 10⁴ C. posadasii FKS/ml in the absence or presence of 2 μg of anti-mouse Dectin-1, 2A11, or control IgG2b MAb/ml. After 18 h of incubation, TNF-α, MIP-2, and IL-6 levels in the culture supernate were measured by ELISA. Data are means ± standard errors for triplicate experiments. *, P < 0.05.

FIG. 6.

Activation of RAW cells with purified β-glucan from C. posadasii spherules. Raw 264.7 cells transfected with the mouse Dectin-1 gene cloned in pFBneo (RAW-Dectin) and cells transfected with vector alone (RAW-pFB) were left unstimulated (Uns) or activated with various concentrations of C. posadasii β-glucan, 10⁷ particles of heat killed S. aureus/ml (Staph), or 10⁶ particles of zymosan/ml. TNF-α levels in the culture supernate were measured by ELISA after 18 h of incubation. Data represent means ± standard errors for triplicate experiments. *, P < 0.05.