Neither dectin-2 nor the mannose receptor is required for resistance to Coccidioides immitis in mice

Abstract

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2(-/-) and MR(-/-) myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.

FIG 1

Comparison of live spherules and FKS. Spherules were incubated overnight with BMDC from B6 mice, and the supernatants were removed to measure the concentration of cytokines in the supernatant. Because of the biohazard of live spherules, the supernatants were sterile filtered through 0.45-μm filters. Supernatants from live spherules and FKS were handled the same way. Due to loss of sample volume from filtering, we could measure only 5 cytokines. There were no differences between the responses of BMDC to live and killed spherules.

FIG 2

Comparison of the responses to spherules by cultured myeloid cells from B6 and MR KO mice. (A) Dose-response curves for elicited macrophages in response to increasing numbers of FKS, determined in duplicate. WT (B6) cells are represented by squares, MR KO cells by circles. (B) Dose-response curves for WT (B6) and MR KO BMDC in response to increasing numbers of FKS. (C) The attachment of FKS to MR KO BMDC (striped bars) was only half that of B6 BMDC (black bars) (*, P < 0.05). In contrast, A-FKS attached equally well to BMDC from the two mouse strains.

FIG 3

Cytokine concentrations in lung lavage fluids from infected MR KO mice. Each bar is the mean from the pooled BAL fluid from 4 mice. There were no significant differences in the MIP-2, IFN-γ, or TNF-α levels. MR-KO mice had higher levels of IL-10, IL-4, IL-17α, IL-22, and IL-23, though the concentration of IL-4 was low in both groups. B6 mice made more IL-6. *, P < 0.05.

FIG 4

Comparisons of CFU in lungs and spleens of B6 and MR KO mice 14 days postinfection (p.i.). This figure combines results from two independent experiments. Each symbol represents one mouse, and the geometric means ± 1 SD are shown by the horizontal lines. Uninfected mice were excluded from analysis.

FIG 5

Dectin-2 and FKS/A-FKS. B6 (WT) mice are represented by square symbols and Dectin-2 KO mice by round symbols. FKS-stimulated cells are solid lines; A-FKS-stimulated cells are dashed lines. (A) Dectin-2 KO macrophages did not make TNF-α, IL-6, or MIP-2 in response to either FKS or A-FKS. They responded equally to Pam3CSK4 (not shown). (B) Secretion of TNF-α, IL-6, IL-1β, and GM-CSF by BMDC was not dependent on Dectin-2, but IL-10 secretion was reduced ∼50% in the absence of Dectin-2. (C) Dectin-2 was not required for attachment of FKS and A-FKS to BMDC.

FIG 6

CFU in lungs and spleens of B6 and Dectin-2 mice and Dectin-2/MR KO mice 14 days p.i. Each point represents a single mouse, and the horizontal lines are the geometric means ± 1 SD. Uninfected mice were excluded from analysis. (A) B6 versus Dectin-2 KO mice had nearly identical numbers of CFU in their lungs and slightly fewer CFU in their spleens, but this was not statistically significant. This figure shows the combined results of two independent experiments. The WT results were previously published (23). (B) There was no significant difference between numbers of CFU recovered from B6 versus Dectin-2/MR double KO mice.

FIG 7

Cytokine concentrations in lavage fluid from infected lungs of B6, Dectin-2 KO, and MR/Dectin-2 KO mice. We pooled equal amounts of lavage fluid from 4 mice in each group and carried out the assays in triplicate. Both KO strains had more IL-10 and IL-4 than B6 controls. Dectin-2 KO mice had more IL-1β than the control. MR/Dectin-2 KO mice had more IL-17A than the other 2 strains. None of these differences reached statistical significance (P > 0.05, analysis of variance [ANOVA]).