CARD9-dependent macrophage plasticity regulates effective fungal clearance
Lu Zhang, Zhichun Tang, Yi Zhang, Wenjie Liu, Haitao Jiang, Li Yu, Kexin Lei, Yubo Ma, Yang-Xin Fu, Ruoyu Li, Wenyan Wang, Fan Bai, Xiaowen Wang
Lu Zhang, Zhichun Tang, Yi Zhang, Wenjie Liu, Haitao Jiang, Li Yu, Kexin Lei, Yubo Ma, Yang-Xin Fu, Ruoyu Li, Wenyan Wang, Fan Bai, Xiaowen Wang
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Research Article Dermatology Immunology Infectious disease

CARD9-dependent macrophage plasticity regulates effective fungal clearance

Abstract

The role of CARD9 in the pathogenesis of various chronic fungal infections has been established; however, the precise mechanisms underlying the pathobiology of these infections remain unclear. We investigated the specific cellular mechanisms by which CARD9 deficiency contributes to the pathogenesis of chronic fungal infections. Using single-cell RNA-seq, we analyzed the immune cell profiles in skin lesions from both murine and human samples. We focused on macrophage differentiation and signaling pathways influenced by CARD9 deficiency. We found that CARD9 deficiency promoted the differentiation of high levels of triggering receptor expressed on myeloid cells 2 (TREM2hi) monocyte–derived macrophages after fungal stimulation, impairing their antifungal functions and inducing exhaustion-like Th1 cells. Mechanistically, NF-κB pathway activation was restricted in CARD9-deficient macrophages, leading to enhanced CREB activation, which, in turn, exerted a positive regulatory effect on Trem2 expression by activating C/EBPβ. Notably, targeting TREM2 enhanced the antifungal immune response in vivo and in vitro, thereby alleviating the severity of CARD9-deficient subcutaneous dematiaceous fungal infection. Our findings highlight the important role of CARD9 in regulating cutaneous antifungal immunity and identify potential targets for immunotherapy in chronic dematiaceous fungal infections.

Authors

Lu Zhang, Zhichun Tang, Yi Zhang, Wenjie Liu, Haitao Jiang, Li Yu, Kexin Lei, Yubo Ma, Yang-Xin Fu, Ruoyu Li, Wenyan Wang, Fan Bai, Xiaowen Wang

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Figure 6

CARD9 negatively regulates Trem2 expression by activating C/EBPβ.

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CARD9 negatively regulates Trem2 expression by activating C/EBPβ. (A) Pr...
(A) Promoter region of WT Trem2, showing 3 predicted C/EBPβ-binding sites at positions. (B and C) Western blot (B) and densitometric (C) analysis of phosphorylated (p-) and total C/EBPβ (left margin) in BMDMs isolated from WT and Card9–/– mice and stimulated for 0–30 minutes (above lanes) with heat-killed P. verrucosa conidia (MOI 10). Each data point represents an independent experimental replicate (n = 3). (D and E) Knockdown of endogenous C/EBPβ by RNA interference in BMDMs, which were transfected with siRNA against murine C/EBPβ and nontargeting control siRNA using Lipofectamine 3000 transfection reagent (Thermo Fisher). Cells were cultured for 48 hours after transfection and then stimulated with heat-killed P. verrucosa conidia (MOI 10) for 24 hours. Cell lysates were subjected to Western blot analysis using indicated antibodies (D) and then quantified using densitometric analysis in Card9–/– group (E). Each data point represents an independent experimental replicate (n = 3). (F) Firefly luciferase activity in human embryonic kidney 293T cells cotransfected with constructs for the overexpression of Cebpb and a construct containing various Trem2 promoter–driven firefly luciferase constructs together with an EF1α promoter–driven Renilla luciferase reporter; results were normalized to those of Renilla luciferase. Ctr, control construct lacking Cebpb cotransfected with a construct containing the Trem2 promoter. (G) Chromatin immunoprecipitation (with control IgG or anti-Cebpb) and PCR analysis of the binding of Cebpb to the Trem2 promoter in BMDMs obtained from WT mice and Card9–/– mice and left unstimulated or challenged for 4 hours in vitro with heat-killed P. verrucosa spores (MOI 10). Data are shown as the mean ± SD. *P < 0.05 and **P < 0.01, and ****P < 0.0001, by multiple unpaired t tests with Holm-Šídák correction (C), or 1-way ANOVA with Tukey’s multiple-comparison test (E–G). PV, Phialophora verrucosa; si-NC, small interfering negative control; mTrem2, mouse Trem2; MT, mutant type.