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 5

CARD9 deficiency induces higher TREM2 expression in macrophages and impairs antifungal infection.

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CARD9 deficiency induces higher TREM2 expression in macrophages and impa...
(A–F) Heatmap of selected gene expression from RNA-seq and Western blot with densitometric analysis of TREM2 in BMDMs (A–C) and THP-1 cells (D–F) stimulated with P. verrucosa for 24 hours (n = 3). (G and H) Western blot (G) and densitometric (H) analyses of phosphorylated and total P65, AKT, GSK3β, and CREB in BMDMs stimulated with P. verrucosa. (I–L) Western blot and densitometric analyses of phosphorylated CREB (I and J) and TREM2 (K and L) in WT BMDMs with or without JSH-23 pretreatment, after P. verrucosa stimulation. (M–O) BMDMs were transfected with a small interfering negative control (si-NC) or si-TREM2 and stimulated with P. verrucosa for 24 hours. Venn diagram (M) and heatmap (N) show the overlap between genes downregulated in Card9–/– si-NC versus WT si-NC and those upregulated in Card9–/– si-TREM2 versus Card9–/– si-NC. The bubble plot shows GO enrichment of the overlap genes in Card9–/– si-TREM2 BMDMs (O). (P and Q) Killing efficacy analysis (P) and ROS production of TREM2-overexressing RAW 264.7 cells and controls with P. verrucosa stimulation for 60 minutes (Q). (R and S) Western blot (R) and densitometric (S) analysis of IL-10 and TGF-β in BMDMs stimulated with P. verrucosa for 72 hours. In A, D, and M, columns represent replicates from independent culture wells (n = 3). In C, F, H, J, L, and S, each point represents an independent replicate. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test (C, F, L, Q, and S), 2-way ANOVA (H), and multiple unpaired t tests with Holm-Šídák correction (J and P). All stimulations used heat-killed P. verrucosa at MOI 10. OE, overexpressing; PV, Phialophora verrucosa; US, unstimulated.