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Dok3–protein phosphatase 1 interaction attenuates Card9 signaling and neutrophil-dependent antifungal immunity
Jia Tong Loh, … , Yue Wang, Kong-Peng Lam
Jia Tong Loh, … , Yue Wang, Kong-Peng Lam
Published June 10, 2019
Citation Information: J Clin Invest. 2019;129(7):2717-2729. https://doi.org/10.1172/JCI126341.
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Research Article Immunology Infectious disease Article has an altmetric score of 3

Dok3–protein phosphatase 1 interaction attenuates Card9 signaling and neutrophil-dependent antifungal immunity

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Abstract

Invasive fungal infection is a serious health threat with high morbidity and mortality. Current antifungal drugs only demonstrate partial success in improving prognosis. Furthermore, mechanisms regulating host defense against fungal pathogens remain elusive. Here, we report that the downstream of kinase 3 (Dok3) adaptor negatively regulates antifungal immunity in neutrophils. Our data revealed that Dok3 deficiency increased phagocytosis, proinflammatory cytokine production, and netosis in neutrophils, thereby enhancing mutant mouse survival against systemic infection with a lethal dose of the pathogenic fungus Candida albicans. Biochemically, Dok3 recruited protein phosphatase 1 (PP1) to dephosphorylate Card9, an essential player in innate antifungal defense, to dampen downstream NF-κB and JNK activation and immune responses. Thus, Dok3 suppresses Card9 signaling, and disrupting Dok3-Card9 interaction or inhibiting PP1 activity represents therapeutic opportunities to develop drugs to combat candidaemia.

Authors

Jia Tong Loh, Shengli Xu, Jian Xin Huo, Susana Soo-Yeon Kim, Yue Wang, Kong-Peng Lam

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Figure 3

Enhanced antifungal effector functions of Dok3–/– neutrophils.

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Enhanced antifungal effector functions of Dok3–/– neutrophils.
(A and B)...
(A and B) Phagocytosis of FITC-labeled (A) unopsonized or (B) opsonized HKCA by bone marrow neutrophils, splenic DCs, and macrophages (MOI 1:2). Gray histogram represents unstimulated control. Symbols represent individual mice (n = 5–7). Data are shown as mean ± SD. **P = 0.01, unpaired 2-tailed Student’s t test. (C and D) NET release after stimulation of purified Dok3+/+ and Dok3–/– neutrophils with hyphae form HKCA (MOI 1:10). (C) Extracellular DNA stained with DAPI 4 hours following stimulation. Scale bars: 20 μm. (D) Quantification of NET release with PI. Percentage of PI+ neutrophils over total number of neutrophils. *P = 0.0003; **P = 0.0001; ***P = 0.0001, 2-way ANOVA, Sidak’s multiple-comparison post test. Data are pooled from 3 independent experiments (n = 3). (E) mRNA expression levels of indicated cytokines in purified Dok3+/+ and Dok3–/– neutrophils after stimulation with HKCA (MOI 1:2). Data are shown as mean ± SD. (n = 3). *P = 0.03; **P = 0.0002; ***P = 0.0006, unpaired 2-tailed Student’s t test. (F) Flow cytometric analysis of indicated cytokine production by Dok3+/+ and Dok3–/– neutrophils following zymosan stimulation. Representative dot plots are shown in Supplemental Figure 3. Graph indicates percentages of IL-6+, TNF-α+ and TGF-β+ neutrophils. Data are pooled from 3 independent experiments (n = 3–4). Data are shown as mean ± SD. *P = 0.05; **P = 0.009; ***P = 0.0004, unpaired 2-tailed Student’s t test. (G) ROS production by Dok3+/+ and Dok3–/– neutrophils. Neutrophils were labeled with DHR123 before stimulation with HKCA (MOI 1:2) (n = 4) or curdlan (100 μg/ml) (n = 3). Fluorescence was measured by flow cytometry. Histograms were pregated on singlet, live Ly6G+Ly6C+ cells. Gray histograms represent unlabeled control. One representative experiment out of 3 independent experiments is shown. Bar graphs indicate fold change in MFI of DHR123 fluorescence in Dok3–/– neutrophils relative to Dok3+/+ neutrophils. Data are pooled from 3 independent experiments. Data are shown as mean ± SD.

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