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

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

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 2

Dok3 negatively regulates fungi killing in neutrophils.

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Dok3 negatively regulates fungi killing in neutrophils. (A) Killing capa...
(A) Killing capacity of bone marrow neutrophils and splenic macrophages and DCs as assessed by overnight coculture with unopsonized (left) or serum-opsonized (right) C. albicans (MOI 1:500). C. albicans were counterstained with crystal violet. Assays were performed in triplicate; more than 5 fields per group were taken. Scale bars: 200 μm. Data are shown as mean ± SD. **P = 0.008, unpaired 2-tailed Student’s t test. (B) Killing capacity of bone marrow neutrophils against unopsonized C. albicans (MOI 1:500 and 1:200). Assays were performed in triplicates; more than 5 fields per group were taken. Data are shown as mean ± SD. **P = 0.008; ***P = 0.0001, unpaired 2-tailed Student’s t test. (C and D) Purified Dok3+/+ neutrophils were treated with (C) HKCA (MOI 1:1) or (D) zymosan (10 μg/ml) for 10 minutes or 30 minutes, respectively. Cell lysates were IP with a Dok3-specific antibody, and precipitates were probed for p-Tyr and Dok3. Images are representative of 2 independent experiments, respectively. (E) Immunoblot analysis of Dok3 protein levels in purified Dok3+/+ neutrophils stimulated for various times with zymosan (10 μg/ml). β-Actin was used as loading control. Image is representative of 3 independent experiments. (F) Dok3 expression in purified Dok3+/+ splenic DCs, splenic macrophages, bone marrow neutrophils, lymph node T cells, and splenic B cells. Total RNA extracted was used for cDNA synthesis before conducting real-time quantitative reverse-transcription PCR (RT-qPCR). Gene expression was normalized against Gapdh. Data are shown as mean ± SD (n = 3).