STAT6/Arg1 promotes microglia/macrophage efferocytosis and inflammation resolution in stroke mice
Wei Cai, Xuejiao Dai, Jie Chen, Jingyan Zhao, Mingyue Xu, Lili Zhang, Boyu Yang, Wenting Zhang, Marcelo Rocha, Toshimasa Nakao, Julia Kofler, Yejie Shi, R. Anne Stetler, Xiaoming Hu, Jun Chen
Wei Cai, Xuejiao Dai, Jie Chen, Jingyan Zhao, Mingyue Xu, Lili Zhang, Boyu Yang, Wenting Zhang, Marcelo Rocha, Toshimasa Nakao, Julia Kofler, Yejie Shi, R. Anne Stetler, Xiaoming Hu, Jun Chen
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Research Article Inflammation Neuroscience

STAT6/Arg1 promotes microglia/macrophage efferocytosis and inflammation resolution in stroke mice

Abstract

Efferocytosis, or phagocytic clearance of dead/dying cells by brain-resident microglia and/or infiltrating macrophages, is instrumental for inflammation resolution and restoration of brain homeostasis after stroke. Here, we identify the signal transducer and activator of transcription 6/arginase1 (STAT6/Arg1) signaling axis as a potentially novel mechanism that orchestrates microglia/macrophage responses in the ischemic brain. Activation of STAT6 was observed in microglia/macrophages in the ischemic territory in a mouse model of stroke and in stroke patients. STAT6 deficiency resulted in reduced clearance of dead/dying neurons, increased inflammatory gene signature in microglia/macrophages, and enlarged infarct volume early after experimental stroke. All of these pathological changes culminated in an increased brain tissue loss and exacerbated long-term functional deficits. Combined in vivo analyses using BM chimeras and in vitro experiments using microglia/macrophage-neuron cocultures confirmed that STAT6 activation in both microglia and macrophages was essential for neuroprotection. Adoptive transfer of WT macrophages into STAT6-KO mice reduced accumulation of dead neurons in the ischemic territory and ameliorated brain infarction. Furthermore, decreased expression of Arg1 in STAT6–/– microglia/macrophages was responsible for impairments in efferocytosis and loss of antiinflammatory modality. Our study suggests that efferocytosis via STAT6/Arg1 modulates microglia/macrophage phenotype, accelerates inflammation resolution, and improves stroke outcomes.

Authors

Wei Cai, Xuejiao Dai, Jie Chen, Jingyan Zhao, Mingyue Xu, Lili Zhang, Boyu Yang, Wenting Zhang, Marcelo Rocha, Toshimasa Nakao, Julia Kofler, Yejie Shi, R. Anne Stetler, Xiaoming Hu, Jun Chen

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Figure 11

Arg1 is essential for efferocytic activity of primary cultured microglia and macrophages.

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Arg1 is essential for efferocytic activity of primary cultured microglia...
NOAH, an arginase inhibitor, or vehicle control was applied to microglia and macrophages for 1 hour before exposure to PI+ dead/dying neurons. (A) Phagocytosis of PI+ dead/dying neurons by microglia or macrophages was quantified as MFI of PI in microglia or macrophages using flow cytometry. Data were collected from 4 independent experiments. *P ≤ 0.05, **P ≤ 0.01 NOAH vs. vehicle, Student’s t test. (B) Protein expression of proinflammatory (IL-6 and TNF-α) and antiinflammatory (IL-10) factors in microglia and macrophages was quantified by flow cytometry at 6 hours after efferocytosis. Data were collected from 4 independent experiments. *P ≤ 0.05, **P ≤ 0.01 NOAH vs. vehicle, Student’s t test. (C) Cell death was quantified by the LDH assay. Data were collected from 6 independent experiments for microglia and 5 independent experiments for macrophage. (D–F) STAT6-KO microglia and macrophages were infected with lentiviral vectors carrying Arg1 cDNA and GFP (Lenti-Arg1) or control lentivirus carrying GFP only (Lenti) for 2d and incubated with PI-labeled post-OGD neurons. Phagocytosis of PI+ dead/dying neurons by Lenti-GFP–transfected WT (WT+Lenti), Lenti-GFP–transfected STAT6-KO (KO+Lenti), or Lenti-Arg1-GFP–transfected STAT6-KO (KO+Lenti-Arg1) microglia and macrophages was quantified 4 hours (microglia) and 1.5 hours (macrophage) after coculture. (D) Representative images of microglia/macrophage (green) phagocytosis of dead/dying neurons (red) overlaid on DIC images. Scale bar: 20 μm. (E) Quantification of the number of engulfed PI+ neurons observed in GFP+ microglia or GFP+ macrophages. (F) Quantification of LDH release in conditioned medium collected from microglia or macrophages after virus transfection. Data represent 6 independent experiments in duplicate. **P ≤ 0.01, ***P ≤ 0.001, 1-way ANOVA.