Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage
Che-Feng Chang, … , J. Christopher Love, Lauren H. Sansing
Che-Feng Chang, … , J. Christopher Love, Lauren H. Sansing
Published December 18, 2017
Citation Information: J Clin Invest. 2018;128(2):607-624. https://doi.org/10.1172/JCI95612.
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Research Article Inflammation Neuroscience

Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage

Abstract

Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.

Authors

Che-Feng Chang, Brittany A. Goods, Michael H. Askenase, Matthew D. Hammond, Stephen C. Renfroe, Arthur F. Steinschneider, Margaret J. Landreneau, Youxi Ai, Hannah E. Beatty, Luís Henrique Angenendt da Costa, Matthias Mack, Kevin N. Sheth, David M. Greer, Anita Huttner, Daniel Coman, Fahmeed Hyder, Sourav Ghosh, Carla V. Rothlin, J. Christopher Love, Lauren H. Sansing

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Figure 3

Engulfment of eryptotic erythrocytes induces macrophage reparative phenotype.

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Engulfment of eryptotic erythrocytes induces macrophage reparative pheno...
(A) Left: Representative flow cytometry plots of TER119 and CD45 expression from brains at days 1 and 3 after ICH. Population TER119+CD45– and TER119–CD45+ quadrants representing eryptotic erythrocytes and apoptotic leukocytes, respectively, and their percentages are shown. Right: Quantification of absolute number of PtdSer-positive RBCs and WBCs in the brains at days 1 and 3 after ICH. n = 3/group. *P < 0.05 versus day 1 group by Student’s t test. (B) Representative flow cytometry shows LIVE/DEAD–annexin V+ population from non–heat-shocked erythrocytes (0 minutes) and 56°C heat-shocked (HS) erythrocytes (5 minutes), and their percentages are shown. n = 3/group. (C) Top: Representative immunofluorescence images show engulfment of PHK-26–labeled HS erythrocytes (red) in normal and thrombin-stimulated CD11b-positive (green) BMDMs with or without annexin V incubation, with higher magnification of the boxed area in the inset. Bottom: Quantification of n = 3/group; each independent experiment includes 2 technical replicates. *P < 0.05 versus control+HS group; #P < 0.05 versus thrombin+HS group by Student’s t test. (D) Gene expression for markers of proinflammatory (Tnf and Cd86) and reparative (Hmox1 and Clec7a) phenotypes from BMDMs with or without HS treatment under normal or thrombin-stimulated conditions. n = 3/group. *P < 0.05 versus thrombin group by 1-way ANOVA and Bonferroni’s post hoc test. (E) Representative phase contrast images showing control and thrombin-stimulated BMDMs treated with HS and beads, with inset of higher magnification of the boxed area showing engulfment. n = 3/group. (F) Gene expression of Tnf and Cd86 in thrombin-stimulated BMDMs is reduced after HS but not bead treatment. n = 3/group. *P < 0.05 versus control group; #P < 0.05 versus thrombin group by Student’s t test. An.V, annexin V; B, beads; C, control; T, thrombin.