Galectin-3 mediates lysosome-related inflammation within monocyte-derived macrophages in a mouse model of ischemic brain injury
Miao Wang, Zhentai Huang, Zhihong Du, Jiajing Shan, Qing Ye, Lingxiao Lu, Ming Jiang, Fei Xu, Ziyang Liu, David J.R. Fulton, Rehana K. Leak, Babak Razani, Jun Chen, Xiaoming Hu
Miao Wang, Zhentai Huang, Zhihong Du, Jiajing Shan, Qing Ye, Lingxiao Lu, Ming Jiang, Fei Xu, Ziyang Liu, David J.R. Fulton, Rehana K. Leak, Babak Razani, Jun Chen, Xiaoming Hu
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Research Article Inflammation Neuroscience

Galectin-3 mediates lysosome-related inflammation within monocyte-derived macrophages in a mouse model of ischemic brain injury

Abstract

Circulating monocyte-derived macrophages (MDMø) rapidly invade the brain after stroke, exerting both detrimental and beneficial effects. Elucidating mechanisms that mediate detrimental properties of MDMø may identify therapeutic strategies to divert MDMø from destructive phenotypes, while preserving their favorable effects. Toward this goal, the current study explores the function of Galectin-3 (GAL3) in MDMø and elucidates mechanisms whereby MDMø-derived GAL3 exacerbates stroke injury. In the acutely injured brain, GAL3 expression was upregulated primarily within MDMø. Global KO of GAL3 reduced brain infarcts in the short term but did not sustain long-term positive outcomes. Using BM chimera mice, macrophage transplantation, and myeloid cell–specific GAL3-KO (LysMCre+/–Lgals3fl/fl) mice, we demonstrated that GAL3 in MDMø mediated acute infarct expansion after stroke. Coculturing brain lysate–treated, BM-derived macrophages (BMDMs) with oxygen glucose deprivation–challenged neurons induced neurotoxicity that was mitigated by the cell-permeable, selective GAL3 inhibitor TD139. GAL3 triggered cathepsin induction and lysosomal leakage in BMDMs, leading to inflammasome activation. Systemic and transient TD139 treatment in the acute injury phase reduced infarcts, tempered neuroinflammation, and improved long-term neurological outcomes. Therefore, MDMø-derived GAL3 represents a drug target that could be accessed in peripheral blood to potentially mitigate post-stroke brain injury.

Authors

Miao Wang, Zhentai Huang, Zhihong Du, Jiajing Shan, Qing Ye, Lingxiao Lu, Ming Jiang, Fei Xu, Ziyang Liu, David J.R. Fulton, Rehana K. Leak, Babak Razani, Jun Chen, Xiaoming Hu

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Figure 4

GAL3 accumulates rapidly in the lysosomes of MDMø after acute ischemic brain injury.

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GAL3 accumulates rapidly in the lysosomes of MDMø after acute ischemic b...
(A) Experimental scheme illustrating the analysis of differentially expressed genes (DEGs) between Lgals3hi and Lgals3lo MDMø 3 d after tMCAO. MDMø were divided into 2 groups based on Lgals3 expression level > 2 or ≤ 2. DEGs were identified by comparing the 2 groups. A volcano plot depicts the DEGs [log (fold change) > 0.59 or < –0.59, Bonferroni adjusted P value < 0.05] between Lgals3hi and Lgals3lo MDMø. (B) KEGG enrichment analysis was performed on the DEGs using Metascape. Shown are the 10 KEGG pathways predicted to be upregulated or downregulated (z score ≥ 5 or z score ≤ 5). Corresponding dot plot shows upregulated genes involved in the lysosome term in the Lgals3hi group. (C) Representative LAMP2 and GAL3 staining in ischemic brains 3 d after tMCAO. Right panel shows Pearson’s correlation analysis of GAL3 and LAMP2 signal. Scale bars: 40 μm, 10 μm (zoom). (D) Fluorescence intensity profiles of GAL3 (red) and LAMP2 (green) signal for objects crossing the white arrows in C. (E) Immunoblot and quantitative analysis of GAL3 expression in lysosome-enriched fraction and the remaining cell lysates (without lysosome) from cultured BMDMs under the indicated conditions. N = 3/condition. (F) Representative images of GAL3 and LAMP2 staining in control and brain lysate–treated BMDMs with or without TD139 (10 μM). (G) The average somal size of BMDMs. N = 6 per condition. (H) The numbers of GAL3+ puncta per cell. (I) The percentages of GAL3+LAMP2+ puncta among total GAL3+ puncta under indicated conditions. N = 22–23 randomly selected cells per condition from 3 independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001. Two-tailed, unpaired Student’s t test (I), 1-way ANOVA and Bonferroni’s test (E and G), or Kruskal-Wallis test (H).