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Objective <p>Myd88 may mediate the inflammatory response after ischemic stroke (IS) by regulating the microglia-associated Toll-like receptor (TLR) signaling pathway, but this requires verification. The present study aims to investigate the molecular mechanism of Myd88 in inflammatory signal transduction after IS through comprehensive single-cell RNA sequencing (scRNA-seq) and experimental verification.</p> Methods <p>For scRNA-seq analysis, cell subpopulations, including Myd88+ microglia, were identified using UMAP dimensionality reduction and cell marker gene annotation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted based on cell marker genes of Myd88+ microglia. The interactions between Myd88+ microglia and other cell subpopulations were inferred by cell communication analysis. MyD88, iNOS, and CD86 expression levels were detected by quantitative real-time PCR (qRT-PCR). The effects of MyD88 knockdown on microglia and endothelial cells (ECs) were verified by enzyme-linked immunosorbent assay (ELISA) and transwell assays.</p> Results <p>Annotation of cell subpopulations revealed that Myd88+ microglia were significantly enriched in IS, showing upregulation of inflammatory and immune cell migration-related pathways, including the TLR signaling pathway. Cell communication analysis indicated close interactions between Myd88+ microglia and endothelial cells. Knockdown of MyD88 alleviated the polarization effect of lipopolysaccharide (LPS) on microglia, reduced the secretion of pro-inflammatory cytokines, and attenuated the inhibitory effects on EC migration and angiogenesis.</p> Conclusion <p>This study identifies a&#xa0;MyD88 microglia subset in cerebral vascular accident (CVA) through scRNA-seq. Combined with ELISA and cell co-culture assays, the findings demonstrate that MyD88 knockdown reduces the production of pro-inflammatory cytokines and alleviates the polarization effect of LPS on microglia and the inhibitory effects on EC migration and angiogenesis. These findings confirm that MyD88 is a&#xa0;key regulatory factor in IS-induced brain injury. Targeting the Myd88 gene expression pathway may be a&#xa0;crucial therapeutic strategy for IS treatment and disease progression inhibition.</p>

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MyD88-induced microglia polarization promotes inflammatory response in ischemic stroke: a single-cell transcriptomic analysis

  • 期刊论文

  • 发表日期：

    2025年12月1日

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  • Huixiao Wang,
  • Zhou Lu,
  • Jiajie Gu,
  • Feng Gao

摘要

Objective

Myd88 may mediate the inflammatory response after ischemic stroke (IS) by regulating the microglia-associated Toll-like receptor (TLR) signaling pathway, but this requires verification. The present study aims to investigate the molecular mechanism of Myd88 in inflammatory signal transduction after IS through comprehensive single-cell RNA sequencing (scRNA-seq) and experimental verification.

Methods

For scRNA-seq analysis, cell subpopulations, including Myd88+ microglia, were identified using UMAP dimensionality reduction and cell marker gene annotation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted based on cell marker genes of Myd88+ microglia. The interactions between Myd88+ microglia and other cell subpopulations were inferred by cell communication analysis. MyD88, iNOS, and CD86 expression levels were detected by quantitative real-time PCR (qRT-PCR). The effects of MyD88 knockdown on microglia and endothelial cells (ECs) were verified by enzyme-linked immunosorbent assay (ELISA) and transwell assays.

Results

Annotation of cell subpopulations revealed that Myd88+ microglia were significantly enriched in IS, showing upregulation of inflammatory and immune cell migration-related pathways, including the TLR signaling pathway. Cell communication analysis indicated close interactions between Myd88+ microglia and endothelial cells. Knockdown of MyD88 alleviated the polarization effect of lipopolysaccharide (LPS) on microglia, reduced the secretion of pro-inflammatory cytokines, and attenuated the inhibitory effects on EC migration and angiogenesis.

Conclusion

This study identifies a MyD88 microglia subset in cerebral vascular accident (CVA) through scRNA-seq. Combined with ELISA and cell co-culture assays, the findings demonstrate that MyD88 knockdown reduces the production of pro-inflammatory cytokines and alleviates the polarization effect of LPS on microglia and the inhibitory effects on EC migration and angiogenesis. These findings confirm that MyD88 is a key regulatory factor in IS-induced brain injury. Targeting the Myd88 gene expression pathway may be a crucial therapeutic strategy for IS treatment and disease progression inhibition.