Elevated Brain Tissue miR-223-3p After Ischemic Stroke Reflects Immune Infiltration Rather Than Intrinsic Upregulation

Ischemic stroke triggers a rapid immune infiltration to the brain, reshaping its cell type composition and possibly confounding the analysis of immune cell enriched molecules. MicroRNAs (miRNAs) act as powerful regulators by fine-tuning messenger RNA (mRNA) expression, thereby modulating cell type specific responses to ischemia. Several bulk tissue analyses suggest elevated levels of anti-inflammatory miRNA, miR-223-3p, in ischemic stroke but pathological relevance of this deregulation remains unclear due to lack of single-cell resolution. We show that miR-223-3p is acutely increased in ischemic stroke patients’ blood and selectively expressed in myeloid cells in mice. Due to myeloid specificity of miR-223-3p, it appears massively elevated in bulk ischemic brain lysates, although the elevation primarily reflects increased myeloid cell abundance rather than per-cell upregulation in the brain. Strikingly, brain infiltrating macrophages exhibited acutely reduced miR-223-3p levels. We hypothesize that these macrophages initially acquire a more proinflammatory phenotype than the brain resident macrophages, and re-analysis of single-cell transcriptomics (GSE234052) further supported a proinflammatory macrophage phenotype after acute cerebral ischemia. Our study exemplifies the inherent limitations of bulk tissue analyses and underscores the need to revisit conclusions drawn from the tissue-level data, especially when immune cell infiltration and enrichment of a molecule is suspected. Overcoming the technical challenges of integrated spatial miRNA and mRNA detection at single-cell resolution will be essential to dissect the regulatory mechanisms underlying ischemic stroke pathology and to inform the development of effective immunomodulatory therapies. Graphical Abstract Increased brain miR-223-3p in ischemic stroke is caused by changes in cell type composition. In healthy brain, miR-223-3p is expressed by microglia and resident macrophages. Ischemia causes infiltration of miR-223-3p-rich peripheral myeloid cells to the brain, increasing the level of miR-223-3p in tissue lysates. In ischemic macrophages, miR-223-3p was decreased while no deregulation was found in microglia.