Abstract
Introduction: Secondary inflammation is a well-established cause of injury in intracerebral hemorrhage (ICH). One potential treatment for ICH limits inflammation through targeted metabolic modulation of immune cells, but leukocyte activation states in ICH are poorly defined. Using single-cell RNA sequencing (scRNA-seq) of patient hematoma evacuates, we characterized the inflammatory and metabolic profile of over a dozen identifiable leukocyte populations. This research provides insight into the dynamics of immune activation in ICH. Methods: Evacuated hematoma and peripheral blood samples from 8 ICH patients (62 ± 2.1 years) were collected via minimally invasive surgery (n=5) or craniotomy (n=3) (Fig. 1a). Collection time ranged from 0 to 4 days after ICH onset (1.5 ± 0.5 days). scRNA-seq was done on the 10X Genomics platform and analyzed using Seurat. Human monocyte-derived macrophage cultures were stimulated with S100A9 and IL-1β, two ICH-relevant inflammatory molecules, or LPS. Echinomycin and 2-deoxy-D-glucose were used to inhibit HIF signaling and glycolysis respectively. Results: Clustering revealed over a dozen transcriptionally distinct CNS-resident and immune cell types, many of which have little prior study in ICH (Fig. 1b). 3,395 differentially expressed genes between hematoma and peripheral blood (p<0.05) were found in CD14 + monocytes and 932 in CD4 + effector T cells. Pathway analysis found upregulation of HIF signaling, glycolysis, and correlated networks of cytokine expression in the hematoma. The findings were significant across all immune populations but most prominent in myeloid cells (Fig. 1c). Inhibition of HIF and glycolysis in human macrophages in-vitro abrogated inflammatory and reparative cytokine production following inflammatory stimulation. In conclusion, this work highlights the interplay of inflammation and metabolism at a single cell level in ICH, contributing to knowledge of the immune response to brain injury.
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