Uncovering a new player in ischemic stroke: a study of intra-arterial interferon-gamma-producing monocytes in hyperacute stroke

Abstract

Stroke triggers a rapid and complex immune response that is not yet fully understood, especially within hours after an ischemic infarct. Our previous study in stroke patients revealed a significant increase in interferon- gamma (IFN-{gamma}) immediately (hyperacute) and downstream of the ischemic ictus, within the arterial compartment. The present study investigated the source, inciting factors, and role of IFN-{gamma} in a preclinical murine model. Stroke was produced using transient middle cerebral artery occlusion, and immune cells within the arterial vasculature distal to the occlusion (pre- and post-occlusion) were characterized using flow cytometry. Compared with the control samples, the post-occlusion samples presented an increase in IFN-{gamma}+ and CD69+ cells, whereas no significant increase was detected in IL17+, IL4+, and CD25+ cells. Further analysis of the IFN-{gamma}+ population revealed two novel attributes. First, interrogation of the identity of these IFN-{gamma}+ cells revealed that the increase in IFN-{gamma} production was largely driven by CD14+ cells in the post- occlusion sample, with negligible contributions from other canonical IFN-{gamma}-producing cells (CD4, CD8). Second, the IFN-{gamma}+ cells exhibited two distinct clusters, an IFN-{gamma}low and an IFN-{gamma}hi population. Further analysis revealed that the IFN-{gamma} low population was largely composed of CD14+ cells, whereas the IFN-{gamma}hi population was dominated by CD4+ T-cells. To explore the conditions driving IFN-{gamma} production, an in vitro ischemia model involving oxygen-glucose deprivation (OGD) was employed. Co-culturing of naive splenocytes with OGD-treated CNS cells and OGD-derived supernatant resulted in a significant increase in IFN-{gamma}+CD14+ cells, as compared to normoxic controls, an effect that coincided with marked loss of DAPI+ and NeuN+DAPI+ cells in mixed cortical (neuronal and glial) cultures. In summary, this study identified intra-arterial CD14+ monocytes as novel early sources of IFN-{gamma} in the hyperacute phase of stroke, a role traditionally attributed to adaptive immune cells. Using in vivo and in vitro ischemia models, the findings reveal that injury-associated signals from CNS cells are sufficient to directly induce IFN-{gamma} production in CD14+ cells, redefining early stroke immunopathology and uncovering a potential target for timely immunomodulation.