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

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-γ) immediately (hyperacute) and downstream of the ischemic ictus, within the arterial compartment. The present study investigated the source, inciting factors, and role of IFN-γ 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-γ(+) and CD69(+) cells, whereas no significant increase was detected in IL17(+), IL4(+), and CD25(+) cells. Further analysis of the IFN-γ(+) population revealed two novel attributes. First, interrogation of the identity of these IFN-γ(+) cells revealed that the increase in IFN-γ production was largely driven by CD14(+) cells in the post-occlusion sample, with negligible contributions from other canonical IFN-γ-producing cells (CD4, CD8). Second, the IFN-γ(+) cells exhibited two distinct clusters, an IFN-γ(low) and an IFN-γ(hi) population. Further analysis revealed that the IFN-γ (low) population was largely composed of CD14(+) cells, whereas the IFN-γ(hi) population was dominated by CD4(+) T-cells. To explore the conditions driving IFN-γ production, an in vitro ischemia model involving oxygen-glucose deprivation (OGD) was employed. Co-culturing of naïve splenocytes with OGD-treated CNS cells and OGD-derived supernatant resulted in a significant increase in IFN-γ(+)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-γ 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-γ production in CD14(+) cells, redefining early stroke immunopathology and uncovering a potential target for timely immunomodulation.