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.