Platelet-derived HMGB1 induces NETosis, exacerbating brain damage in the photothrombotic stroke model.

Following cerebral ischemia, neutrophil extracellular traps (NETs) contribute significantly to brain damage by exacerbating delayed immune cell infiltration and vascular injury. They are detected both in brain tissue and within blood vessels. Danger-associated molecular pattern (DAMP) molecules have been implicated in inducing NETosis after cerebral ischemia. This study investigated the role of High mobility group box 1 (HMGB1), a prototype DAMP molecule, in NETosis induction following photothrombotic stroke (PTS), with a particular focus on neutrophil-platelet interactions. In PTS, thrombi consist primarily of aggregated platelets and neutrophils, lacking significant fibrin content. Triphenyltetrazolium chloride (TTC) staining revealed rapid but progressive expansion of the infarct area in the PTS model, commencing within 1 h and continuing until 24 h. Concomitant with this, peripheral neutrophils isolated following PTS exhibited progressive NETosis, particularly intravascular NETosis. This was evidenced by significant increase in citrullinated histone H3 (CitH3), a marker of NETosis, as early as 1 h post-PTS. Furthermore, serum levels of free DNA gradually and significantly increased, further supporting the induction of NETosis following PTS. Intranasal administration of BBCA, a peptidylarginine deiminase (PAD) inhibitor, effectively suppressed the induction of intravascular NETosis. Importantly, BBCA administration, both 30 min before and 4 h after PTS surgery, significantly reduced infarct volumes at 24 h and improved neurological outcomes. These findings underscore the crucial role of NETosis in both the initiation and progression of ischemic brain damage in this model. Following PTS, HMGB1 rapidly accumulated in serum, detectable as early as 1 h. Immunofluorescence staining revealed initial localization of HMGB1 in neurons, followed by its accumulation within activated neutrophils and platelets within blood vessels. Functional inhibition of HMGB1 by intranasal administration of an HMGB1 A box 4 h post-PTS significantly suppressed NETosis induction, reduced infarct volume, and improved neurological deficits, confirming the pivotal role of HMGB1 in NETosis induction. Notably, we observed a rapid platelet activation and concomitant HMGB1 induction within activated platelets after PTS. Co-culture experiments using naïve PMNs-platelets isolated following PTS demonstrate that extracellular HMGB1, particularly one derived from platelets, plays a critical role in activating neutrophils and inducing intravascular NETosis via a TLR4-dependent manner. Collectively, these findings highlight the critical role of NETosis not only in the initial stages of thrombus formation but also in the subsequent progression of ischemic brain damage in the PTS animal model. HMGB1, particularly platelet-derived HMGB1, emerges as a key mediator to this process. Therefore, targeting NETosis through modulation of HMGB1 presents a promising multipotent therapeutic strategy for mitigating ischemic brain damage.