OC 72.1 - Platelet HMGB1 mediates pathological NET formation in Ischemic Stroke

Wednesday, July 13, 2022

10:15 AM – 10:30 AM

Location: ExCel Center, ICC Capital Suite Room 7&12

Presentation Format: In-Person

Presenting Author(s)

Frederik Denorme, PhD

Postdoc
University of Utah
Salt Lake City, Utah, United States

Co-Author(s)

Irina Portier, PhD

Postdoc
University of Utah
Salt Lake City, Utah, United States
YK

Yasuhiro Kosaka

Research Scientist
University of Utah Molecular Medicine Program
Salt Lake City, Utah, United States
JR

John L. Rustad

Laboratory Technician
University of Utah
Salt Lake City, Utah, United States
MC

Mark J. Cody

Laboratory Technician
University of Utah
Salt Lake City, Utah, United States
CY

Christian C. Yost

Professor
University of Utah
Salt Lake City, Utah, United States
Matthew D. Neal, MD

Associate Professor
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
JM

Jennifer J. Majersik

Professor
University of Utah
Salt Lake City, Utah, United States
RC

Robert A A. Campbell, PhD

Professor
Molecular Medicine Program, University of Utah, Salt Lake City, UT, USA; Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA
Salt Lake City, Utah, United States

Background: Ischemic stroke provokes a strong inflammatory response which is associated with worse outcomes in patients. Classic anti-inflammatory strategies have been unsuccessful in clinical trials for ischemic stroke, implying other mechanisms contribute to injurious inflammation in ischemic stroke.

Aims: In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and if they contribute to ischemic stroke outcomes.

Methods: Brain tissue and plasma from ischemic stroke patients and healthy matched controls were analyzed for the presence of NETs (n=28 per group). Flow cytometry was used to characterize platelet function. For murine stroke studies, mice were subjected to transient middle cerebral artery occlusion.

Results: NET forming neutrophils were found in brain tissue of all stroke patients examined while NETs were absent in healthy brain tissue. Specific markers of NET formation including citrullinated histone H3 (H3cit) and MPO-DNA complexes were significantly elevated in plasma from stroke patients compared to controls. Interestingly, H3cit and MPO-DNA complexes significantly correlated with worse long-term stroke outcomes (r=0.45, p=0.024 and r=0.507, p=0.01, respectively).

Next, we observed increased plasma and platelet-surface expressed high mobility group box 1 (HMGB1) in ischemic stroke patients compared to matched controls, and HMGB1 correlated with plasma NETs (r=0.433, p=0.0019). Blocking HMGB1 in vitro prevented platelet-induced NET formation. Mechanistically, depleting platelets in mice before stroke reduced plasma HMGB1 levels as well as NET formation and improved outcomes after ischemic stroke (p < 0.005). Treatment of thrombocytopenic mice with recombinant HMGB1 increased stroke-induced NETs to similar levels of non-platelet depleted mice and exacerbated stroke outcomes. In agreement with these results, platelet-specific HMGB1 knockout mice had significantly reduced NETs after stroke and greatly improved stroke outcomes (p < 0.001).

Conclusion(s): Our results support a pathological role for platelet derived HMGB1 in mediating NET formation in ischemic stroke and warrant further investigation into targeting NETs to improve stroke outcomes.