TLR7 (Toll-Like Receptor 7) Facilitates Heme Scavenging Through the BTK (Bruton Tyrosine Kinase)–CRT (Calreticulin)–LRP1 (Low-Density Lipoprotein Receptor–Related Protein-1)–Hx (Hemopexin) Pathway in Murine Intracerebral Hemorrhage

Abstract

Background and Purpose— Heme and iron are considered to be key factors responsible for secondary insults after intracerebral hemorrhage (ICH). Our previous study showed that LRP1 (low-density lipoprotein receptor–related protein-1)–Hx (hemopexin) facilitates removal of heme. The TLR7 (Toll-like receptor 7)–BTK (Bruton tyrosine kinase)–CRT (calreticulin) pathway regulates the expression of LRP1-Hx. This study is designed to clarify whether TLR7 activation facilitates heme scavenging and to establish the potential role of the BTK-CRT-LRP1-Hx signaling pathway in the pathophysiology of ICH. Methods— ICH was induced by stereotactic, intrastriatal injection of type VII collagenase. Mice received TLR7 agonist (imiquimod) via intraperitoneal injection after ICH induction. TLR7 inhibitor (ODN2088), BTK inhibitor (LFM-A13), and CRT agonist (thapsigargin) were given in different groups to further evaluate the underlying pathway. Mice were randomly divided into sham, ICH+vehicle (normal saline), ICH+Imiquimod (2.5, 5, and 10 μg/g), ICH+ODN2088, ICH+LFM-A13, ICH+thapsigargin, and ICH+ODN2088+thapsigargin. Imiquimod was administered twice daily starting at 6 hours after ICH; ODN2088 was administered by intracerebroventricular injection at 30 minutes, and LFM-A13 or thapsigargin was administered by intraperitoneal injection at 3 hours after ICH induction. Neurological scores, cognitive abilities, as well as brain edema, blood-brain barrier permeability, hemoglobin level, brain expression of TLR7/BTK/CRT/LRP1/Hx were analyzed. Results— Low dosage imiquimod significantly attenuated hematoma volume, brain edema, BBB permeability, and neurological deficits after ICH. Imiquimod also increased protein expressions of TLR7, BTK, CRT, LRP1, and Hx; ODN2088 reduced TLR7, BTK, CRT, LRP1, and Hx expressions. Conclusions— TLR7 plays an important role in heme scavenging after ICH by modulating the BTK-CRT-LRP1-Hx pathway. TLR7 may offer protective effects by promoting heme resolution and reduction of brain edema after ICH.