Hydroxychloroquine Attenuates Myocardial Ischemic and Post-ischemic Reperfusion Injury by Inhibiting the Toll-like Receptor 9 – Type I Interferon Pathway

Abstract Background: Hydroxychloroquine (HCQ) is a toll-like receptor (TLR) 7 and 9 inhibitor that has been used clinically for its anti-inflammatory effects. We hypothesized that HCQ would attenuate myocardial ischemia/reperfusion (IR) injury by inhibiting the TLR9 – type I interferon (IFN-I) pathway. Methods: The left coronary artery of wild-type (WT) C57BL/6 and congenic TLR9-/- mice was occluded for 40 minutes to induce myocardial ischemia, with or without 60 minutes of subsequent reperfusion (40’/0’ or 40’/60’). Treatment with TLR9 inhibitors ODN-2088 or HCQ, or with TLR9 agonist ODN-1826 was administered either before ischemia or before reperfusion to determine effect on infarct size (IS). Myocardial IS as a percentage of risk region was measured by TTC-Blue staining. After 40’/0’, cardiac perfusate (CP) was collected from harvested hearts and used to treat either intact WT mice after 20 minutes of ischemia or isolated splenocytes. IFNa and IFNb levels were measured in plasma and splenocyte culture supernatant, and HMGB1 and cfDNA levels were measured in CP. Results: After 40’/60’ of IR, WT mice treated with HCQ or ODN-2088 had significantly reduced IS. Both 40’/0’ ischemic injury and 40’/60’ IR injury were similarly attenuated in TLR9-/- mice and HCQ-treated WT mice. HCQ did not further reduce IS in TLR9-/- mice. TLR9-/- and HCQ-treated WT mice had significantly lower IFNa and IFNb in CP after 40’/0’ ischemic injury and in plasma after 40’/60’ IR. 40’/0’ CP or ODN-1826 significantly increased IS in WT mice undergoing 20’/60’ IR. CP-treated WT splenocytes produced significantly higher IFNa and IFNb in culture supernatant at 2 hours. HCQ significantly reduced the production of IFNa and IFNb. Conclusions: The TLR9 – IFN-I-mediated inflammatory response is an important contributor to both ischemic and post-ischemic myocardial IR injury. HMGB1 and cfDNA released from ischemic myocardium activated the intra-myocardial TLR9 – IFN-I inflammatory pathway during ischemia and the extra-myocardial TLR9 – IFN-I inflammatory pathway during reperfusion. Hydroxychloroquine reduces production of IFN-I and attenuates myocardial IR injury, likely by inhibiting the TLR9 – IFN-I pathway.