Abstract 474: Macrophage Par2 Contributes To Influenza A Virus Pathology And Is A Potential Therapeutic Target To Treat Influenza A Virus Infection.

Abstract

Proteinase-activated receptor 2 (PAR2) was shown to enhance proinflammatory toll-like receptor 3 (TLR3) responses, while dampening TLR3-dependent antiviral response in epithelial cells. Moreover, Par2 -/- mice exhibited improved survival after influenza A virus (IAV) infection. The objective of the study is to identify PAR2 expressing cells contributing to IAV pathology, and test if PAR2 inhibition is a potential new target to treat IAV. Infection with H1N1 IAV (PR8) was analyzed in global ( Par2 -/- ), myeloid ( Par2 fl/fl ;LysM Cre+ ), neutrophil ( Par2 fl/fl ;MRP8 Cre+ ) and lung epithelial cell (EpC) Par2 deficient ( Par2 fl/fl ;SPC Cre+ ) mice and respective controls ( Par2 +/+ and Par2 fl/fl ). The effect of PAR2 activation on poly I:C activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM) and murine macrophage-like J774A.1 cells. Lastly, we analyzed PAR2 inhibition in wild-type (WT) mice using mouse-anti PAR2 antibody (SAM11) and small molecule PAR2 inhibitor ENMD-1068. Par2 -/- and Par2 fl/fl ;LysM Cre+ mice exhibited increased survival, reduced proinflammatory mediators and cellular infiltration in bronchoalveolar lavage fluid (BALF) 3 days post infection (dpi) compared to control mice. Reduced IAV pathology resulted in significantly better lung function (PenH, airway resistance) in Par2 -/- mice compared to Par2 +/+ mice 7 dpi. Neither Par2 fl/fl ;MRP8 Cre+ or Par2 fl/fl ;SPC Cre+ mice showed any survival benefit compared to Par2 fl/fl . In vitro studies showed that Par2 -/- BMDM produced less IL6 and IL12p40 than Par2 +/+ BMDM after poly I:C stimulation. J774A.1 cells stimulated with PAR2 agonist and poly I:C showed an increase in NFκB activation compared to poly I:C alone. Prophylactic PAR2 inhibition with SAM11 before and daily after IAV infection, decreased BALF inflammation and lung virus load compared to control IgG treated mice 3dpi. Therapeutic PAR2 inhibition with ENMD-1068 daily from 3 dpi resulted in improved lung function (PenH) compared to vehicle treated mice 7 dpi. Global or macrophage cell but not neutrophil or lung EpC Par2 deficiency was associated with reduced IAV-induced lung pathology and mortality. Additionally, PAR2 inhibition experiments showed PAR2 may be a therapeutic target to reduce IAV pathology.