PB0724 - TF-FVIIa-PAR2-β-arrestin signaling regulates monocyte/macrophage responses to double stranded RNA

Background: G-protein coupled protease activated receptors (PARs) are cleaved by coagulation proteases and connect hemostasis with the innate immune response. Tissue factor (TF) is the receptor for the coagulation proteases FVIIa and FXa which cleave PAR2, followed by signaling via G proteins or via recruitment of ß-arrestin for cytosolic mitogen activated protein kinase (MAPK) activation. TF-FVIIa signaling stimulates MAPK and activates cancer cell migration, but functions of TF signaling in immune cell migration are unknown.

Aims: We hypothesized that the TF-FVIIa-PAR2-β-arrestin axis regulates monocyte/macrophage migration in inflammation. We studied the role of TF signaling by inducing inflammation with the double stranded RNA Poly(I:C), which mimics RNA generated during viral infections and activates TLR3 or cytosolic RNA sensors (RIG-I/MAVS).

Methods: We analyzed migration of isolated primary peritoneal macrophages and bone marrow monocytes on defined extracellular matrices after Poly(I:C) stimulation. We challenged mouse strains with mutations in the TF-FVIIa-PAR2-β-arrestin axis with Poly(I:C) in vivo and determined lung inflammation.

Results: Poly(I:C) stimulated migration of monocytes/macrophages dependent on MAPK activation and RIG-I/MAVS, but independent of TLR3. TF, myeloid-cell expressed FVIIa, and cleavage of PAR2 were specifically required for integrin αMβ2-dependent migration on fibrin, but not for integrin β1-dependent migration on fibronectin. To further analyze this pathway, we generated PAR2A365A368 mutant mice deficient in β-arrestin coupling. Macrophages from these mice showed reduced cytosolic, but same nuclear MAPK phosphorylation and were defective in migration on fibrin following Poly(I:C) stimulation. In addition, loss of the TF-FVIIa-PAR2-β-arrestin axis markedly reduced lung inflammation, as indicated by alveolar infiltration of monocytes and macrophages after intranasal Poly(I:C) challenge.

Conclusion(s): These results identify biased PAR2 signaling through coupling to β-arrestin as a crucial pathway for regulating innate immune cell migration and implicate cell intrinsic TF-FVIIa-PAR2 signaling in RNA-triggered inflammation with potential implications for viral infections in vivo.