Inflammation and Tissue Damage Mediated By PAR-1 Expression Is Cell Type-Specific and Varies Depending on the Inflammatory Challenge

Protease activated receptor-1 (PAR-1) is a G protein-coupled receptor activated by cleavage of the extracellular domain, revealing an encrypted ligand. PAR-1 can be activated by several proteases, including thrombin and activated protein C, coupling hemostatic proteases to cell signaling. PAR-1 activation is a significant determinant of the inflammatory response in multiple settings. However, the fact that PAR-1 is expressed on many cell types makes a mechanistic understanding of the effector mechanisms challenging. We hypothesized that the effector cells expressing PAR-1 that drive inflammatory functions are contextually dependent on the type of inflammatory challenge. To test this hypothesis, we used recently-generated mice carrying a conditional (floxed) PAR-1 allele (PAR-1^fl/fl) interbred with established transgenic mice expressing Cre recombinase in a cell type-specific manner in two experimental mouse models, an acetaminophen (APAP) hepatotoxicity model and a Citrobacter rodentium infectious colitis model. Cre recombinase was expressed under the control of the CMV promoter (global PAR-1 deletion), the Tie2 promoter (endothelial and hematopoietic cell PAR-1 deletion), and the LysM promoter (myeloid PAR-1 deletion). An established hepatocyte-tropic AAV Cre recombinase vector was used to delete hepatocyte-associated PAR-1.

To define the cell type-specific role of PAR-1 in APAP induced liver damage, mice were challenged with APAP and euthanized 24 hours later for analyses. Control mice exposed to toxic APAP doses had a 10-100 fold increase in serum alanine transaminase (ALT) levels and ~50% hepatocyte necrosis based on morphometric analyses of liver tissue sections. Consistent with previous results, APAP-challenged mice with global PAR-1 deletion (CMV Cre/PAR-1^fl/fl) demonstrated significantly lower serum ALT levels relative to control animals (PAR-1^fl/fl) and significantly less hepatocyte necrosis. Neither AAV-Cre mediated deletion of PAR-1 in hepatocytes nor LysM Cre-mediated deletion in myeloid cells had any significant impact on APAP-induced liver injury. In contrast, deletion of PAR-1 in the endothelial and hematopoietic compartments (Tie2 Cre) phenocopied global PAR-1 deletion, resulting in a significant diminution in serum ALT and a 30% decrease in APAP-induced hepatocyte necrosis. Together, these results suggest that either a non-myeloid leukocyte and/or endothelial cells are the effector cells expressing PAR-1 that promote liver damage in this experimental context.

To define the role of PAR-1 in the setting of infectious colitis, mice were challenged with C. rodentium by oral gavage and euthanized 14 days later for analyses. Global deletion of PAR-1 resulted in significantly less weight loss, longer colonic crypts, and a local diminution in multiple inflammatory cytokines (IFNγ, IL-1β, IL-17A, IL-22, and IL-6). In contrast to what was observed in APAP-induced liver injury, cell type-specific deletion of PAR-1 in myeloid cells (LysM Cre) resulted in a phenocopy of global PAR-1 deficiency, including blunted weight loss and longer colonic crypt lengths. The difference in colonic crypt length appeared to be due, at least in part, to genotype-dependent differences in colonic epithelial apoptosis, demonstrated by diminished staining for cleaved caspase 3 in crypts harvested from LysM Cre/PAR-1^fl/fl mice relative to controls. Deletion of myeloid-associated PAR-1 also resulted in a local diminution in several cytokines (IFNγ, IL-1β, IL-17A, and IL-6) similar to global PAR-1 deficiency.

Together, these results strengthen previous data showing the importance of PAR-1 in mediating multiple inflammatory pathologies. These data also highlight the finding that the specific effector cells expressing PAR-1 that promote inflammatory processes may differ depending on the type of inflammatory challenge. A detailed effector cell-specific understanding of the role of PAR-1 is critical to understanding the mechanisms coupling hemostatic proteases to inflammation and tissue injury and for defining the potential of PAR-1 as a target to mitigate these pathologies.