PB0723 - Protease-activated Receptor 4 differentially modulates TLR3-dependent responses in vitro and in vivo.

Background: PAR4 is expressed by immune cells, and lung epithelial cells. Recent studies showed an important role for PAR4 in modulating the inflammatory in different cell-types. We showed that global PAR4 deficiency was associated with increased influenza A virus pathology in mice. PARs can modulate toll-like receptor 3 (TLR3) and TRPV4 activity.

Aims: To investigate the role of PAR4 in TLR3-dependent responses.

Methods: In vitro studies with murine lung epithelial cells (EpC E10), macrophages (J774-Dual or Raw264.7-Dual cells) were performed. Cells were stimulated with poly IC and PAR4 activators. Sterile lung inflammation was induced in vivo by single intranasally application of polyIC (100μg in 50μL PBS) or repeated daily application for 3 days to PAR4-/- mice (ΔPAR4) or mice with PAR4 deficiency in dendritic cells and alveolar macrophages (PAR4ΔCD11c). In addition, mice were injected with 8mg/kg polyIC and responses analyzed 2hrs later. Lastly, PAR4-/- mice were infected with mouse-adapted SARS-CoV2.

Results: Experiments showed that PAR4 stimulation during polyIC incubation enhanced IFN-response in E10 and J774 cells compared to polyIC alone. PAR4 stimulation reduced NFκB-pathway activation and CXCL1 expression during polyIC stimulation. Reduced NFκB-pathway activation during co-stimulation of PAR4 and TLR3 was due to PAR4-dependent inhibition of TRPV4. PAR4-/- mice exhibited increased levels of CXCL1, CCL2 and CCL5 levels in the BALF one day after polyIC application compared to controls. Moreover, PAR4ΔCD11c mice exhibited similar increase in proinflammatory cells in the BALF, plasma or spleen after repeated i.n. poly IC application or single polyIC injection, respectively. Importantly, PAR4-/- mice exhibited increased SARS-CoV2 virus load in the lung compared to controls.

Conclusion(s): PAR4 enhances antiviral TLR3:IRF3 responses by an unknown mechanism. Interestingly, PAR4 reduces the activity of the Ca2+ channel TRPV4. TRPV4 activity is linked to increased NFκB pathway activation. PAR4-dependent TRPV4 inhibition may explain why PAR4 activation reduces the TLR3:NFκB proinflammatory responses in vitro and vivo.