Abstract Rationale Alveolar macrophages are an innate immune cell critical for bacterial clearance. However, persistent macrophage activation leads to ongoing lung injury or acute respiratory distress syndrome (ARDS). Our group found that the mechanosensitive, cation channel, TRPV4, protects the lung from bacterial pneumonia associated lung injury in vivo. This study seeks to find the molecular mechanism by which TRPV4 exerts its protective effect. Methods The lung injury response was assessed by bronchoalveolar lavage fluid cytokines in control (Trpv4fl/fl) and alveolar macrophage-specific Trpv4 KO (Trpv4fl/fl X Cd11cCre) mice after intratracheal installation of Pseudomonas aeruginosa. Post-translational modification of TRPV4 and TRPV4-NF-κB complex identification was determined by mass spectrometry. In silico modeling was performed using AlphaFold. To define the role of the TRPV4 phosphorylation site, co-immunoprecipitation was performed upon overexpressing TRPV4 full-length and TRPV4 phosphorylation mutants (S159A: phosphodeficient and S159D: phosphomimetic) in macrophages. mRNA and cellular localization were analyzed by qPCR and immunofluorescence respectively. The effect of matrix stiffness was assessed by plating macrophages on polyacrylamide hydrogels of pathophysiologic-range lung stiffness (1-25 kPa). NF-ĸB promoter activity was assessed using NF-κB reporter cells. Results Alveolar macrophage-specific Trpv4 KO mice had increased pro-inflammatory cytokine expression in alveolar macrophages and increased lung injury upon P. aeruginosa pneumonia in vivo, suggesting macrophage TRPV4 functions to inhibit critical mechanisms of lung injury. TRPV4 was found to inhibit NF-κB promoter activity and bind to components of the NF-κB complex, as shown by multiple independent complementary techniques. Wild-type TRPV4 overexpression results in a matrix stiffness-dependent inhibition of NF-κB promoter activity in macrophages. In silico modeling of TRPV4-NF-κB/p65 interaction demonstrates favorable binding kinetics that are enhanced by modeling phosphorylation of TRPV4 at Serine 159 (S159) in its intracellular amino-terminal region. Enhanced binding of phosphorylated S159 TRPV4 and NF-κB was confirmed by co-immunoprecipitation and mass spectrometry. Furthermore, expression of TRPV4 phosphomimetic (S159D) mutant localizes TRPV4 to the cytoplasm, inhibits NF-κB promoter activity and decreases pro-inflammatory gene expression in LPS treated macrophages. Conversely, phosphodeficient TRPV4 mutant exhibits plasma membrane localization, and does not bind to NF-κB. These data demonstrate that TRPV4 S159 phosphorylation is the key event for binding and inhibition of NF-κB activity. Conclusions Collectively, these data demonstrate macrophage TRPV4 downregulates NF-κB activity in a matrix stiffness-dependent manner. Phosphorylation of TRPV4 at S159 is the critical event driving TRPV4’s protective effect. The NF-κB interaction with phosphorylated TRPV4 at S159 is a novel therapeutic target for ameliorating ARDS after bacterial pneumonia. This abstract is funded by: NIH
Boulton et al. (Fri,) studied this question.