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The Mechanosensitive TRPV4 Ion Channel Mediates the Pro-resolution/Anti-fibrotic Macrophage Response to LPS Rachel G. Scheraga, MD Cleveland Clinic Cleveland, OH Bacterial pneumonia in Idiopathic pulmonary fibrosis (IPF) • Recurrent pneumonia in IPF patients causes progressive decline in lung function • Infection-associated lung injury leads to edema and inflammatory cell infiltration causing increased matrix stiffness and secretion of cytokines Molyneaux et al. Am J Respir Crit Care Med, 2014 Murray et al. Nat Rev Immunol , 2001 Wynn et al. Nature, 2013 Macrophage phagocytosis • Critical to lung host defense and lung injury, repair, and fibrosis • Requires orchestration of macrophage surface receptors, the particle itself, and extracellular matrix Rougerie et al. Immunol. Rev, 2013 Wynn et al. Nature, 2013 Blakney et al. J Biomed Mater Res A, 2012 Transient receptor potential vanilloid 4 (TRPV4) • Mechanosensitive cation-permeable channel that is activated by chemical and physical stimuli, including plasma membrane stretching • Ubiquitous cell type and tissue expression • Our lab is the first to investigate the significance of TRPV4 in lung fibrosis Montell et al. Cell, 2002; Everaerts et al; Prog Biophys Mol Biol, 2010 Hypothesis TRPV4 integrates the extracellular matrix stiffness and LPS signals to mediate infection-associated lung injury and repair TRPV4 mediates macrophage phagocytosis of IgG-coated beads UT LPS 400 * 300 * 200 si R H C N O K TR PV 4 T W A 100 0 TRPV4 KO-UT * 4 WT-LPS + TR PV WT-UT Fluorescence Intensity (AU) 500 TRPV4 KO-LPS Scheraga et al. J Immunol, In press TRPV4 mediates macrophage phagocytosis of E. coli particles AM BMDM 80 C H N 4 PV TR TR PV si 4 R K T O 40 A 60 * 80 60 40 O 100 100 K 120 4 * * 120 TR PV * 140 T 160 UT LPS W UT LPS Fluorescence Intensity (AU) (Normalized to WT UT) 140 W Fluorescence Intensity (AU) (Normalized to WT UT) 180 + + Scheraga et al. J Immunol, In press 40 20 ss G la kP a 25 kP a 8 kP a 0 80 60 # # # # 40 20 0 ss 60 100 G la + 120 WT-No HC (TRPV4 inhibitor) TRPV4 KO-No HC WT+HC 25 kP a 80 Phagocytosis, % Induction by LPS (Fluorescence Intensity per cell) + 100 1 Phagocytosis, % Induction by LPS (Fluorescence Intensity per cell) The TRPV4-mediated phagocytic effect depends on matrix stiffness Scheraga et al. J Immunol, In press ** 200 150 100 50 0 WT KO LPS-stimulated IL-10 (% Increase above UT) LPS-stimulated IL-1b (% Increase above UT) TRPV4 modulates an anti-inflammatory cytokine profile in response to LPS 120 100 80 * 60 40 20 0 WT KO Scheraga et al. J Immunol, In press TRPV4 mediates macrophage phagocytosis of IgG-coated beads in live mice Saline LPS WT # beads/macrophage 20 KO + IT SALINE IT LPS 15 10 * 5 0 WT KO Scheraga et al. J Immunol, In press # Macs # PMNs # Lymph 400 * 300 * 200 100 Day 3 O K T WT TRPV4 KO * 600 500 400 300 200 100 PV 4 W TR PV 4 W K T O 0 700 Total protein (mg/mL) [ g/ml] 500 TR Absolute # BAL cells (x100/mL) TRPV4 limits lung injury and initiates resolution in a bacterial pneumonia model Day 8 0 Day 3 Day 8 Conclusions • These data suggest : - TRPV4 mediates the phagocytic and cytokine profile response to infection - stiffness-dependent manner • May play a role in progressive fibrosis seen in patients with IPF after infection Acknowledgements Cleveland Clinic - Pathobiology Dr. Mitchell Olman Dr. Brian Southern Dr. Lisa Grove Susamma Abraham Kathryn Niese Cleveland Clinic - Immunology Dr. Thomas Hamilton Dr. Chenyang Zhao Dr. Olman’s Lab Questions?