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A6231 - Host Matricellular Protein Thrombospondin-1 Limits Extracellular Matrix Proteolysis During Acute Pseudomonas Aeruginosa-Induced Lung Injury
Author Block: Y. Qu1, T. F. Olonisakin1, W. Bain1, M. Hulver1, Z. Xiong1, M. E. Zegans2, R. M. Shanks3, J. Bomberger4, J. M. Pilewski1, A. Ray1, Z. Cheng5, P. Ray1, J. Lee1; 1Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2Department of Microbiology and Immunology, Geisel School of Medicine,Dartmouth College, Hanover, NH, United States, 3Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, United States, 4Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, United States, 5Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada.
Rationale: Pseudomonas aeruginosa (PA) is a Gram-negative extracellular bacterial pathogen that secretes exoproducts including proteases to disarm the host. However, host factors that protect against pathogen-triggered matrix proteolysis during acute lung infection are poorly understood. Thrombospondin-1 is a host matricellular protein released extracellularly following tissue injury and displays inhibitory activity against serine proteases such as neutrophil elastase (NE). We tested the hypothesis that thrombospondin-1 counters proteolytic injury in the lungs during acute PA intrapulmonary infection. Methods: PA14, PA14 lasB::Tn5 containing a transposon insertion within the lasB gene, and PA14ΔxcpQ deletion mutant of the type II secretion system XcpQ outer membrane protein were used. Thbs1-/- and WT mice were inoculated with PA strains at an inoculum of 106 CFU and mice were harvested 20 h post-infection. Lung tissue homogenate CFU, MPO, and cytokines were measured. BAL was also measured for cell counts and differential, total protein concentrations, and NE activity. In some experiments, a synthetic dipeptide LasB inhibitor N-mercaptoacetyl-Phe-Tyr-amide (LasBI) was administered intraperitoneally 2h post-infection. In other experiments, purified TSP-1 from platelets or platelet releasates from WT and Thbs1-/- mice were used to assess ability of TSP-1 to counter PA elastolytic activity in vitro and exaggerated inflammation in vivo of WT and Thbs1-/- mice. Cell-free supernatants (SN) from PA strains were tested for total protease, elastase, and LasB activity using fluorogenic substrates in the presence or absence of LasBI or purified TSP-1. Results: Following acute intrapulmonary PA infection, Thbs1-/- mice show higher lung bacterial burden, increased microvascular permeability, increased BAL NE activity, as well as higher lung IL-17A, GM-CSF, and IL-6 concentrations compared to WT mice. Administration of TSP-1 reduces lung bacterial burden, airspace neutrophil recruitment, lung NE activity, IL-17A, GM-CSF, and IL-6 concentrations in Thbs1-/- mice. We identified TSP-1 as a potent inhibitor of Pseudomonas elastase activity of which the type II secretion endo-metalloprotease LasB is the predominant source. Although TSP-1 is cleaved into two fragments by PA14 SN but not by SN from PA14 lasB::Tn5 lacking LasB activity, TSP-1 retains ability to substantially inhibit PA elastolytic activity in vitro. Administration of LasB inhibitor post-infection or genetically disabling the PA type II secretion system or LasB gene decreases lung bacterial burden and counters the exaggerated neutrophilic response in Thbs1-/- mice. Conclusions: Host matricellular protein TSP-1 limits microbial-triggered proteolytic injury by countering the actions of P. aeruginosa virulence factor LasB during acute intrapulmonary infection and tempering pathogenic neutrophilic inflammation.