Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis
Yanyan Qu, Tolani Olonisakin, William Bain, Jill Zupetic, Rebecca Brown, Mei Hulver, Zeyu Xiong, Jesus Tejero, Robert M.Q. Shanks, Jennifer M. Bomberger, Vaughn S. Cooper, Michael E. Zegans, Hyunryul Ryu, Jongyoon Han, Joseph Pilewski, Anuradha Ray, Zhenyu Cheng, Prabir Ray, Janet S. Lee
Yanyan Qu, Tolani Olonisakin, William Bain, Jill Zupetic, Rebecca Brown, Mei Hulver, Zeyu Xiong, Jesus Tejero, Robert M.Q. Shanks, Jennifer M. Bomberger, Vaughn S. Cooper, Michael E. Zegans, Hyunryul Ryu, Jongyoon Han, Joseph Pilewski, Anuradha Ray, Zhenyu Cheng, Prabir Ray, Janet S. Lee
View: Text | PDF
Research Article Pulmonology

Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis

Abstract

Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1–deficient (Thbs1–/–) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1–/– mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger.

Authors

Yanyan Qu, Tolani Olonisakin, William Bain, Jill Zupetic, Rebecca Brown, Mei Hulver, Zeyu Xiong, Jesus Tejero, Robert M.Q. Shanks, Jennifer M. Bomberger, Vaughn S. Cooper, Michael E. Zegans, Hyunryul Ryu, Jongyoon Han, Joseph Pilewski, Anuradha Ray, Zhenyu Cheng, Prabir Ray, Janet S. Lee

×

Figure 5

The majority of protease activity secreted by Pseudomonas aeruginosa (PA) exhibits the profile of a metalloprotease and TSP-1 shows dose-dependent inhibition of PA exoprotease activity.

Options: View larger image (or click on image) Download as PowerPoint
The majority of protease activity secreted by Pseudomonas aeruginosa (PA...
(A) PA cell-free supernatant (SN) was tested for total protease activity by measuring the hydrolysis of fluorogenic casein substrate over time in the presence or absence of inhibitor. Increasing width of arrowheads indicates increasing molar concentrations of inhibitors. Although SN from Klebsiella pneumoniae (Kp) grown under the same conditions shows no detectable protease activity, that from PA exhibits robust protease activity that is inhibited by EDTA (20, 50, and 100 μM). The effects of serine protease inhibitor aprotinin (20, 50, and 100 μM), and cysteine protease inhibitor E-64 (20, 50, and 100 μM) on PA protease activity are shown for comparison. (B) Recombinant human TSP-1 (rhTSP-1) dose-dependently inhibits PA protease activity (rhTSP-1 at 78, 156, and 312 nM). This is compared with the inhibitory activity of EDTA (50 μM, 500 μM, and 5 mM), and specific LasB inhibitor (LasBI) N-mercaptoacetyl-Phe-Tyr-amide (1, 10, and 100 μM). PA SN in the absence of inhibitor is the reference control set as 100% protease activity. (C) Human purified TSP-1 (200 pM, 1 nM, and 78 nM) dose-dependently inhibits PA LasB activity as measured by hydrolysis of specific LasB substrate aminobenzoyl-Ala-Gly-Leu-Ala-p-nitro-benzyl-amide. This is compared with the inhibitory activity of LasBI (78 nM, 312 nM, and 20 μM). (D) Purified LasB protein (pLasB) was incubated with human TSP-1 from thrombin-activated platelet releasates (PRs) at 1 μg or 10 μg total protein concentrations and elastase activity measured by hydrolysis of elastin substrate over time. LasBI (500 nM, 50 μM, and 100 μM) was incubated with pLasB protein and the degree of percentage pLasB elastase activity inhibition is shown as a comparison. (A–D) Assays were performed in triplicate and a representative study of 3 independent experiments is shown. Lines indicate the median.