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Thrombospondin-1 Restrains Neutrophil Granule Serine Protease Function and Regulates the Innate Immune Response During Klebsiella Pneumoniae Infection

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Thrombospondin-1 Restrains Neutrophil Granule Serine Protease Function and Regulates the Innate Immune Response During Klebsiella Pneumoniae Infection ARTICLES nature publishing group Thrombospondin-1 restrains neutrophil granule serine protease function and regulates the innate immune response during Klebsiella pneumoniae infection Y Zhao1, TF Olonisakin1, Z Xiong1, M Hulver1, S Sayeed1,MTYu1, AD Gregory1, EJ Kochman1, BB Chen1, RK Mallampalli1,2, M Sun3, RL Silverstein4, DB Stolz3, SD Shapiro1, A Ray1, P Ray1 and JS Lee1,5 Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1 À / À ) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild- type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1 À / À mouse lungs compared with WTcontrols. Lung NE activity was increased in thbs1 À / À mice following K. pneumoniae challenge, and thbs1 À / À neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1 À / À neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793–801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal. INTRODUCTION cell–matrix interactions.2 Thus, it is not surprising that TSP-1 Thrombospondin-1 (TSP-1) is a matricellular protein that has been implicated in a number of important biological functions exists as a trimer of identical B150–180 kDa subunits tethered such as embryogenesis, wound repair, tumor growth and together by disulfide bonds.1,2 A major source of TSP-1 is metastasis, angiogenesis, hemostasis, and inflammation.2,5,8–11 platelet a-granules, with TSP-1 being released upon platelet Others have previously shown that platelet proteins such as activation.3–5 TSP-1 also exists in plasma at low concentrations TSP-1 are found in the airspace of patients with acute under basal conditions, and is made by numerous cells respiratory distress syndrome and TSP-1 concentrations including myeloid-derived cells such as neutrophils.6,7 Given correlate with composite injury scores that quantify the degree its adhesive nature, TSP-1 can bind to the surface of platelets, of lung injury.12 The original description of mice deficient extracellular matrix, and a number of other cells including in TSP-1 (thbs1 À / À ) showed spontaneous development of fibroblasts, smooth muscle cells, endothelium, neutrophils, and non-infectious pneumonia and the predisposition to develop macrophages, providing a multiplicity of potential cell–cell and inflammation due to impaired homeostasis.13 We recently 1Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 2The Medical Specialty Service Line, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania, USA. 3Department of Cell Biology and Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 4Department of Medicine, Medical College of Wisconsin and Blood Research Institute, Blood Center of Wisconsin, Milwaukee, Wisconsin, USA and 5Vascular Medicine Institute, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. Correspondence: JS Lee ([email protected]) Received 16 July 2014; accepted 31 October 2014; published online 10 December 2014. doi:10.1038/mi.2014.120 896 VOLUME 8 NUMBER 4 | JULY 2015 | www.nature.com/mi ARTICLES demonstrated that, although thbs1 À / À mice do not sponta- stoichiometric predictions demonstrating 1 mol of TSP-1 neously develop non-infectious pneumonia, these mice exhibit interacting with 1 mol of plasmin.23 This prompted further a defect in their ability to resolve from injurious stimuli to the studies showing that TSP-1 binds and competitively inhibits the lungs.14 We have also shown that interleukin (IL)-10 is essential enzymatic activity of purified NE and cathepsin G (CG) for recovery of lung inflammation during the late phase of in vitro.3,4 These findings suggest a regulatory role for TSP-1 bacterial infection.14,15 In an experimental model of lung injury, during inflammation, but how TSP-1 modulates neutrophil we further show that the bridging function of TSP-1 is required function during the innate immune response to bacterial for optimal triggering of macrophage IL-10 production pathogens is unclear. following contact recognition of apoptotic neutrophils that 14,15 is necessary for effective resolution of inflammation. It RESULTS remains unclear, however, what the role of TSP-1 is, if any, Increased bacterial clearance from the lungs, reduced during bacterial pneumonia, an important cause of morbidity splenic dissemination, and enhanced survival following and mortality worldwide and a well-known risk factor for acute intratracheal instillation of Klebsiella pneumoniae in mice respiratory distress syndrome.16 deficient in TSP-1 Neutrophils are innate immune effector cells of microbial To investigate the role of TSP-1 in pulmonary host defense, killing and are critical for pulmonary host defense against thbs1 À / À mice were inoculated with the bacterial pathogen K. pathogen. Two major modes of intracellular killing are operant pneumoniae at 4 Â 103 colony-forming units (CFUs). At 48 and in neutrophils: (1) oxygen-dependent mechanism involving the 72 h following intratracheal inoculation, thbs1 À / À mice recruitment and activation of the NADPH oxidase complex, the showed reduced bacterial burden in the lungs compared with generation of oxygen free radicals and superoxide, resulting in wild-type (WT) mice, as measured by CFU/lung (Figure 1a). myeloperoxidase-mediated halogenation to form hypochlor- Consistent with the findings in the lungs, thbs1 À / À mice ous acid; (2) the release of cytosolic granule contents within the showed reduced splenic dissemination compared with WT phagosome, comprised of neutrophil serine proteases, the mice (Figure 1b). Thus, pulmonary host defense is enhanced activation of these proteases within the phagosome,17–19 and during bacterial pneumonia in the absence of TSP-1 that is the contribution of antimicrobial proteins.20 Neutrophil associated with reduced systemic dissemination. We next elastase (NE), a key granule serine protease, degrades outer determined whether the enhanced bacterial clearance in membrane protein A on the surface of Gram-negative thbs1 À / À mice confers a survival advantage during bacteria21 and is an important contributor to the oxygen- bacterial pneumonia with K. pneumoniae. At an inoculum 4 independent arm of microbial killing. Although effective of 1.2 Â 10 CFU, lethal dose (LD50) was achieved at 72 h in WT neutrophil microbial killing is required to thwart collateral mice (Figure 1c). Kaplan–Meier curve followed by the log-rank tissue damage and organ injury induced by microbial–host test showed enhanced survival in thbs1 À / À mice compared interactions, the host also requires mechanisms to curtail its with WT mice (P ¼ 0.02, n ¼ 20 per group). The median microbial killing arsenal to prevent subsequent collateral tissue survival for thbs1 À / À mice was 312 h (13 days), compared with damage and organ injury. Indeed, unbridled neutrophil 72 h (3 days) for WT mice. A kinetics study examining protease activity is associated with lung injury or acute responses of WT and thbs1 À / À mice 24, 48, 72, and 96 h respiratory distress syndrome in humans.22 Thus, a fine following intratracheal inoculation (1.8 Â 103 CFU) indicated balance is required for effective neutrophil microbial-killing that, by 72 and 96 h, thbs1 À / À mice were clearing bacteria from activity on one hand and the curtailment of an over-vigorous the lungs faster than WT mice (Supplementary Figure 1 host inflammatory response on the other. online). Consistent with our prior findings,24 TSP-1 is a competitive inhibitor of serine proteases such as thbs1 À / À mice show deficiency in IL-10, which is required plasmin, preventing the cleavage of fibrinogen in vitro with for optimal resolution of lung inflammation during the late 10 * * 10 * 100 9 9 WT 8 8 80 thbs1–/– 7 7 6 6 60 scale) 5 scale) 5 10 4 10 4 40 % survival Lung CFUs 3 3 (log WT (log WT Spleen CFU 2 2 –/– 20 1 thbs1–/– 1 thbs1 0 0 0 48 72 48 72 01020 30 40 Time (h) Time (h) Time (days) Figure 1 thbs1 À / À mice show enhanced lung bacterial clearance, reduced splenic dissemination, and enhanced survival following intratracheal K. pneumoniae inoculation. Colony-forming units (CFU) obtained from (a) lung tissue homogenate and (b) splenichomogenate cultures of WT and thbs1 À / À mice 48 and 72 h after intratracheal (i.t.) inoculation with K. pneumoniae, n ¼ 7–10 mice per group. Kruskal–Wallis test with Dunn’s multiple comparisons, *Po0.05. (c) A Kaplan–Meier survival curve of WT and thbs1 À / À mice following i.t. infection with K. pneumoniae (n ¼ 20 mice per genotype, P ¼ 0.02). The median survival for WT mice was 72 h (3 days) and 312 h (13 days) for thbs1 À / À mice. WT, wild type. MucosalImmunology | VOLUME 8 NUMBER 4 | JULY 2015 897 ARTICLES phase after infection.15 Taken together, TSP-1 deficiency remove extracellular, membrane-attached bacteria. At time 0, improves early lung bacterial clearance, reduces systemic neutrophils from thbs1 À / À and WT mice ingested similar dissemination, and confers increased survival during bacterial numbers of bacteria as indicated by CFU following lysis of cells pneumonia induced by K. pneumoniae. However, thbs1 À / À (Figure 3a).
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