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Pneumolysin Induces 12-Lipoxygenase–Dependent Neutrophil Migration During Streptococcus Pneumoniae Infection

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Pneumolysin Induces 12-Lipoxygenase–Dependent Neutrophil Migration During Streptococcus Pneumoniae Infection Pneumolysin Induces 12-Lipoxygenase− Dependent Neutrophil Migration during Streptococcus pneumoniae Infection This information is current as Walter Adams, Rudra Bhowmick, Elsa N. Bou Ghanem, of September 24, 2021. Kristin Wade, Mikhail Shchepetov, Jeffrey N. Weiser, Beth A. McCormick, Rodney K. Tweten and John M. Leong J Immunol published online 27 November 2019 http://www.jimmunol.org/content/early/2019/11/26/jimmun ol.1800748 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/11/26/jimmunol.180074 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 27, 2019, doi:10.4049/jimmunol.1800748 The Journal of Immunology Pneumolysin Induces 12-Lipoxygenase–Dependent Neutrophil Migration during Streptococcus pneumoniae Infection Walter Adams,*,† Rudra Bhowmick,*,1 Elsa N. Bou Ghanem,*,2 Kristin Wade,‡,3 Mikhail Shchepetov,x,4 Jeffrey N. Weiser,{ Beth A. McCormick,‖ Rodney K. Tweten,‡ and John M. Leong* Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)–dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in Downloaded from triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the a-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non–pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX–dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX– http://www.jimmunol.org/ dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX–deficient mice indicates that 12-LOX–independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indi- cate that PLY is an important factor in promoting hepoxilin A3–dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion. The Journal of Immunology, 2020, 204: 000–000. he Gram-positive bacterium Streptococcus pneumoniae is A hallmark of a S. pneumoniae lung infection is a robust the leading cause of community-acquired pneumonia and proinflammatory host response characterized by a massive influx T also causes several other infections, including otitis me- of neutrophils (polymorphonuclear leukocytes [PMNs]) into the by guest on September 24, 2021 dia, bacteremia, and meningitis. Asymptomatic colonization alveoli. PMNs, which confront the invading S. pneumoniae with a by S. pneumoniae has been estimated to be as high as 95% in number of antibacterial effector mechanisms, are beneficial for the children and 40% in adults and is considered to be an important host during early stages of the infection (6). Indeed, murine in- prerequisite for invasive disease (1, 2). In the United States fection studies have found that decreased neutrophil recruitment alone, there are ∼900,000 cases of pneumococcal pneumonia leads to higher bacterial loads in the lungs by 12–24 hours after annually, with a mortality rate of 5–7%, making the disease both pulmonary challenge with S. pneumoniae (7, 8). However, a strong a significant health and financial burden (3, 4). According to the PMN response to control bacterial outgrowth during the first World Health Organization, S. pneumoniae pneumonia accounts phase of infection can also lead to epithelial barrier disrup- for ∼500,000 deaths in children under 5 years old in developing tion, pulmonary edema, and significant lung damage (9, 10), countries (5). and mice containing high numbers of pulmonary PMNs several *Department of Molecular Biology and Microbiology, Tufts University, Boston, MA This work was supported by National Institutes of Health Award 5R37AI037657- 02111; †Department of Biological Sciences, San Jose State University, San Jose, CA 22 (to R.K.T.), by Award Number K12GM074869 from the National Institute of 95192; ‡Department of Microbiology and Immunology, University of Oklahoma General Medical Sciences (to W.A.), and American Lung Association Senior Re- Health Sciences Center, Oklahoma City, OK 73104; xDepartment of Microbiology, search Training Fellowship RT 194942 N (to R.B.). University of Pennsylvania, Philadelphia, PA 19104; {Department of Microbiology, ‖ Address correspondence and reprint requests to Prof. John M. Leong, Tufts New York University School of Medicine, New York, NY 10016; and Department of University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. E-mail Microbiology and Physiological Systems, University of Massachusetts Medical address: [email protected] School, Worcester, MA 01655 The online version of this article contains supplemental material. 1Current address: School of Chemical Engineering, Oklahoma State University, Stillwater, OK. Abbreviations used in this article: AA, arachidonic acid; ABTS, 2,29-azinobis- 3-ethylbenzotiazoline-6-sulfonic acid; BALF, bronchoalveolar lavage fluid; 2Current address: Department of Microbiology and Immunology, University at bcn, baicalein; CDC, cholesterol-dependent cytolysin; Ci-Di-Cy, cinnamyl- Buffalo School of Medicine, Buffalo, NY. 3,4-dihydroxy-a-cyanocinnamate; cPLA2a, cytosolic phospholipase A2a; H292, hu- 3 Current address: Manufacturing Sciences and Technology Department, Cytovance man pulmonary mucoepidermoid carcinoma-derived NCI-H292; HXA3, hepoxilin Biologics, Oklahoma City, OK. A3; ILY, intermedilysin; i.t., intratracheal(ly); 12-LOX, 12-lipoxygenase; MPO, myeloperoxidase; PFO, perfringolysin O; PI, propidium iodide; PLY, pneumo- 4Current address: Pathology Department, Children’s Hospital of Philadelphia, lysin; PLY , PLY cholesterol binding site toxoid; PLY , PLY early prepore Philadelphia, PA. CBS EP toxoid; PLYLP, PLY late prepore toxoid; PMN, polymorphonuclear leukocyte; ORCIDs: 0000-0001-7168-8090 (J.N.W.); 0000-0001-9293-9899 (R.K.T.); 0000- RD2, RedDot2; SLO, streptolysin O; WT, wild-type. 0003-0240-6402 (J.M.L.). Ó Received for publication May 29, 2018. Accepted for publication October 16, 2019. Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800748 2 PLY ELICITS 12-LIPOXYGENASE–DEPENDENT NEUTROPHIL MIGRATION days postinfection suffer bacteremia and succumb to infection dilutions on Tryptic Soy Agar plates supplemented with 5% sheep blood (11, 12). These findings suggest that for a favorable host out- agar (Northeast Laboratory Services, Winslow, ME). Bacillus subtilis come the timing and degree of PMN recruitment must be carefully strains were grown overnight at 37˚C in Luria Broth. The TIGR4 ply mutant (Dply) was a gift from A. Camilli, and D39 and 23F WT Dply orchestrated. mutants and revertant strains were a gift from J. Weiser. Strains D39 and S. pneumoniae encodes pneumolysin (PLY), a 53-kDa member 23F WT and Dply mutants have been previously described (30–34). The of a large family of cholesterol-dependent cytolysins (CDCs) that D39 revertant strain was generated by transformation of the D39Dply form ∼25-nm diameter pores in eukaryotic membranes (13). mutant with D39 chromosomal DNA and screening for hemolytic trans- formants. Insertion of WT ply at the original locus was confirmed by CDCs have been identified in over 40 bacterial species and include sequencing. intermedilysin (ILY) of S. intermedius, streptolysin O (SLO) of S. pyogenes, and perfringolysin O (PFO) of Clostridium perfringens Bacterial growth conditions (14). Pore formation by CDCs is a multistep process involving S. pneumoniae strains TIGR4 (serotype 4), D39 (serotype 2), E134 membrane binding, oligomerization, and membrane insertion, and (serotype 23F) were grown in Todd Hewitt Broth (BD Biosciences) several PLY toxoids that are defective at discrete steps have been supplemented with 0.5% yeast
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