Title: Administration of a CXC Chemokine Receptor 2 (CXCR2) Antagonist, SCH527123, Together with Oseltamivir Suppresses NETosis and Protects Mice from Lethal Influenza and Piglets from Swine-Influenza Infection
Abstract: Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza–infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections. Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza–infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections. Influenza virus infections during pandemic outbreaks and yearly seasonal epidemics cause significant morbidity and mortality rates globally.1Short K.R. Kroeze E.J. Fouchier R.A. Kuiken T. 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Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.Am J Pathol. 2011; 179: 199-210Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar Herein, we evaluated the therapeutic efficacy of a CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide) alone or in combination with antiviral agent, oseltamivir (which inhibits viral neuraminidase and prevents progeny virus release from infected cells), in models of lethal influenza-infected mice and sublethal swine influenza-infected piglets. SCH527123 plus oseltamivir significantly improved survival in lethal influenza-challenged mice, and attenuated lung pathology in swine influenza-infected piglets. Thus, SCH5277123 plus oseltamivir represents a promising combination treatment against influenza pneumonia. Influenza viruses A/Puerto Rico/8/34, H1N1 (A/PR/8/34), and A/swine/Iowa/15/30 (A/SW/15/30) were obtained from ATCC (Manassas, VA). Viral titers were determined by median tissue culture infectivity dose (TCID50) assay via infection of Madin-Darby canine kidney (ATCC) cells.18Narasaraju T. Edwin Y. Ramar perumal S. Ng H.H. Poh W.P. Audrey-Ann L. Phoon Meng C. van Rooijen N. Chow V.T. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.Am J Pathol. 2011; 179: 199-210Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar Female BALB/c mice (6- to 8-week–old) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in micro-isolator cages in an Animal Biosafety Level 2 animal facility with food and water ad libitum. For pig influenza infections, 3- to 4-week–old piglets were obtained from a healthy herd at Oklahoma State University. Piglets were influenza-seronegative, tested in preserum via neutralization assay for absence of neutralizing antibodies against swine influenza A/SW/15/30 virus and a pandemic isolate, swine A/New York/18/2009 virus (randomly selected).39Rowe T. Abernathy R.A. Hu-Primmer J. Thompson W.W. Lu X. Lim W. Fukda K. Cox N.J. Katz J.M. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays.J Clin Microbiol. 1999; 37: 937-943Crossref PubMed Google Scholar Piglets from infection and infection plus treatment groups were housed in the same room, whereas mock-infected piglets were housed in a separate room. All piglet studies were performed in an Animal Biosafety Level 2 facility, in a negatively pressurized animal room with 100% high-efficiency particulate air filtered exhaust, at Oklahoma Animal Disease and Diagnostic Laboratory, with food and water ad libitum. All mice (protocol number VM 17-32) and piglet (protocol number VM 16-5) studies were approved by the Institutional Animal Care and Use Committee of Oklahoma State University, and were performed in strict accordance with their recommendations. These experiments were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Mice were anesthetized with a mixture of ketamine (7.5 mg/kg) and xylazine (0.1 mg/kg) by i.p. injection. Mice were infected with lethal doses of A/PR/8/34 (2500 TCID50 in 50 μL by intranasal route, equivalent to 10 median lethal dose, determined in our laboratory) diluted in phosphate-buffered saline (PBS).17Sharma G. Sharma D.C. Fen L.H. Pathak M. Bethur N. Pendharkar V. Peiris M. Altmeyer R. Reduction of influenza virus-induced lung inflammation and mortality in animals treated with a phosophodisestrase-4 inhibitor and a selective serotonin reuptake inhibitor.Emerg Microbes Infect. 2013; 2: e54Crossref PubMed Scopus (26) Google Scholar Equal volumes of PBS were inoculated into control mice. Bronchoalveolar lavage (BAL) fluids were collected at 12 hours post infection (hpi) and 1, 2, 3, 4, 5, 6, 7, and 8 days post infection (dpi) from lethal influenza-challenged mice (five to six mice at each time point). For collection of BAL fluids, control and infected mice lungs were lavaged twice with intratracheal administrations of 0.5 mL of PBS,18Narasaraju T. Edwin Y. Ramar perumal S. Ng H.H. Poh W.P. Audrey-Ann L. Phoon Meng C. van Rooijen N. Chow V.T. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.Am J Pathol. 2011; 179: 199-210Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar and the recovery of the BAL was >90% for all animals. The BAL cells were separated, as described earlier,18Narasaraju T. Edwin Y. Ramar perumal S. Ng H.H. Poh W.P. Audrey-Ann L. Phoon Meng C. van Rooijen N. Chow V.T. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.Am J Pathol. 2011; 179: 199-210Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar and differential cell counts were performed using modified Giemsa staining. Different cell types in BAL were identified (Supplemental Figure S1). One of the advantages of Giemsa method after cytospin is that it preserves exact cellular picture in BAL as there are no washing/resuspension steps involved. Cell-free BAL fluids were stored at −80°C for determining MPO activity and neutrophil elastase (NE) levels. Whole lungs were fixed from mice infected at different time intervals, as described above, and histopathology analysis was performed (Table 1).Table 1Semiquantitative Histopathology ScoresPhasePathologic lesionsCON1 dpi3 dpi5 dpi6 dpi8 dpiInflammatory phaseNecrotizing bronchitis–++++++++++++++Bronchiolar inflammation–+++++++++++∗P < 0.01 versus 3 dpi.Alveolar damage–+++++++++++++Interstitial inflammation–++++++++++++∗P < 0.01 versus 3 dpi.Hemorrhagic exudative phaseHemorrhage––+++++++++∗P < 0.01 versus 3 dpi.Pulmonary edema––++++++++++∗P < 0.01 versus 3 dpi.Fibrin deposition–––+++++++∗P < 0.01 versus 3 dpi.Blinded scoring (N.T.) of inflammatory phase and hemorrhagic exudative phase was evaluated by pathologic lesions, including necrotic bronchioles, bronchiolar inflammation, alveolitis, interstitial inflammation, pulmonary edema, hemorrhage, and fibrin deposition. n = 5 mice per group.–, No lesions; +, minimum; ++, mild; +++, moderate; ++++, severe; CON, control; dpi, days post infection.∗ P < 0.01 versus 3 dpi. Open table in a new tab Blinded scoring (N.T.) of inflammatory phase and hemorrhagic exudative phase was evaluated by pathologic lesions, including necrotic bronchioles, bronchiolar inflammation, alveolitis, interstitial inflammation, pulmonary edema, hemorrhage, and fibrin deposition. n = 5 mice per group. –, No lesions; +, minimum; ++, mild; +++, moderate; ++++, severe; CON, control; dpi, days post infection. MPO activity in the BAL fluids collected from control and infected mice between 12 hpi and 8 dpi was determined, as described previously.20Ashar H.K. Mueller N.C. Rudd J.M. Snider T.A. Achanta M. Prasanthi M. Pulavendran S. Thomas P.G. Ramachandran A. Malayer J.R. Ritchey J.W. Rajasekhar R. Chow V.T. Esmon C.T. Narasaraju T. Role of extracellular histones in influenza virus pathogenesis.Am J Pathol. 2018; 188: 135-148Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Levels of NE in BAL were measured using NE enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). The MPO/DNA enzyme-linked immunosorbent assay kit was used for measuring cell-free NETs, as described previously.21Sivasami P. Rudd J.M. Ashar H.K. Maram P. Thomas P.G. Akhilesh R. Malayer J. Chow V.T. Teluguakula N. Platelets-neutrophil interaction is critical in NETs induction and exacerbation of pulmonary pathology during influenza pneumonia.Am J Res Cell Mol Biol. 2019; 61: 689-701PubMed Google Scholar For identifying NETs or surfactant protein C (SP-C; alveolar type II epithelial marker)-positive cells, BAL from control or infected mice at 12 hpi to 8 dpi was stained with anti–citrullinated histone 3 (citH3) or anti–SP-C antibodies, respectively, followed by fluorescence-labeled secondary antibodies. At least 200 cells were counted in each sample to determine cells positive for citH3 and SP-C. CXCR2 ligands, including mouse keratinocyte chemoattractant/IL-8 and macrophage inflammatory protein, in the plasma samples of control or infected mice between 12 hpi and 8 dpi were evaluated by enzyme-linked immunosorbent assay (Roche, Indianapolis, IN), according to the manufacturer's instructions. Murine lethal influenza (A/PR/8/34; 2500 TCID50) infection model was used. Mice were divided into five groups: i) mock infected, ii) infected, iii) infected + oseltamivir treated; iv) infected + SCH527123 treated, and v) infected + oseltamivir + SCH527123 treated (Table 2). SCH527123 was administered alone or in combination with oseltamivir (equivalent to human dose),40Nguyen J.T. Smee D.F. Barnard D.L. Julander J.G. Gross M. de Jong M.D. Went G.T. Efficacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant influenza A viruses.PLoS One. 2012; 7: e31006Crossref PubMed Scopus (71) Google Scholar to assess the additive effects of SCH527123. Initial studies on dosage and timing of oseltamivir treatment indicated that administration of oseltamivir (30 mg/kg; Roche; orally using an oral gavage) starting at 24 hpi or 48 hpi, followed by four additional doses at 1-day intervals did not yield any protection. However, oseltamivir treatment starting at 12 hpi (20 mg/kg) followed by four additional dose treatments achieved about 30% survival in lethal influenza-challenged mice (Supplemental Figure S2). On the basis of these results, oseltamivir administered at 12 and 24 hpi (10 mg/kg) and at 2, 3, 4, and 5 dpi (20 mg/kg) was used throughout the experiments.Table 2Animal Groups from CON, Influenza Infection, and Treatment GroupsGroup designationInfection/treatmentMice, nCONPhosphate-buffered saline10INFInfection15INF + OSLInfection and oseltamivir10INF + SCHInfection and SCH5271235INF + OSL + SCH (3 doses)Infection, oseltamivir, and SCH527123 (3 doses)10INF + OSL + SCH (4 doses)Infection, oseltamivir, and SCH527123 (4 doses)5INF + OSL + SCH (5 doses)Infection, oseltamivir, and SCH527123 (5 doses)10The infection and treatment experiments were repeated at least twice, with five mice in each group per experiment. SCH527123 treatment did not show any protection, and was thus tested only once. The infection and treatm