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

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.¹ Seasonal influenza-associated deaths have increased in recent years, with an estimated of >600,000 fatalities annually.² A significant proportion of hospitalized patients with influenza develop complications of acute respiratory distress syndrome, characterized by widespread alveolar-capillary injury, inflammation, edema, and parenchymal hemorrhage.3, 4, 5, 6, 7, 8 These pathologic manifestations are driven by virus-inflicted cytopathic effects as well as exaggerated host immune responses.9, 10, 11 Vaccination is the logical choice for controlling the virus. However, because of unrelenting emergence of new strains and their mutative ability, vaccination presents a major challenge during influenza outbreaks.^12,13 In such cases, treatment primarily depends on antiviral therapy. Administration of antiviral drugs may not always be effective, as considerable lung pathology is mediated by exaggerated host-immune responses in addition to virus-inflicted cytotoxicity.14, 15, 16, 17Previously, we established that massive neutrophil influx, their induced neutrophil extracellular traps (NETs), and extracellular histones (ECHs) aggravate pulmonary pathology in severe influenza.18, 19, 20, 21, 22, 23 Aberrant neutrophil activity and accumulation of NETs are also documented in patients with severe influenza.^24,25 Neutrophils are recruited to the site of injury/infection via chemokine signaling, mediated through chemokine receptors. Among various chemokine receptors, CXC chemokine receptor 2 (CXCR2) plays a critical role in modulating neutrophil functionality during influenza.²⁶ Numerous clinical studies have also tested CXCR2 antagonists for their efficacy in reducing inflammation and organ injuries in acute and chronic diseases.27, 28, 29, 30, 31 Recently, human phase 2 trials evaluated the safety and efficacy of a CXCR2 antagonist, danirixin, alone or in combination with oseltamivir in influenza-infected patients.^27,28 Although administration of danirixin was found to be safe and well-tolerated, no differences in the clinical scores were observed between patients given oseltamivir alone and those given danirixin plus oseltamivir.²⁷ Furthermore, there was inconsistency in neutrophil numbers among different treatment groups. This inconsistency may be attributed to the absence of rational determination of the optimal timing and dosing of danirixin, to achieve the fine balance of suppressing excessive neutrophil influx, without compromising the beneficial host immunity by neutrophils. Moreover, the underlying mechanistic roles of targeting CXCR2 and its pathogenic association with influenza pneumonia have not been established.NETs are large extracellular web-like chromatin strands that were initially proposed to have a defense mechanism against invading pathogens.³² However, excessive release of NETs aggravates tissue injury and death, as reported in several disease conditions.33, 34, 35, 36 NETosis is regulated by various granule and nuclear proteins.³⁷ Myeloperoxidase (MPO) and neutrophil elastase (NEs) are released from azurophilic granules, anchor chromatin scaffolds in NETs, and mediate histone degradation during NETosis.³⁸ We reported earlier that blocking MPO decreases NETs, but signaling mechanisms in influenza-induced NETosis remain unclear.^17,18 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.