Inhibition of Reactive Oxygen Species Production Ameliorates Inflammation Induced by Influenza A Viruses via Upregulation of SOCS1 and SOCS3

Abstract

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/ 1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. IMPORTANCEHighly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Widespread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combinati...

Metabolic host response and therapeutic approaches to influenza infection

“…ROS-producing enzymes induced by influenza infection mainly include NADPH oxidase (Nox) and xanthine oxidase, upregulation of which causes the impaired defensive function of antioxidants [16]. An increase in ROS production, along with impaired antioxidant function, ultimately leads to a profound change in redox homeostasis of the cell [16][17][18][19]. Nox2 is a phagocytic enzyme that is involved in the production of ROS induced by influenza virus [19][20][21][22], and impaired Nox2 expression results in a lack of increased RNS and ROS production following influenza infection [20].…”

PA-X-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic H5N1 avian influenza virus in mice

“…However, the potential pathogenic mechanism for PA-X-associated attenuation of acute lung injury is currently unknown. Previous histological and pathological indicators strongly suggest a key role of an excessive host response in mediating ALI, and various effector arms of the host response can act deleteriously to initiate or exacerbate pathological damage in this viral pneumonia, including cytokines and chemokines [50][51][52][53], cell death [54][55][56][57][58][59][60], oxidative stress (ROS production) [38,[42][43][44][45], complement response [30][31][32][33][34][35][36][37] and other factors.…”

“…During influenza virus infection, a rapid influx of inflammatory cells often results in an increased ROS production [73]. A number of studies have demonstrated that ROS plays a role in the occurrence of the acute lung injury induced by influenza virus [39,40,42,44,47] and it is an attractive and promising target for therapeutic intervention [38,43,45,46,74]. In this study, significantly higher expression level of ROS was detected in the PA-X-deficient virus-infected cells compared to the parental virus at 24 h p.i (Fig.…”

“…Accumulated studies have shown that ROS contributes to the AIL and plays a role in influenza virus pathogenesis [38][39][40][41][42][43][44][45][46][47]. To evaluate the role of PA-X in ROS production, we compared the ROS expression level in MDCK cells inoculated with the parental virus and PA-X-deficient viruses.…”

Role of Nrf2 and Autophagy in Acute Lung Injury

“…ROS may be produced by inflammatory cells (macrophages and neutrophils) trying to fight infections, or by dysfunctional mitochondria or enzymatic systems in alveolar epithelial cells and capillary endothelial cells after lung tissue damage by hyperoxia, electrophilic xenobiotics, and shear stress (Brune et al 2013; Warabi et al 2007; Bernard et al 2014; Reddy et al 2009c). Antioxidants have been shown to reduce the severity of ALI/ARDS in multiple mouse models that use different initiating insults, highlighting the central role of ROS in the pathophysiology of ALI/ARDS (Aggarwal et al 2012; Howard et al 2014; Ye et al 2015; Husari et al 2014; Shohrati et al 2014; Zhao et al 2014; Lingaraju et al 2015; Hu et al 2015; Yilmaz et al 2014; Yamamoto et al 2012; Campos et al 2012). …”