Activation of Interleukin-32 Pro-Inflammatory Pathway in Response to Influenza A Virus Infection

Abstract

BackgroundInterleukin (IL)-32 is a recently described pro-inflammatory cytokine that has been reported to be induced by bacteria treatment in culture cells. Little is known about IL-32 production by exogenous pathogens infection in human individuals.Methods and FindingsIn this study, we found that IL-32 level was increased by 58.2% in the serum samples from a cohort of 108 patients infected by influenza A virus comparing to that of 115 healthy individuals. Another pro-inflammatory factor cyclooxygenase (COX)-2-associated prostaglandin E2 was also upregulated by 2.7-fold. Expression of IL-32 in influenza A virus infected A549 human lung epithelial cells was blocked by either selective COX-2 inhibitor NS398 or Aspirin, a known anti-inflammatory drug, indicating IL-32 was induced through COX-2 in the inflammatory cascade. Interestingly, we found that COX-2-associate PGE2 production activated by influenza virus infection was significantly suppressed by over-expression of IL-32 but increased by IL-32-specific siRNA, suggesting there was a feedback mechanism between IL-32 and COX-2.ConclusionsIL-32 is induced by influenza A virus infection via COX-2 in the inflammatory cascade. Our results provide that IL-32 is a potential target for anti-inflammatory medicine screening.

Negative feedback regulation of IL‐32 production by iNOS activation in response to dsRNA or influenza virus infection

“…The luciferase reporter vector (pGL3) containing an IL-32 promoter region (À746/125) pIL-32-Luc, pGL3 containing an iNOS promoter region (À8.3 K/190) phiNOS-Luc, renilla internal control vector pRL-TK (Promega), IL-32 expression vector Flag2A-IL-32, expression constructs containing ten IV genes and IL-32-specific siRNA expression plasmid siRNA-IL32 were documented in our previous studies [1,7,8].…”

“…In vitro, IV infection of A549 lung epithelial cells resulted in release of IL-32 and activation of COX-2 expression [1]. As a proxy of virus infection we used stimulation of the cells with dsRNA1IFN-g, a combination previously shown to act synergistically when administered intratracheally [2] and in mouse peritoneal macrophages in vitro [3].…”

“…As a proxy of virus infection we used stimulation of the cells with dsRNA1IFN-g, a combination previously shown to act synergistically when administered intratracheally [2] and in mouse peritoneal macrophages in vitro [3]. We demonstrated that production of IL-32 depends on prior COX-2 activation, but also that IL-32 can exert negative feedback control over activation of COX-2 [1].…”

“…NO is synthesized from L-arginine by NOS in numerous mammalian cells and tissues and plays a critical role in signal modulation during immune responses, chronic inflammation and carcinogenesis. Three isoforms of NOS have been identified until now: two constitutively expressed and calciumdependent isoforms endothelial NOS and neuronal NOS; one inducible and calcium-independent isoform (iNOS), which catalyzes the production of high amount of NO in response to diverse stimuli [4].Increased IL-32 and iNOS expression stimulated by viral infections has been reported in previous investigations [1,5,6]. However, the function of IL-32 in the pro-inflammatory network is still unclear.…”

“…Twenty-four hours post transfection, cells were serum-starved for another 24 h before being harvested. Luciferase activities were then measured and renilla luciferase activities were determined as internal control for transfection efficiency as previously described [1,10]. Assay results were expressed as RLU (relative luciferase activity unit) or as LUC (luciferase activity).…”