Necroptosis Underlies Neutrophilic Inflammation Associated with the Chronic Rhinosinusitis with Nasal Polyps (CRSwNP).

Necroptosis is an inflammatory cell death associated with a variety of chronic diseases. Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease accompanied by eosinophil and neutrophil infiltration. The role of necroptosis in the pathogenesis of CRSwNP remains elusive. Cell death, including apoptosis, pyroptosis and necroptosis in control sinonasal mucosa and CRSwNP, were analyzed by immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF) staining for cleaved caspase 3, cleaved gasdermin D and p-MLKL, respectively. Correlations between necroptosis, inflammatory cytokines and neutrophil infiltration were assessed and a possible role of necroptosis in CRSwNP was evaluated. Primary nasal polyp cells (DNPCs) were stimulated with damage-associated molecular patterns (DAMPs) including ATP or IL-1α and their expression of inflammatory cytokines was analyzed using RT-PCR. The expression of TNF-α and IFNs in nasal polyps was measured by ELISA; human monocyte THP-1 cells were treated with TNF-α or IFN-γ and cell death was measured by LDH release. Necroptosis, rather than apoptosis or pyroptosis, was overtly activated in both eosinophilic and non-eosinophilic CRSwNP as evidenced by the presence of prominent phosphorylation of MLKL compared to controls. The abundance of DAMPs (IL-1α, HMGB1), inflammatory cytokines (IL-6) and chemokines (IL-8, CXCL-1) were all increased especially in non-eosinophilic CRSwNP. The extent of necroptosis was positively correlated with the abundance of DAMPs and cytokines, and neutrophil infiltration in CRSwNP. In DNPCs, ATP and IL-1α induced the expression of IL-8 and CXCL-1. Macrophage was found to be the predominant cell type positive for p-MLKL in CRSwNP. Concomitant treatment with TNF-α and IFN-γ, which were abundantly present in CRSwNP, triggered marked necroptosis in THP-1 cells. Necroptosis induced by TNF-α and IFN-γ may facilitate the production and release of a myriad of proinflammatory cytokines and entailed neutrophil infiltration to exacerbate inflammation in CRSwNP.