Characterizing The Neutrophilic Inflammation In Chronic Rhinosinusitis With Nasal Polyps

Background:Chronic rhinosinusitis with nasal polyps（CRSw NP）is a severely underrated and insufficiently treated disease characterized by inflammation of the sinonasal mucosa and polyp formation.Chinese CRSw NPs demonstrate different immunopathological characteristics from Caucasian CRSw NPs.Based on the tissue eosinophils distribution,CRSw NP is classified into eosinophilic and non-eosinophilic type.About half of Chinese CRSw NP patients presented non-eosinophilic inflammation and enhanced infiltration of neutrophils was previously shown in both eosinophilic and non-eosinophilic nasal polyps（NPs）in Chinese patients.Neutrophilic inflammation has been reported to associate with a poor response to corticosteroid therapy in patients with CRSw NP.The mechanisms underlying neutrophilic inflammation in chronic rhinosinusitis with nasal polyps remain poorly investigated.Objective:This study aimed to characterize the neutrophilic inflammation,explore the association between tissue neutrophilia and difficult-to-treat disease in patients with CRSw NP and examine the factors that contribute to tissue neutrophilia in CRSw NPs.Methods:The numbers of neutrophils and active caspase-3-positive apoptotic neutrophils in sinonasal tissues were assessed by immunofluorescence staining.The 95^thpercentile of neutrophil numbers in control tissues was chosen as a cut-off to define neutrophil-high（Neu-high）or neutrophil-low（Neu-low）nasal polyps（NPs）.The levels of34 cytokines,chemokines,and immunoglobulins in sinonasal tissues were analyzed by Bio-Plex assay.Purified human peripheral blood neutrophils were incubated with nasal tissue homogenates,and the apoptotic neutrophils,live neutrophils and necrotic neutrophils were assessed by flow cytometry.Results:The cut-off for Neu-high NPs was>10 myeloperoxidase positive cells/high-power filed.Compared with Neu-low NPs,Neu-high NPs had higher levels of IL-1β,IL-1Ra,IL-6,IL-8,granulocyte colony stimulating factor（G-CSF）,MCP-1,and MIP-1α,but lower levels of IL-5,IL-13,Ig E and eosinophils.Principal component and multiple correspondence analysis revealed a mixed type 1,type 2,and type 3 endotype for Neu-low NPs,and a predominant type 1 and type 3 endotype for Neu-high NPs.Neu-high NPs had lower percentages of apoptotic neutrophils than Neu-low NPs.A positive correlation was found between the protein levels of IL-1β,IL-1Ra,IL-6,IL-8,G-CSF,MCP-1 and MIP-1αand MPO⁺neutrophils in NPs.In comparison with Neu-low NPs,the percentage of active caspase-3⁺MPO⁺apoptotic neutrophils in total neutrophils was significantly lower in Neu-high NPs.The numbers of neutrophils correlated with G-CSF and IL-6 levels in NPs.Tissue homogenates from Neu-high NPs,but not those from Neu-low NPs suppressed neutrophil apoptosis in vitro,which could be reversed by anti-G-CSF treatment.Tissue neutrophil numbers associated with difficult-to-treat disease one year after surgery.Conclusion:Neu-high NPs and Neu-low NPs have obvious immunopathological characteristics.Neutrophil infiltration is associated with neutrophil chemotaxis and survival in nasal polyp tissue.Local G-CSF is a key factor affecting the apoptosis of neutrophils in nasal polyps.Both blood and tissue eosinophil and neutrophil abundance were associated with difficult-to-treat disease.G-CSF and granulocyte colony-stimulating factor receptor（G-CSFR）may provide novel therapeutic targets for difficult-to-treat disease in patients with neutrophilic CRSw NP.