Effects Of Interleukin-33 On Inflammatory Response In Chronic Rhinosinusitis With Nasal Polyps And Its Mechanism

Background:Chronic rhinosinusitis with nasal polyps(CRSw NP)is an inflammatory disease of unknown aetiology characterized by eosinophilic inflammation and overabundance of T-helper(Th)2 inflammatory cytokines.It has been suggested that microbial triggers may overstimulate the inflammatory response of Th2 cells in CRSw NP,leading to chronic disease manifestations.On the other hand,no direct association was found between the occurrence and development of nasal polyps,and specific pathogens or exogenous stimuli.It can therefore be assumed that host immune response is also a contributor to long-term inflammation in CRSw NP.Overexpression of Th2 cytokines,such as Interleukin IL-4,IL-5,IL-13 and IL-22,is believed to play an important role in eosinophilic mucosal inflammation in CRSw NP.This involves cell stimulation via the Interleukin 1 Receptor-like 1 Protein,which belongs to the IL-1 receptor family,but binds IL-33.Exogenous and endogenous stimuli(mechanical injury,infection,smoking,products of epithelial cell damage and necrosis)cause epithelial cells to release IL-33,which leads to subsequent inflammation.Interestingly,biological effects of IL-33 depend on its length.As mentioned above,IL-33 is cleaved by caspase-1,which is activated in apoptotic cells.Cleavage by caspase-1 reduces the biological activity of IL-33.In contrast,the full-length IL-33 is inflammagenic.Therefore,cell apoptosis inactivates IL-33 and prevents the development of the Type 2 immune response.Animal models are very useful for studies of gene and protein function.In this study,we utilized a murine model to test the involvement of IL-33 in the occurrence and development of CRSw NP.These observations were further supported by studies of patients’ specimens.Objective:1.To investigate the expression of inflammatory cytokine in patients with CRSw NP.2.To study the expression of IL-33 and the function of IL-33 on the release of inflammatory factors in CRSw NP mice.3.To clarify the role of IL-33 on the CRSw NP mice by regulating NF-κB signaling pathway topromote the expression of inflammatory factors.Methods:1.Detect the expression of inflammatory cytokine in patients with CRSw NP.(1)Our study included 35 patients and 10 control individuals enrolled inthe Department of Otorhinolaryngology-Head and Neck Surgery,First Affiliated Hospital of Harbin Medical University between April 2016 and August 2016.Thirty-five patients were diagnosed with CRSw NP,and 10 individuals with maxillary sinus cysts served as controls.Neither patients nor control individuals had a history of allergic diseases or positive skin prick test.None of study individuals received antibiotics or corticosteroids for 4 weeks prior to the enrollment.(2)Verifying the expression of IL-4,IL-5,IL-13,IL-22,CCL-11 and CCL-24 in tissuses between two groups by q RT-PCR and Western blot.2.Studying the function of IL-33 on the release of inflammatory factors in CRSw NP mice(1)The m RNA expression level of IL-33 in the CRSw NP model was detected by q RT-PCR.(2)The protein expression level of IL-33 in the CRSw NP model was detected by Western blot.(3)The expression level of Ig E in the CRSw NP model was detected by ELISA after IL-33 blocked.(4)The m RNA expression level of IL-4,IL-5,IL-13,IL-22,CCL-11 and CCL-24 in the CRSw NP model was detected by q RT-PCR after IL-33 blocked.3.The role of IL-33 on the CRSw NP mice by regulating NF-κB signaling pathway topromote the expression of inflammatory factors(1)The m RNA expression level of NF-κB,MyD88 and TLR7 in the CRSw NP model was detected by q RT-PCR after IL-33 blocked.(2)The protein expression level of NF-κB,My D88 and TLR7 in the CRSw NP model was detected by Western blot after IL-33 blocked.Results:1.Various inflammatory factors were high expression in CRSw NP patients The m RNA expression of IL-4,IL-5,IL-13,IL-22,IL-33,CCL-11 and CCL-24 in CRSw NP group were higher than that in control group,and the difference between the two groups was statistically significant(P<0.05).The expression level of Th2 cytokines was detected by Western blot.The protein expression of IL-4,IL-5,IL-13,IL-22,IL-33,CCL-11 and CCL-24 in CRSw NP group were higher than that in control group,and the difference between the two groups was statistically significant(P<0.05).2.IL-33 was high expression in CRSw NP mice and promoted the release of various inflammatory factors The tested mice showed signs of inflammation.This was obvious on hematoxylin-eosin staining,which showed nasal polyp tissue.Randomly selected 5 high magnification fields(x 400)demonstrated influx of inflammatory cells,including eosinophils.Meanwhile,the s Ig E expression levels in control group,CRSw NP group and IL-33 blockade group were analyzed by Elisa.The expression level of serum total Ig E in control mice was significantly lower than that in CRSw NP mice and IL-33 blockade inhibited the serum total Ig E expression level compared with CRSw NP group.The result showed CRSw NP model was successfully constructed.In order to detect the effects of IL-33 blockade,the m RNA and protein expression levels of IL-13 in control group,CRSw NP group and IL-33 blockade group at 12 weeks after CRSw NP model establishment were analyzed and Th2 cells counts were detected in all groups.After the CRSw NP model constructed at 12 weeks,the IL-33 expression levels were significantly increased.Expression of IL-33 m RNA and protein in control group and IL-33 blockade group were significantly lower than that in CRSw NP group at 12 weeks after CRSw NP model establishment.Meanwhile,IL-33 blockade suppressed Th2 cells production.We next quantified m RNA levels of Th2 cytokines.The m RNA expression levels of IL-4,IL-5,IL-13,IL-22,CCL-11,and CCL-24 in control group,CRSw NP group and IL-33 blockade group at 12 weeks after CRSw NP model establishment were analyzed by q RT-PCR.CRSw NP mice showed upregulated levels of IL-4,IL-5,IL-13,IL-22,CCL-11,and CCL-24 after the CRSw NP model constructed at 12 weeks and IL-33 blockade reverted this trend.3.IL-33 on the CRSw NP mice by regulating NF-κB signaling pathway topromote the expression of inflammatory factors we wanted to test whether the expression of the pro-inflammatory transcription factor inflammation-related factors was modulated in CRSw NP mice.The q RT-PCR assay was used to analyze the m RNA levels of NF-κB,My D88 and TLR7 in all groups.Our results demonstrated that the m RNA levels of NF-κB,My D88 and TLR7 in CRSw NP group were significantly higher than those in control group.However,after IL-33 blockade,the m RNA levels of NF-κB,My D88 and TLR7 in IL-33 blockade group was obviously lower than that in CRSw NP group.To further confirm the mechanisms about the protein expression of NF-κB,My D88 and TLR7,the Western blot analysis was conducted.Similar with the results ofq RT-PCR detection,we found that the CRSw NP mice remarkably increased the protein expression of NF-κB,My D88 and TLR7.However,after IL-33 blockade,the protein expression of NF-κB,My D88 and TLR7 in IL-33 blockade group was clearly lower than that in CRSw NP group.Conclusion:Our study provides evidence that CRSw NP is associated with overexpression of IL-33,with subsequent activation of Th2 immune response by NF-κB signaling pathway.Thus,IL-33 may be considered a potential drug target in CRSw NP.