The New Role Of Eosinophils In Chronic Rhinosinusitis:Exploration With Co-Stimulators CD40-CD40L And ICOS-ICOSL

Chronic rhinosinusitis(CRS)is a chronic inflammatory disease with high heterogeneity and unclear governance effects.Based on the extent of tissue eosinophilia,CRS can be classified into eosinophilic chronic rhinosinusitis(ECRS)and noneosinophilic(Non-eCRS)subtypes.Compared with Non-eCRS,ECRS is always associated with worse disease severity,a higher ratio of recurrence and revision surgery and a higher risk of comorbid diseases.Thus,researches on this type are of great significance.Eosinophils is the hallmark of development of ECRS.CD40-CD40 ligand(CD40L)and inducible co-stimulator(ICOS)-ICOS ligand(ICOSL)are important positive co-stimulators in the interaction between T cells and B cells,which involve in many immune diseases.Researches have showed that the target therapy against these costimulators can alleviate the progression of diseases.Besides the respective roles,the activation of ICOS-ICOSL pathway can promote the expression of CD40L,which in turn strengthen the CD40-CD40L interaction to provide a co-stimulatory signal for B cell.Recently,studies have demonstrated that there is CD40 expression mainly on eosinophils in nasal polyps as well as the ICOSL expression on eosinophils through RNA sequencing.In addition,the activation of CD40-CD40L pathway promotes the survival and cytokines secretion of eosinophils.However,it’s unknow that whether the CD40-CD40L and ICOSICOSL pathways contribute to the development of CRS or target eosinophils.Objective:The goal of this study is to investigate the possible involvement of CD40CD40L and ICOS-ICOSL in the development of CRS through eosinophils,which can give the theoretical guideline for immunotherapy.Methods:We enrolled 31 CRS patients treated with functional endoscopic sinus surgery(FESS)from April 2021 to May 2021.in the otolaryngology department of the first affiliated hospital of Soochow University retrospectively.Immunohistology was performed to evaluate the expression level of CD40,CD40L,UCOS,ICOSL in nasal tissues and the correlation analysis were performed in CRS patients.At the same time,the co-localization of CD40 or ICOSL with eosinophils was evaluated by immunofluorescence.In the functional experiments,peripheral blood from CRS patients was stimulated with recombinant human CD40L(rhCD40L)or recombinant human ICOSL(rhICOSL)for 24h.Then,we analyzed the proportion of activated eosinophils(CD45~-CD16~-CD69~+)by flow cytometry.Moreover,we isolated eosinophils from healthy donators to explore the effect of cytokines.With or without the 24h or 48h stimulation of TNF-α.IL-5,p38 mitogen-activated protein kinase(MAPK)inhibitor SB203580,the expressions of CD40 and ICOSL on eosinophils w ere assessed by flow cvtometry.Results:Through the research,the following results were obtained:1.The expression level of CD40,ICOS,ICOSL were significantly higher in ECRS as compared to Non-eCRS.CD40 and ICOSL were predominantly located on eosinophils in nasal tissues.2.There were significant correlations among expression level of CD40,CD40L,ICOS,ICOSL:CD40 expression was positively correlated with ICOS and ICOSL expression.CD40L expression was positively correlated with ICOS.At the same time,ICOS expression was also positively correlated with ICOSL.In respect to clinical characteristics,these four co-stimulators all correlated with tissue eosinophils.And except for CD40L,CD40.ICOS and ICOSL were statistically associated with blood eosinophils.Additionally,ICOS and ICOSL expression in nasal tissues correlated with disease activity assessed by Lund-Mackay score.3.In the stimulation of rhCD40L and rhICOSL,activated eosinophils in peripheral blood from ECRS patients increased.4.Compared with medium condition.TNF-α rather than IL-5 stimulation enhanced the CD40 expression on eosinophils isolated from healthy donators,which could be strengthen by TNF-α combined with IL-5.The regulation was time independent.When compared with the TNF-α+IL-5 group.the adding of specific p38 MAPK inhibitor SB203580 downregulated the CD40 expression significantly.Neither TNF-α nor IL-5 regulated ICOSL expression of eosinophils.Conclusion:The increased expression of CD40.ICOS and ICOSL in ECRS group compared with Non-eCRS as well as the correlation among these co-stimulators may indicate their potential role in CRS development.The further co-localization and correlation with eosinophils suggest that eosinophils may be one of targets on which these co-stimulators take effect.Importantly,CD40-CD40L pathway enhances eosinophils activation and TNF-α and IL-5 can upregulate the CD40 expression on eosinophils which is partly mediated by p38 MAPK pathway.This demonstrate that TNF-α and IL-5 may reinforce the function of eosinophils in a new manner.