Study On Mechanism Of CSF1R Regulating Chronic Sinusitis With Nasal Polyps Through MTOR Pathway

Chronic rhinosinusitis(CRS)is a persistent inflammation of the mucous membranes in the nasal cavity and paranasal sinuses,which lasts for over 12 weeks.The symptom of Chronic rhinosinusitis with nasal polyposis(CRSwNP)include nasal congestion,runny nose,and loss of sense of smell.Despite not being a life-threatening condition,CRS has significant negative impacts on patients’ daily life.Despite remarkable progress in pharmacological and surgical treatment of CRSwNP,the recurrence rate of CRSwNP remains high,indicating the underlying pathophysiological mechanisms behind CRSwNP remain to be fully elucidated.The colony-stimulating factor 1 receptor(CSF1R),also known as macrophage colony-stimulating factor receptor(M-CSFR),is a receptor that can be activated by two ligands: colony-stimulating factor 1(CSF-1)and interleukin 34(IL-34).The CSF1 R signaling pathway is required for the survival,proliferation,and differentiation of macrophages,myeloid cells,and others.In addition,CSF1 R signaling is associated with many diseases and may be a therapeutic target for diseases such as cancer,neurodegeneration,and inflammatory bone diseases.The mammalian target of rapamycin(mTOR)protein kinase is a member of the phosphatidylinositol 3-kinase-related kinase family.mTOR is linked to other proteins and serves as a central component of two different protein complexes(mTOR complex 1 and mTOR complex 2),which regulate different cellular processes.As a core component of both complexes,mTOR functions that regulates cell growth,cell proliferation,cell motility,cell survival,protein synthesis,autophagy,and transcription.In addition,mTOR signaling pathway plays a crucial role in many important biological events.However,the mechanism of action of CSF1 R in CRSwNP and the relationship between the roles of CSF1 R and mTOR in CRSwNP have not been reported.whether CSF1 R can regulate the development of CRSwNP through mTOR is unclear.Objective.To investigate the mechanism of action of CSF1 R in regulating CRSwNP.To investigate whether CSF1 R can regulate the development of CRSwNP through mTOR.Methods.In this study,we utilized gene expression microarrays and transcriptome sequencing datasets from the GEO database to investigate the pathophysiological mechanisms underlying CRSwNP.We selected nasal polyp tissue from CRSwNP patients as the disease group and normal patient tissue as the control group.We performed gene expression differential analysis using the R packages limma and DESeq2 and utilized the Wilcoxon non-parametric test to analyze the differential expression of the single gene CSF1 R.Based on the median expression value of the CSF1 R gene,we divided CRSwNP patients into high and low expression groups and conducted GO and KEGG enrichment analysis using the R package cluster Profiler.Next,we selected CRSwNP patients with nasal polyps and control group samples to detect the expression of CSF1 R and mTOR at both the transcript and protein levels using RT-q PCR and Western blot.We also constructed CSF1 R sh RNA lentivirus to knock down CSF1 R expression in human nasal epithelial cells(HNEp C),as well as CSF1 R overexpression lentivirus to overexpress CSF1 R in HNEp C cells.We then examined the expression levels of CSF1 R and mTOR in lentivirus-transfected HNEp C cells via RT-q PCR and Western blot,and measured the expression and secretion levels of the inflammatory cytokine TSLP in cells and cell culture supernatants using RT-q PCR and ELISA,respectively.Results.1.A total of 1291 and 1913 differentially expressed genes were identified in the datasets GSE36830 and GSE136825,respectively,and upregulation of CSF1 R expression was found in CRSwNP.2.Enrichment analysis of the high CSF1 R expression group revealed enrichment in multiple immune and cellular signaling pathways,including immune response,T cell signaling pathway,leukocyte proliferation,and chemokine signaling pathway.3.Tissue sample analysis confirmed that CSF1 R and mTOR were upregulated in CRSwNP and their gene expression levels were positively correlated.Western blot analysis also showed upregulation of mTOR and phosphorylated mTOR in CRSwNP.4.In nasal mucosal epithelial HNEp C cells,knockdown of CSF1 R resulted in decreased expression of CSF1 R and mTOR,while overexpression of CSF1 R resulted in increased expression of CSF1 R and mTOR.5.Knockdown of CSF1 R expression in HNEp C cells reduced the secretion of TSLP by nasal epithelial cells,while overexpression of CSF1 R increased TSLP secretion.These results suggest that the upregulation of CSF1 R in CRSwNP may be involved in the regulation of TSLP in epithelial cells,which in turn affects the occurrence of inflammation.Conclusions and implications.CSF1R can regulate TSLP secretion through mTOR and promote the development of chronic rhinosinusitis with nasal polyps.This study provides new insights that will help us better understand the pathogenesis of CRSwNP and lay the foundation for future therapeutic studies.