The Efficacy And Mechanism Of Adenoid-derived Mesenchymal Stem Cells In The Treatment Of Allergic Rhinitis

Background:Allergic rhinitis(AR)has become a global public health issue with increasing incidence rates.Despite the current treatment principles of AR,which include environmental control,drug treatment,allergen-specific immunotherapy and health education,some patients still cannot achieve good symptom control.Therefore,alternative treatments for AR are urgently needed.Mesenchymal stem cells(MSCs)have been studied extensively due to their powerful immunosuppressive ability.Previous researches have demonstrated that MSCs derived from different sources such as bone marrow,adipose tissue,umbilical cord blood and tonsils have inhibitory effects on type 2 inflammation in diseases such as AR and asthma.Furthermore,the secretome of MSCs,also known as conditioned medium(CM),has similar therapeutic effects to MSCs,suggesting that the immunomodulatory effects of MSCs depend mainly on their paracrine pathways.Exosomes(exo)are important components of the paracrine system and play a critical role in intercellular communication by carrying their cargo,such as long non-coding RNAs(lnc RNAs).MSCs derived from immune tissues seem to have stronger immunomodulatory abilities.Current researches on the treatment of AR with MSCs or MSC-exo mainly focus on observing symptoms in animal models and detecting immune indicators,while exploration of their mechanisms in regulating systemic immunity and local mucosal immunity is still limited.To address these issues,it is important to extract MSCs from immune tissues that have been overlooked,to verify their therapeutic effects on AR,and to explore their mechanisms in regulating systemic and local mucosal immunity.Our proposed approach involved extracting MSCs from adenoids(a MSC),constructing an AR mouse model using OVA,and evaluating the therapeutic effects of a MSC and a MSC-CM on AR.Transcriptomic analysis was carried out on the nasal mucosa of the mouse model to identify potential target sites of a MSC and a MSC-CM on local mucosal immunity.Furthermore,we isolated a MSC-exo and co-cultured them with CD4~+T cells differentiated into Th2 cells in vitro,to investigate their potential impact on T cell differentiation in the systemic immune system and target sites.These efforts hold significant clinical implications.Objectives:(1)To obtain MSCs from adenoid tissue and isolate exosomes from a MSC-CM.(2)To evaluate the therapeutic effects of a MSCs and a MSC-CM on mouse models of AR.(3)To investigate the effects of a MSCs and a MSC-CM on the transcriptome of the local nasal mucosa in AR.(4)To explore the inhibitory role of a MSC-exo on Th2 differentiation by examining the Linc00632/EZH2/GATA-3 pathway.Method:(1)Adenoid tissue specimens were obtained and a MSCs were isolated and cultured using an enzymatic digestion method.Flow cytometry was used to analyze the surface markers of MSCs,including CD73,CD90 and CD105,as well as the negative marker CD45.To assess differentiation potential,reagents were used to induce the cells to differentiate into osteoblasts,adipocytes and chondrocytes.Exosomes were extracted from a MSC-CM by ultracentrifugation and identified through transmission electron microscopy,Nanosight particle size analysis and western blotting.(2)AR model was induced in BALB/c mice by OVA,and they were then randomly divided into three groups for different interventions:(1)MSC treatment group:mice received tail vein injections of a MSCs,qd x4;(2)MSC-CM treatment group:mice received tail vein injections of a MSC-CM,qd x 4;(3)Control group:mice received tail vein injections of PBS,qd x 4.The numbers of sneezing and nose rubbing in the mice were observed,and they were euthanized immediately after the final challenge.Serum OVA-specific Ig E was detected by ELISA,the Th1/Th2and Th17/Treg ratios in spleen tissues were detected by flow cytometry,the inflammatory status of the nasal mucosa was evaluated by pathological section staining,and the transcription levels of IL-4,IL-13and IFN-γin the nasal mucosa were detected by RT-q PCR.(3)The nasal mucosa from each group of mice was subjected to transcriptome sequencing.Differentially expressed genes(DEGs)were identified by bioinformatics analysis,and the DEG gene sets were subjected to cluster analysis,enrichment analysis,and protein-protein interaction network analysis.The target genes screened by bioinformatics were validated by RT-q PCR.(4)CD4~+T cells were isolated from human peripheral blood and induced to differentiate into Th2 cells in vitro.Co-culture with a MSC-exo was performed to investigate the effect of MSC-exo on Th2differentiation.The expression of Linc00632 was detected in the nasal mucosa of patients with AR and healthy controls.Knockdown and overexpression systems of Linc00632 were constructed,as well as EZH2knockdown and GATA-3 overexpression systems,to explore the role of the Linc00632/EZH2/GATA-3 pathway in the effect of MSC-exo on Th2differentiation.Immunoprecipitation was used to validate the interaction between Linc00632 and EZH2.Results:(1)Cells isolated and cultured from human adenoid tissue samples displayed the classic spindle-shaped morphology of MSCs and exhibited stable proliferation under microscopic observation.These cells expressed the surface markers of MSCs CD73,CD90,and CD105,while not expressing the negative marker CD45.Through induced differentiation culture,the cells were able to differentiate into osteoblasts,adipocytes,and chondrocytes,demonstrating the successful isolation of a MSCs.The exosomes isolated from a MSC-CM were verified by TEM,Nanosight particle size analysis,and WB,meeting the laboratory standards.(2)AR mice treated with a MSC and a MSC-CM experienced a notable alleviation of nasal symptoms,along with a decrease in serum OVA-specific Ig E levels,Th2/Th1 and Th17/Treg ratios in the spleen.Moreover,there was a notable reduction in nasal mucosal inflammation and a downregulation of inflammatory cytokines m RNA expression in the nasal mucosa.(3)Transcriptome analysis of nasal mucosal tissue from AR mice treated with a MSC or a MSC-CM revealed 446 differentially expressed genes(DEGs).Gene Ontology(GO)enrichment analysis of these DEGs showed that they were mainly located in the Z-disc and I-band and involved in biological processes such as cell differentiation and development.KEGG enrichment analysis revealed that they were enriched in the oxidative phosphorylation signaling pathway.Protein-protein interaction network analysis indicated that Fos had the strongest interaction relationship.RT-q PCR confirmed that Fos m RNA expression in the nasal mucosa was decreased in the a MSC or a MSC-CM treatment group.(4)In vitro experiments showed that a MSC-exo were effectively taken up by CD4~+T cells and had the ability to inhibit Th2 differentiation.This inhibitory effect was found to be mediated by Linc00632.Further studies revealed that Linc00632 interacted with EZH2 and inhibited GATA-3 expression,resulting in the inhibition of Th2 differentiation.Immunoprecipitation experiments provided additional evidence for this mechanism.Conclusions:(1)Adenoid tissue-derived MSCs(a MSCs)were successfully obtained and characterized as a new member of the MSC family.Additionally,a MSC-exo was obtained successfully.(2)a MSCs have a therapeutic effect on AR mice,indicating their potential as a therapeutic approach for AR.(3)Fos may play a role in inhibiting local mucosal inflammation in AR treated with a MSC,providing a potential mechanism for their therapeutic effect.(4)a MSC-exo-derived Linc00632 was found to inhibit GATA-3expression by interacting with EZH2,thus affecting Th2 differentiation.These results suggest that a MSC-exo could be used as a potential therapeutic approach for treating type 2 inflammatory diseases.Overall,this study provides insights into the therapeutic potential of a MSCs and their exosomes,and sheds light on the molecular mechanisms underlying their therapeutic effect in AR and other type 2 inflammatory diseases.