Study On The Mechanism Of Dental Pulp Stem Cells Blocking Oral Submucous Fibrosis By Regulating Epithelial-T Cell Interaction

BackgroundOral submucous fibrosis(OSF)represents a chronic,debilitating oral pathology with oncogenic potential,afflicting diverse regions of the oral mucosa and significantly deteriorating patient quality of life,alongside their psychophysical well-being.The etiopathogenesis remains elusive,constraining therapeutic approaches and their efficacy.Unraveling the pathogenic mechanism of OSF and devising novel intervention strategies are thus imperative to enhance patient outcomes and life quality.Central to OSF pathophysiology is a disruption in collagen metabolism within the oral submucosa,precipitated by arecoline.As the first protective barrier of the oral mucosa,the destruction of the integrity of epithelial cells plays a crucial role in the occurrence and development of the disease.During the process of chewing betel nut,the epithelial barrier of oral mucosa is damaged.After the stimulation of arecoline,a variety of receptors on the surface of epithelial cells are activated,and the secretion of a variety of cytokines is stimulated,leading to changes in the local microenvironment and collagen metabolism disorders to promote the process of fibrosis.Fibrosis is a pathological process that can widely occur in human organs.In addition to oral mucosa,fibrosis can also occur in various organs of the whole body,eventually leading to organ structural damage and dysfunction.At present,several studies have shown that Mesenchymal stem cells(MSCs)have anti-inflammatory,anti-fibrosis and immunomodulatory effects,which can effectively block the progress of fibrosis in the liver,lung,kidney and other organs.Given the similar pathological basis,it is of great clinical significance to explore whether it can be used for the treatment and block of OSF.Dental pulp stem cells(DPSCs)exhibit capabilities in immunomodulation,self-renewal,and pluripotency,with the merits of accessibility and minimal immunogenicity.Human dental pulp mesenchymal stem cell injection,independently developed by academician Wang Songling and his team from Capital Medical University,has been approved for clinical trial by the Center for Drug Evaluation of China National Medical Products Administration.The current clinical trial results show that the injection is safe,effective and quality controllable in human administration.In summary,we hypothesize that betel nut consumption disrupts the homeostasis of the oral mucosal epithelial barrier,instigating chronic inflammation and consequentially,OSF,through alterations in the local immune milieu.DPSCs can play a therapeutic role by improving the immune microenvironment to slow down the process of OSF and relieve the symptoms of OSF.AimEmploying clinical samples of OSF,an arecoline-stimulated epithelial cell model,a rat model of OSF,and DPSCs intervention experiments,this study aims to elucidate:1.To explore the interaction between oral mucosal epithelial cells and immune cells in the process of OSF disease in clinical samples,and to clarify the pathogenic mechanism from the perspective of local microenvironment changes.2.Verifying the epithelial-immune cell interactions through an in vitro cellular model of OSF epithelial cells,examining the pro-fibrotic influence of the local microenvironment on fibroblasts,substantiating and expanding upon single-cell RNA sequencing findings.3.To verify the pathogenic role of local microenvironment changes in OSF and the interaction between epithelial cells and immune cells in an animal model to lay a foundation for further exploration of the role of DPSCs.4.Investigating the inhibitory effects and mechanisms of DPSCs on OSF through animal intervention studies and in vitro assays.By further analyzing the pathogenic mechanism of OSF,the above studies will further clarify the therapeutic potential and mechanism of DPSCs and provide a theoretical and experimental basis for promoting the establishment of a new treatment strategy for OSF mediated by DPSCs.Methods1.Single-cell transcriptomics of OSF clinical specimens.Pathological and corresponding control tissues were procured from OSF patients followed by histopathological characterization and validation via hematoxylin and eosin,Masson’s trichrome,immunohistochemical,and immunofluorescence staining.A single-cell RNA suspension was prepared for analysis using the 10×Genomics platform.We performed subgroup clustering,subdivision,trajectory analysis,cell-cell communication assessment,and KEGG pathway analysis.2.The effect of OSF cell model on T cells and fibroblasts.The concentration and time of arecoline solution on human oral keratinocytes(HOK)were determined by scratch test and CCK8,and the success of the OSF epithelial cell model was further verified by immunofluorescence staining.Immunofluorescence was used to detect the expression of Epi1.2 epithelial subpopulation marker gene KRT19 and its ligand MIF in the model.CD3~+T cells were isolated by gradient centrifugation and co-cultured with OSF epithelial cells.Immunofluorescence was used to detect the expression of ligands between Epi1.2epithelial cells and T cells.The activation and proliferation of T cells were detected by flow cytometry after adding an epithelial ligand antagonist,and the migration of T cells was detected by Transwell.Enzyme-linked immunosorbent assay(ELISA),immunofluorescence staining and q RT-PCR were used to detect the expression of inflammatory factors andα-SMA in the supernatant of each co-culture cell system.3.Establishment and characterization of the OSF model.The OSF rat model was established by local application of arecoline solution(smearing method)and local submucosal injection of arecoline solution combined with smearing method(compound method).The parameters were measured and recorded once a week.The tissue sections were characterized and verified by HE,Masson,immunohistochemistry and immunofluorescence staining.Immunohistochemistry and immunofluorescence staining were used to verify the epithelial-immune cell interaction in the OSF animal model.4.Blocking effect and mechanism of DPSCs on OSF disease model.The surface markers of DPSCs were detected by flow cytometry,and the multilineage differentiation ability of DPSCS was detected.The experimental animals were randomly divided into control groups,OSF,OSF+PBS,OSF+Glucocorticoids(GC)/PBS(positive control group)and OSF+DPSC/PBS.The drugs of each group were injected locally for 4 and 8weeks.The therapeutic effect and mechanism of each drug on the rat model were evaluated by measuring clinical characteristic parameters,HE,Masson,Van Gieson,Sirius red,scanning electron microscopy(SEM),atomic force microscopy(AFM),immunohistochemistry and immunofluorescence staining.DPSCs supernatant(DPSC-CM)was used to act on the co-culture system of OSF cell model and T cells in vitro,and immunofluorescence was used to verify the mechanism of DPSC-CM blocking the progression of OSF lesions.Results1.Single-cell RNA sequencing of OSF specimens.Revealed preliminary disease architecture,distinguishing six cell phenotypes.Notably,the OSF cohort exhibited a higher prevalence of epithelial cells,keratinocytes,and T cells compared to controls.Epithelial cells clustered into four subgroups(Epi1.1,Epi1.2,Epi1.3,Mel),categorized by marker gene expression.Epi1.2 cells were exclusive to the OSF group,with pseudotemporal analysis suggesting differentiation predominantly from other subgroups.Moreover,this subset expressed a variety of inflammatory factors(IL6st,CD63,CD74,IL1r2,IL33,CCL28,CXCL14,RAI14,DDIT4,VGLL4,CD46,EIF2AK2 and REL)and genes related to extracellular matrix production(such as TRIM2 and REL).Cell communication analysis showed that Epi1.2 epithelial cells interacted with T cells through MIF-(CD74/CXCR4)ligand-receptor pair to alter the local immune microenvironment and promote the progression of fibrotic diseases.2.Effects of OSF cell model on T cells and fibroblasts.The OSF epithelial cell model was induced by 60μg/m L arecoline solution for 24 h.Immunofluorescence results showed that the model expressed the marker gene KRT19 and its ligand MIF of the Epi1.2epithelial cell subset,indicating the existence of the Epi1.2 epithelial cell subset.The protein expression levels of KRT19 and MIF were increased in OSF group(P<0.01).T cells also express the CD74/CXCR4 receptor complex.The expression of MIF-(CD74/CXCR4)protein was significantly up-regulated by ligand-receptor in the co-culture of OSF epithelial cells and T cells(P<0.05),and the expression levels of CD69 and CD25 were significantly increased(P<0.05),and the proliferation and migration of T cells were significantly enhanced(P<0.05).The activation,proliferation and migration of T cells were significantly decreased in the presence of MIF antagonist(P<0.05).ELISA results showed that compared with the HOK group,the expression of TGF-βin the supernatant of the OSF group was up-regulated(P<0.0001)and down-regulated IL-1βexpression(P<0.01);Compared with the T cell group,the expression of TNF-α,TGF-βand IL-6 was up-regulated in the OSF+T cell group(P<0.001).On the clear liquid after incubation with fibroblasts,immunofluorescence staining and q RT-PCR results showed that compared with the control group,OSF+T cells,T cells and OSF group of alpha-SMA protein and significantly raised the level of gene expression(P<0.01).After the addition of MIF antagonist,the expression levels ofα-SMA protein and gene in fibroblasts were lower than those in the supernatant of OSF epithelial cells+T cells co-culture group(P<0.05).3.Establishment and characterization of the OSF disease model.Both smear and compound application methodologies consistently generated stable leukoplakic lesions within the buccal mucosa in rats accompanied by diminished mouth opening.Histological analyses revealed that compared with the control group,the epithelial layer of OSF rats was notably thinner,with reduced rete ridges,decreased collagen deposition in the lamina propria,fewer blood vessels,and an increase in myofibroblast populations.Clinical characterization and histological staining results showed that both methods could successfully construct OSF disease models(P>0.05).The expression of MIF-(CD74/CXCR4)was verified in this model because the smear method was closer to the development process of clinical OSF.The results of immunohistochemistry and immunofluorescence showed that the expression of KRT19~+MIF~+epithelial cell subset(Epi1.2)and CD74~+CXCR4~+T cells was significantly up-regulated in the model(P<0.01).4.Blocking effect and mechanism of DPSCs on OSF disease model.In the OSF rat model,compared with OSF,OSF+PBS and OSF+GC groups,the DPSCs treatment group had a significant reduction in the area of white lesions on the buccal mucosa at 4and 8 weeks after local submucosal injection of DPSCs(P<0.001)and increased mouth opening(P<0.05).HE,Masson and VG staining showed that OSF+GC and OSF+DPSC groups had increased epithelial thickness and number of nails,and decreased collagen fiber content(P<0.01),and there was no significant difference between the two groups.The results of Sirius red staining showed that the OSF+DPSC group had less deposition of typeⅢcollagen fibers than other intervention groups(P<0.001).The results of immunohistochemistry showed that GC and DPSCs treatment could significantly increase the number of blood vessels in the buccal submucosa of OSF rats compared with OSF and OSF+PBS groups(P<0.05)and reduced myofibroblasts protein expression(P<0.05),but OSF+DPSC group had a better therapeutic effect than OSF+GC group at 8 weeks(P<0.05).At 4 week,compared with OSF group and OSF+PBS group,SEM results showed that the porosity of mucosal tissue in OSF+GC group and OSF+DPSC group increased significantly(P<0.01),OSF+DPSC group was better than OSF+GC group(P<0.0001);AFM results showed that the hardness of OSF+DPSC group was lower than that of other intervention groups(P<0.05).In addition,the results of immunohistochemical staining showed that compared with the OSF group,OSF+PBS group and OSF+GC group,OSF+DPSC group expressed less KRT19~+MIF~+epithelial cell subsets and CD74~+CXCR4~+T cells(P<0.001).In vitro,compared with OSF+T cells,the ligand-receptor interaction between Epi1.2 epithelial cells and T cells decreased after DPSC-CM treatment(P<0.05).Conclusion1.Epi1.2,a subset of epithelial cells,exhibits pro-inflammatory and pro-fibrotic characteristics in OSF,characterized by KRT19~+MIF~+expression.Interaction with T cells through the MIF-(CD74/CXCR4)axis modulates the local immune microenvironment,augmenting fibrosis.2.Epi1.2 epithelial cells exist in the OSF epithelial cell model,and T cells were activated after co-culture with the cell model.The expression of pro-inflammatory and pro-fibrotic cytokines was up-regulated in the cell supernatant of the co-culture system,and the supernatant could significantly activate fibroblasts into myofibroblasts,confirming the experimental results of single-cell RNA sequencing.3.KRT19~+MIF~+epithelial cells(Epi1.2)and CD74~+CXCR4~+T cells are present in the animal model constructed by the smear method,which lays the experimental foundation for further exploration of new treatment methods.4.Local submucosal injection of DPSCs could effectively improve clinical symptoms and histological features of OSF,and down-regulate the expression of KRT19~+MIF~+epithelial cells(Epi1.2)and CD74~+CXCR4~+T cells.This study reveals the immune factors in the pathogenesis of OSF and creatively identifies a pro-inflammatory and pro-fibrotic epithelial cell subset Epi1.2 in OSF disease,which interacts with T cells to alter the local immune microenvironment and promote the progression of fibrosis.In addition,we pioneered the use of human dental pulp mesenchymal stem cells to block the progression of OSF in rats.We elucidated the therapeutic effect of DPSCs on MIF-(CD74/CXCR4)through ligand-receptor,providing new insights and theoretical support for the mechanism and treatment of OSF disease.