Molecular Mechanism Of Autoimmune Inner Ear Disease

Hearing impairment or loss is common disease in otorhinolaryngology.It is estimated by the WHO that there were 360 million suffer from hearing disability globally in 2013.The patients with hearing loss concomitantly manifested tinnitus,dizziness,insomnia,anxiety,depression and even afraid of communicating with others,all which cause serious distress for patients,and influence their quality of life.Thus,how to prevent and manage hearing loss has been a public health problem globally.According to the etiology,hearing loss can be induced by genetic,infection,drug toxic,trauma,aging,noise and autoimmune.Among them,autoimmune inner ear disease(AIED)is one of the few causes of rapidly progressive,but reversible if timely and appropriate treatment is given.Hereby,it is of important clinical significance to investigate the pathogenesis of AIED and to explore how to timely diagnose and treat AIED.Recently,endolymphatic sac(ES)is suggested as the constitutive immune defense system of inner ear.Once the inner ear is stimulated by the antigen,the immunocompetent cells in ES can be proliferated rapidly and release the pro-inflammatory cytokines to mediate the recruitment of inflammatory cells in systemic circulation,leading to the magnification of the immunization in ES and ultimately completing the antigen capture,phagocytosis,and clearance.If excessive immune response is induced,the pathological changes of cochlea tissue can be caused,eventually leading to the occurrence of AIED.These indicate targeting the pro-inflammatory genes may be underlying approaches for treatment of AIED.However,rare studies were performed to investigate the genes in ES.Due to the incapability of biopsy in inner ear,the diagnosis of AIED at present mainly depends on the comprehensive judgment of clinical symptoms,laboratory examination,audiological imaging and so on.Among them,blood detection is the main method,including specific antigen,immune cells(lymphocyte)and secreted cytokines(i.e.TNF).However,the biomarker for diagnosis of AIED remains limited and further investigation is needed.MicroRNAs(miRNAs),endogenous,noncoding RNAs(approximately 22 nt in length),have been implicated to involve in the development,progression and prognosis of a serial of diseases via binding to its mRNA targets.There are studies to demonstrate the diagnostic performance of miRNAs for autoimmune thyroid disease and autoimmune pancreatitis,but the AIED-related miRNAs remain rarely reported.Based on the above problems,this thesis mainly carries out the following three aspects of work:Part Ⅰ:Purpose:To screen inflammation related genes in ES by using microarray data,which may provide some target for AIED.Methods:The microarray dataset under accession no.E-MEXP-3022 was obtained from the European Bioinformatics Institute database,including three biological replicates of ES plus dura tissues and three replicates of pure dura tissues form rats.The raw data were pro-pressed by the Robust Multichip Average(RMA)method;differentially expressed genes(DEGs)were screened using the Linear Model for Microarray data method,with the threshold value of P<0.05 且|log2FC|>1.Function enrichment analysis of upregulated and downregulated DEGs was performed using the Database for Annotation,Visualization and Integrated Discovery online tool(DAVID),including gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Protein-protein interaction(PPI)network was constructed using the interaction data from the Search Tool for the Retrieval of Interacting Genes/Proteins(STRING)database via the Cytoscape.CytoNCA was used to calculate the topological features,including degree,the betweenness and subgraph centrality for each gene in PPI network.ClusterONE was adopted to extract the functionally associated and densely interconnected modules,followed which the function enrichment analysis was predicted for genes in each module.To further confirm the importance of identified genes,our DEGs were overlapped with the inflammation related genes screened by the study of M(?)ller et al.in 2015.Results:Based on the set threshold,a total of 612 genes were identified as DEGs between ES plus dura tissues and pure dura tissues including 396 up-and 216 downregulated genes.GO enrichment analysis indicated 116(59 BP,35 CC and 22 MF)GO terms were enriched for upregulated DEGs,including GO:0007155-cell adhesion,with a5-integrin(Itgal)and secreted phosphoprotein 1(Spp1);GO:0031295-T cell co-stimulation,with Ccl21 and Ccl19;and GO:0070555-response to IL-1,with Ccl21 and Ccl19.For downregulated DEGs,77(including 48 BP,11 CC,and 18 MF)GO terms were enriched.In line with the upregulated DEGs,the majority of downregulated DEGs was associated with GO:0007155-cell adhesion[i.e.transforming growth factor(Tgf)-βi];GO:0002224-toll-like receptor signaling pathway(i.e.Tlr2,Tlr7 and Tlr8);and GO:0042346-positive regulation of nuclear factor(NF)-κB import into the nucleus(i.e.Tlr2 and Tlr7).Furthermore,pathway analyses also indicated upregulated DEGs participated in six pathways,including rno04530:Tight junction[i.e.claudin(Cldn)-4];rno04512:Extracellular matrix(ECM)-receptor interaction(i.e.Itgal and Sppl);and rno04670:Leukocyte transendothelial migration(i.e.Cldn4).Downregulated DEGs were enriched in seven pathways,including inflammation-associated diseases,rno05144:Malaria(i.e.Tgf-(33 and Tlr2).Following the mapping of DEGs into protein interactions pairs,a PPI network was constructed,including 338 nodes and 771 interaction relationships.Key nodes(Itgal and Sppl)in the PPI network were screened by calculating their topological features.Following ClusterONE analysis,5 significant submodules were created.Function enrichment analysis revealed that the genes participating in module 1 were closely associated with inflammation,including Ccl21 and Ccl19,whereas the genes in module 2 and 5 were significantly enriched in cell adhesion pathways,including Cldn4.A total of 6 genes were identified to be shared between our DEGs and the study of M(?)ller et al.in 2015,including Tlr7,Tlr2,Tlr8,Lpo,Gzm-m and Muc1.Function enrichment analysis of these genes showed Tlr7,Tlr2,Tlr8 and Gzm-m were enriched in immune or inflammatory response-associated GO terms or pathways.Conclusion and Significance:The present study revealed abnormal expression of important inflammatory-associated genes,including Itgal,Sppl,Ccl21,Ccl19,Cldn4,Tlr2,Tlr7 and Tlr8 in ES may describe the underlying mechanism for the development of AIED.Part Ⅱ:Purpose:To screen AIED related biomarkers by using miRNA next generation sequencing.Methods:The inner ear antigen was prepared from the inner ear tissue of guinea pigs.Inner ear tissue antigen was emulsied with an equal volume of complete Freund’s adjuvant,which was subcutaneously injected into the multi-points in the neck and back of animals to induce AIED.After immunization for one or two weeks,the AIED model was confirmed by histopathological analysis of the cochlea via standard hematoxylin and eosin(H&E)staining.The serum samples of three AIED and three control mice were obtained and subjected to miRNA deep sequencing analysis.FASTQC software was used for preliminary quality control analysis of the fastq files.miRDeep2 software was applied for the qualitative,quantitative and normalized analysis of miRNAs.The differentially expressed miRNAs(DE-miRNAs)between control and AIED were identified by the Student’s t-test method.miRNAs were considered to be differentially expressed at the threshold value of p<0.05,fold change(FC)>1.5 or<1/1.5 and average expression>log2(5 RPM).The target genes of DE-miRNAs were predicted by using acknowledged online software—TargetScan with Mouse selected as species.Then,a miRNA-target gene interaction network was constructed and visualized using the Cytoscape software.To explain the underlying functions of target genes of DE-miRNAs,GO terms and KEGG pathway enrichment analyses were performed using the clusterProfiler package in R.Results:H&E staining indicated inflammatory reactions in the inner ears of animals were evident in groups after immunization,exhibiting infiltration of inflammatory cells(mainly lymphocytes)in the cochlear axis.The number of the spiral ganglion cells was also obviously reduced and distributed mussily.Shrinkage occurred in the organ of Corti,with a small amount of necrosis displayed in the outer hair cells.Vacuolar degeneration was occasionally visible in the hair cells of cristae ampullaris.These changes were not present in the control mice,indicating our AIED model may be established successfully.After quality control analysis,a total of 22 miRNAs were identified as DE-miRNAs between AIED and control mice based on the given threshold,including 10 upregulated DE-miRNAs and 12 downregulated DE-miRNAs.Based on the TargetScan prediction,1958 genes were identified as the targets for the 22 DE-miRNAs.Then,a miRNA-mRNA interaction network was constructed,in which 1696 genes and 22 miRNAs were included.Function enrichment analysis showed only target genes of 8 miRNAs were enriched for GO terms(miR-10b-3p,let-7j,miR-1943-5p,miR-196b-5p,miR-338-3p,miR-455-5p,miR-8112,miR-92b-3p)and KEGG pathways(miR-10b-3p,let-7c-5p,let-7g-5p,let-7j,miR-181 c-5p,miR-181d-5p,miR-222-3p,miR-8112),among which the target genes of miR-10b-3p,let-7j and miR-8112 were all enriched in two methods.The results showed miR-10b-3p may be especially important for AIED because its target gene was inflammatory chemokine(such as Ccl12,chemokine[C-C motif]ligand 12).miR-8112 might be involved in AIED by influencing Wnt signaling pathway(such as Wnt9b;wnt 3a;Wnt2b).The target genes of let-7j were enriched in Glycosphingolipid biosynthesis-lacto and neolacto series and Mucin type O-glycan biosynthesis pathways(i.e.Galnt2,polypeptide N-acetylgalactosaminyltransferase 2;Galnt12).Conclusion and Significance:Our present study preliminarily reveals miR-10b-3p,miR-8112 and let-7j may be potential biomarkers for AIED models.They may be involed in AIED by targeting their inflammatory target genes.Part Ⅲ:Purpose:Integrated analysis of transcriptome in ES and miRNA exprsssion profiles in serum to explore underlying target for diagnosis and treatment of AIED.Methods:Venn diagram was performed to screen the common genes in 612 DEGs and Es and 1968 target genes of DE-miRNAs.Function enrichment analysis of common genes was performed using DAVID database via selecting rat and mouse as the species,including GO and KEGG pathway.P<0.05 was set as the threshold value.Results:Venn diagram analysis indicated there were 44 common genes in 612 DEGs in ES and 1968 target genes of DE-miRNAs.These 44 genes were enriched into 10 GO terms using rat as the species,while 13 GO terms were enriched using mouse as the species.Among them,GO:0005578～proteinaceous extracellular matrix and GO:0031012-extracellular matrix the common,in which SBSPON,VWF,WNT16,ADAMTS8,SFRP1,LAMC2,AD AMTS 12 and PLSCR1 were involved.These genes were regulated by miR-1943-3p,miR-532-3p,let-7g-5p,miR-340-3p,miR-874-3p and miR-511-3p,respectively.There was a negative expression trend between miR-511-3p and its target gene ADAMTS12,indirectly explaining their negative regulation relationship.Furthermore,GO:0031295～T cell co-stimulation was also enriched in using rat as the species,suggesting its related genes(EFNB3,DPP4)may be important for AIED.EFNB3 could be regulated by miR-1943-5p,while DPP4 could be regulated by miR-196-5p.The expressions of these two interaction pairs were negatively related.Conclusion and Significance:miR-511-3p-ADAMTS12,miR-1943-5p-EFNB3 and miR-196-5p-DPP4 may be involed in AIED by participating in extracellular matrix and T cell costimulation.Thus,they may be underlying targets for diagnosis and treatment of AIED.