Genome‐wide DNA‐methylation profiles in human bone marrow mesenchymal stem cells on titanium surfaces

The characteristics of titanium (Ti) have been shown to influence dental implant fixation. Treatment of surfaces using the sandblasted, large‐grit, acid‐etched (SLA) method is widely used to provide effective osseointegration. However, the DNA methylation‐associated mechanism by which SLA surface treatment affects osseointegration of human bone marrow mesenchymal stem cells (hBMSCs) remains elusive. Genome‐wide methylation profiling of hBMSCs on SLA‐treated and machined smooth Ti was performed using Illumina Infinium Methylation EPIC BeadChip at day 7 of osteogenic induction. In total, 2,846 CpG sites were differentially methylated in the SLA group compared with the machined group. Of these sites, 1,651 (covering 1,066 genes) were significantly hypermethylated and 1,195 (covering 775 genes) were significantly hypomethylated. Thirty significant enrichment pathways were observed, with Wnt signaling being the most significant. mRNA expression was identified by microarray and combined with DNA‐methylation profiles. Thirty‐seven genes displayed negative association between mRNA expression and DNA‐methylation level, with the osteogenesis‐related genes insulin‐like growth factor 2 (IGF2) and carboxypeptidase X, M14 Family Member 2 (CPXM2) showing significant up‐regulation and down‐regulation, respectively. In summary, our results demonstrate differences between SLA‐treated and machined surfaces in their effects on genome‐wide DNA methylation and enrichment of osteogenic pathways in hBMSCs. We provide novel insights into genes and pathways affected by SLA treatment in hBMSCs at the molecular level.     

DNA methylation profiling in different phases of temporomandibular joint osteoarthritis in rats

ObjectiveTemporomandibular joint osteoarthritis (TMJOA) is a complex disease with strong genetic and epigenetic components in its pathogenesis. The aim of this study was to evaluate DNA methylation in mandibular head cartilage in different phases of experimentally-induced TMJOA in rats.DesignDNA methylation was evaluated using microarrays in the mandibular head cartilage of early, intermediate and late stage experimentally-induced TMJOA, and of the normal age-matched control groups. Genes with differentially methylated CpG sites were analyzed to reveal the over-represented gene ontologies and pathways at different stages, and were compared with published expression profiles to assess their overlappings. The DNA methylation patterns of the target genes were validated by methylated DNA immunoprecipitation qPCR in additional independent cartilage samples and mRNA levels were analyzed by real-time PCR.ResultsWe observed 9489 differentially methylated regions between the TMJOA and controls. A total of 440 consistently altered genes were revealed in all three stages; most (80%) were hypomethylated and many were associated with cell cycle regulation. We also detected different DNA methylation changes in early and late stage TMJOA (R_early = 0.68, R_late = 0.47), while the differences between age-matched healthy cartilage were subtle. Strong inverse changes between methylation status and mRNA levels were confirmed in Adamts5, Chad, Cldn11 and Tnf.ConclusionsOur data reveals dynamic DNA methylation patterns during the progression of TMJOA, with a different host of genes and pathways. The changes of cartilage DNA methylation patterns might contribute to understand the etiologic mechanisms of TMJOA epigenetically.     

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren's syndrome-like disease

Oral Diseases (2011) 17 , 801–807 Objective: Sjögren’s syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS‐like disease. Methods: Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll‐like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland–infiltrating cells were characterized by flow cytometry. Results: Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)‐treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. Conclusions: Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS‐like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.     

非综合征型唇腭裂DNA甲基化谱的生物信息学分析

目的:针对非综合征型唇腭裂（non-syndrome cleft lip/cleft, NSCL/P）DNA甲基化谱,采用生物信息学技术筛选NSCL/P异常甲基化位点和异常甲基化区域,探讨DNA甲基化与NSCL/P发病机制的关系。方法:从基因表达综合数据库（Gene Expression Omnibus, GEO）的数据集下载原始数据,纳入67例NSCL/P患者和59例无出生缺陷者的全血甲基化数据。数据分析包括①探针过滤、质控、归一化等数据清洗;②差异甲基化位点和差异甲基化区域等差异甲基化分析;③对差异甲基化区域所在的基因进行基因本体（Gene Ontology,GO）富集分析、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）信号通路富集分析,明确异常DNA甲基化对生物功能学的影响。采用R 3.4.3统计学软件进行数据清洗、筛选差异甲基化位点和差异甲基化区域,采用DAVID 6.8工具对差异甲基化区域进行GO和KEGG富集分析。结果:NSCL/P患者和正常对照者差异显著的甲基化位点共814个（校正P<0.001,|Δβ|>0.125）,其中NSCL/P组与对照组相比,高甲基化位点178个,低甲基化位点636个;差异显著的甲基化区域共386个（P<0.05）,其中高甲基化区域204个,低甲基化区域182个。GO富集分析显示,富集于7个生化过程相关的差异性甲基化基因共38个,富集于3个分子功能相关的差异性甲基化基因共163个,富集于3个细胞成分有关的差异性甲基化基因共114个（P<0.01）。KEGG通路分析显示,9个信号通路出现差异性甲基化基因富集,涉及59个基因（P<0.05）。结论:DNA甲基化异常可能是影响NSCL/P发生、发展的重要表观遗传学机制,为诊断标志物筛选和靶向干预提供了线索。     

Review of genes potentially related to hyposecretion in male non-obese diabetic (NOD) mice,a Sjögren's syndrome model

BackgroundSjögren's syndrome (SS) is known to cause dry eyes and mouth due to inflammation of the lacrimal and salivary glands. However, some reports imply that other factors trigger dry eyes and mouth. We previously investigated various factors using RNA-sequencing analysis of lacrimal glands from male non-obese diabetic (NOD) mice, an SS model. In this review, we described (1) the exocrine features of male and female NOD mice, (2) the up- and down-regulated genes in the lacrimal glands of male NOD mice as revealed by our RNA-sequencing data, and (3) comparisons between these genes and data in the Salivary Gland Gene Expression Atlas.HighlightsMale NOD mice exhibit a steady worsening of lacrimal hyposecretion and dacryoadenitis, whereas females exhibit a complex pathophysiological condition that includes diabetic disease, salivary hyposecretion, and sialadenitis. Ctss, an up-regulated gene, is a potential inducer of lacrimal hyposecretion and is also expressed in salivary glands. Two other up-regulated genes, Ccl5 and Cxcl13, may worsen the inflammation of SS in both the lacrimal and salivary glands. The genes Esp23, Obp1a, and Spc25 were detected as down-regulated, but judging the relationship between these genes and hyposecretion is difficult as only limited information is available. Another down-regulated gene, Arg1, is involved in lacrimal hyposecretion, and it also has the potential to cause salivary hyposecretion in NOD mice.ConclusionIn NOD mice, males may be better than females at evaluating the pathophysiology of SS. Some regulated genes revealed by our RNA-sequencing data might be potential therapeutic targets for SS.     

Sjögren's syndrome

Sjögren's syndrome (SS) is a chronic autoimmune disease affecting the exocrine glands, primarily the salivary and lacrimal glands. It has been suggested that exogenous agents may trigger SS in genetically predisposed individuals. However, at present, the etiology of SS is far from being understood, and no direct evidence for any of these triggers has been presented. The salivary and lacrimal glands from patients with SS harbor unique and highly selected T‐ and B‐cell populations. Disturbance in glandular cell apoptosis may be one possible explanation for the sicca symptoms in SS. However, discrepancies between glandular destruction and salivary flow give rise to processes causing glandular dysfunction preceding or triggering glandular cell destruction. Recent reports suggested autoantibodies inhibiting neuronal innervation of acinar cells and defective water transport to be implicated in salivary secretion deficiency observed in SS. Several types of autoantibodies have been suggested to contribute to the pathogenesis of SS. However, how the tolerance to these structures is broken down is unknown at present. Studies on B‐cell activating factor indicated that diminished apoptosis and disturbed B‐cell maturation could be responsible for the occurrence of autoreactive B‐cells and B‐cell hyperreactivity. B‐cell activation may also provide a basis for lymphoma development observed in up to 5% of the patients with SS.     

Oral manifestations and their treatment in Sjögren′s syndrome

Sjögren's syndrome (SS) is a complex, chronic, systemic, autoimmune disease that mainly affects the exocrine glands, especially the salivary and lacrimal glands, leading to dryness of the oral and ocular mucosae. Several factors have been studied that could explain the glandular hypofunction primarily related to water transport. Recent reports have shown alterations in secretory route and trafficking in labial salivary glands, explaining alterations in the saliva quality. The decrease in salivary flow and qualitative alterations in saliva could explain many of the oral manifestations. The exocrine manifestations and systemic involvement significantly impact the patient's perception of health‐related quality of life. For this reason and given its systemic nature, the treatment of these patients should be multidisciplinary. This review addresses some particular oral health aspects of SS patients and focuses on relevant topics concerning the treatment and prevention of common oral disorders associated with this disease.     

Follicular dendritic cells confirm lymphoid organization in the minor salivary glands of primary Sjögren’s syndrome

Background: Sjögren’s syndrome (SS) is an autoimmune chronic inflammatory disorder affecting the salivary and lacrimal glands. The aim of this study was to explore immunophenotypic features of chronic inflammatory reactions in the minor salivary glands in patients with primary SS (pSS). Methods: Formalin‐fixed, paraffin‐embedded labial minor salivary gland tissue sections from randomly selected patients with pSS (n = 60) were investigated for the expression of CD21, CD23, CD35 and IgD by immunohistochemistry. Results: Based on the distribution and staining pattern of CD21, CD23, CD35 and IgD in lymphoid aggregates, several stages of chronic inflammatory reactions were observed. In 12/60 (20%) patients, lymphoid infiltrates with germinal centre (GC)‐like features such as extensive networks of CD21‐, CD23‐ and CD35‐positive cells were observed in the minor salivary gland tissue. Smaller networks and /or focal infiltrates with scattered CD21⁺, CD23⁺ and CD35⁺ cells were observed in the remaining 48/60 (80 %) cases. When dividing patients according to the presence (GC+) or the absence (GC?) of GC in the minor salivary glands, the mean focus score was significantly higher in the GC+ patients (P < 0.05). Double staining of the minor salivary glands revealed focal infiltrates with follicular dentritic cell networks and B cells resembling normal GCs in tonsillar tissue. Conclusion: A particular cellular profile was demonstrated in a sub‐group of patients with pSS and could be linked to serological aberrations. These findings warrant further prospective studies.     

Salivary glands – `an unisex organ’?

Oral Diseases (2010) 16 , 577–585 Usually no distinction is made between female and male salivary glands although cyclic changes of and/or differences in serum and salivary sex steroid concentrations characterize women and men. Moreover, sexual dimorphism is well recognized in salivary glands of rodents. Salivary glands contain estrogen and androgen receptors and are, according to modern high throughput technologies, subjected to gender differences not explainable by gene dose effects by the X chromosome alone. Because sex steroids are lipophilic, it is often thought that approximately 10% of them passively diffuse from plasma to saliva. Indeed, saliva can find use as sample material in sports medicine, pediatrics, veterinary medicine and behavioral sciences. Last but not least, humans and other primates are unique in that they have a reticular zone in their adrenal cortex, which produces dehydroepiandrosterone and androstendione pro‐hormones. These are processed in peripheral tissues, not only in female breast and uterus and male prostate, but also in salivary glands by an intracrine enzymatic machinery to active 17β‐estradiol, dihydrotestosterone and others, to satisfy and buffer against a constantly changing needs caused by circadian, menstrual, pregnancy and chronobiological hormonal changes in the systemic circulation. Female dominance of Sjögren’s syndrome and certain forms of salivary gland cancer probably reflect these gender‐based differences.     

Effect of Myd88 deficiency on gene expression profiling in salivary glands of female non-obese diabetic (NOD) mice

ObjectivesSjögren's syndrome (SS) is a chronic autoimmune disease characterized by inflammatory lesions in the salivary and lacrimal glands, which are caused by distinct lymphocytic infiltrates. Female non-obese diabetic (NOD) mice spontaneously develop inflammatory lesions of the salivary glands with SS-like pathological features. Previous studies have shown that MyD88, a crucial adaptor protein that activates innate immune signaling, affects lymphocytic infiltration, but its detailed role remains unclear. In this study, we investigated the role of MyD88 through gene expression profiling in the early phase of pathogenesis in the salivary glands of female NOD mice.MethodsSubmandibular glands collected from 10-week-old female wild-type and Myd88-deficient NOD mice were used for RNA preparation, followed by microarray analysis. The microarray dataset was analyzed to identify Myd88-dependent differentially expressed genes (DEGs). Data generated were used for GO enrichment, KEGG pathway, STRING database, and INTERFEROME database analyses.ResultsMyd88 deficiency was found to affect 230 DEGs, including SS-associated genes, such as Cxcl9 and Bpifa2. Most of the DEGs were identified as being involved in immunological processes. KEGG pathway analysis indicated that the DEGs were putatively involved in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, the DEGs included 149 interferon (IFN)-regulated genes.ConclusionsMyD88 is involved in the expression of specific genes associated with IFN-associated immunopathological processes in the salivary glands of NOD mice. Our findings are important for understanding the role of MyD88-dependent innate immune signaling in SS manifestation.     

DNA methylation patterns of genes related to immune response in the different clinical forms of oral lichen planus

Background

The oral lichen planus is a chronic inflammatory disease. Although its aetiology is not well understood, the role of T lymphocytes in its inflammatory events is recognised. Identifying the epigenetic mechanisms involved in the pathogenesis of this immune‐mediated condition is fundamental for understanding the inflammatory reaction that occurs in the disease. The purpose of this work was to evaluate the methylation pattern of 21 immune response‐related genes in the different clinical forms of oral lichen planus.

Methods

A cross‐sectional study was performed to analyse the DNA methylation patterns in three distinct groups of oral lichen planus: (i) reticular/plaque lesions; (ii) erosive lesions; (iii) normal oral mucosa (control group). After DNA extraction from biopsies, the samples were submitted to digestions by methylation‐sensitive and methylation‐dependent enzymes and double digestion. The relative percentage of methylated DNA for each gene was provided using real‐time polymerase chain reaction arrays.

Results

Hypermethylation of the STAT5A gene was observed only in the control group (59.0%). A higher hypermethylation of the ELANE gene was found in reticular/plaque lesions (72.1%) compared to the erosive lesions (50.0%).

Conclusion

Our results show variations in the methylation profile of immune response‐related genes, according to the clinical type of oral lichen planus after comparing with the normal oral mucosa. Further studies are necessary to validate these findings using gene expression analysis.     

DNA甲基化在调节干细胞成骨分化中的作用

DNA甲基化和去甲基化是表观遗传学的重要机制之一,在细胞分化、增殖、衰老等方面具有重要的调控作用。干细胞在成骨分化过程中成骨特异性基因发生去甲基化进而表达上调,而与干细胞多能分化潜能相关的基因发生高甲基化进而表达抑制。DNA甲基化和去甲基化的动态变化和平衡,对于协调基因表达的时序性和抑制不和谐的分化表型具有重要作用,是干细胞成骨分化的重要保证。成骨分化中甲基化修饰机制的异常不仅会影响干细胞的正常成骨分化功能,并且与多种骨骼常见疾病的发生发展具有密切的关系。本文综述了干细胞成骨分化过程中受DNA甲基化修饰调控的相关基因和调控机制的新进展,以及DNA甲基化修饰异常可能导致的骨骼疾病。     

表观遗传在牙发生和牙再生中的作用及意义

表观遗传是指DNA序列不发生变化,基因表达却发生了可遗传改变的一种遗传方式,主要涉及DNA甲基化和组蛋白的不同翻译后修饰,决定了特定的基因表达形式。DNA甲基化常引起基因表达抑制,而脱甲基化则引起基因表达开放。组蛋白有众多的共价修饰形式,根据修饰的种类、位点及个数的不同,引起基因沉默或激活。表观遗传修饰是细胞定向分化和重编程中基因特异性表达的重要调控方式,在机体发生中扮演着重要的角色。在牙发生过程中,表观遗传与传统的基因表达调控协同,调节细胞增殖、分化和迁移相关基因的时空表达,最后导致牙的形成。诠释牙发生过程中的表观遗传调控机制,无疑可为牙再生提供关键的线索和思路。     

Fcγ receptors mediate internalization of anti-Ro and anti-La autoantibodies from Sjögren''s syndrome and apoptosis in human salivary gland cell line A-253

Background: The presence of serum anti‐Ro and anti‐La autoantibodies directed against the ribonucleoproteins Ro and La has been associated with Sjögren’s syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. There is increasing evidence of the direct involvement of autoantibodies in the pathogenesis of tissue injury and correlation of their presence with clinical manifestations in SS. The focus of this work was to explore the cellular apoptotic pathway triggered by binding and penetration of anti‐Ro and anti‐La autoantibodies in human salivary gland cell line A‐253 and to identify the membrane receptors through which anti‐Ro and anti‐La could exert their effect. Methods: Anti‐Ro and anti‐La autoantibodies were purified from IgG fractions, obtained from eleven healthy volunteers and patients with primary Sjögren’s syndrome, using Sepharose 4B‐Ro and Sepharose 4B‐La affinity columns. Flow cytometry, RT‐PCR, western blot and confocal microscopy analysis were used to visualize the FCγRI, FCγRII and FCγRIII receptors on the A‐253 cell membrane. DNA laddering and western blot analysis of caspases activation were studied to evaluate in A‐253 cells treated with anti‐Ro and anti‐La autoantibodies. Results: The results yeilded the evidence of the presence of members of the Fcγ receptors (FcγRs) family on the cell membrane of the human salivary gland cell line A‐253. Furthermore, we demonstrated that, in the A‐253 cell line, anti‐Ro and anti‐La autoantibodies can access the cells probably through Fcγ receptors, and trigger apoptotis. Conclusions: We conclude that anti‐Ro and anti‐La autoantibodies have pathogenic effects that could depend on binding to Fcγ receptors.     

Cytokines in Sjögren's syndrome

Cytokines play a central role in the regulation of immunity and are often found to be deregulated in autoimmune diseases. Sjögren's syndrome is a chronic autoimmune disease characterized by inflammation and loss of secretory function of the salivary and lachrymal glands. This review highlights the current knowledge of the expression and the function of pro- and anti-inflammatory cytokines both locally and systemically in Sjögren's syndrome patients. In the salivary glands, saliva and serum of these patients, many pro-inflammatory cytokines are upregulated. Concomitantly, most anti-inflammatory cytokines are not detectable or are expressed at low levels. Besides a role in inflammation, cytokines are also thought to be involved in salivary gland dysfunction by directly interfering with the epithelial cells in the glands. Future research on the role of novel cytokines in Sjögren's syndrome in combination with a better understanding of the effect of cytokines on exocrine dysfunction will aide the identification of the best therapeutic targets for Sjögren's syndrome.     

Loss of PKCδ results in characteristics of Sjögren's syndrome including salivary gland dysfunction

Oral Diseases (2011) 17 , 601–609 Objectives: Chronic infiltration of lymphocytes into the salivary and lacrimal glands of patients with Sjögren’s Syndrome (SS) leads to destruction of acinar cells and loss of exocrine function. Protein kinase C‐delta (PKCδ) is known to play a critical role in B‐cell maintenance. Mice in which the PKCδ gene has been disrupted have a loss of B‐cell tolerance, multiple organ lymphocytic infiltration, and altered apoptosis. To determine whether PKCδ contributes to the pathogenesis of SS, we quantified changes in indicators of SS in PKCδ?/? mice as a function of age. Salivary gland histology, function, the presence of autoantibodies, and cytokine expression were examined. Materials and methods: Submandibular glands were examined for the presence of lymphocytic infiltrates, and the type of infiltrating lymphocyte and cytokine deposition was evaluated by immunohistochemistry. Serum samples were tested by autoantibody screening, which was graded by its staining pattern and intensity. Salivary gland function was determined by saliva collection at various ages. Results: PKCδ?/? mice have reduced salivary gland function, B220+ B‐cell infiltration, anti‐nuclear antibody production, and elevated IFN‐γ in the salivary glands as compared to PKCδ+//+ littermates. Conclusions: PKCδ?/? mice have exocrine gland tissue damage indicative of a SS–like phenotype.