Saliva microbiome in primary Sjögren's syndrome reveals distinct set of disease-associated microbes.

OBJECTIVE: This study systematically aims to evaluate the salivary microbiome in patients with primary Sjögren's syndrome (pSS) using 16S rRNA sequencing approach. METHODS: DNA isolation and 16S rRNA sequencing was performed on saliva of 37 pSS and 35 control (CC) samples on HiSeq 2500 platform. 16S rRNA sequence analysis was performed independently using two popular computational pipelines, QIIME and less operational taxonomic units scripts (LoTuS). RESULTS: There were no significant changes in the alpha diversity between saliva of patients and controls. However, four genera including Bifidobacterium, Lactobacillus, Dialister and Leptotrichia were found to be differential between the two sets, and common between both QIIME and LoTuS analysis pipelines (Fold change of 2 and p < .05). Bifidobacterium, Dialister and Lactobacillus were found to be enriched, while Leptotrichia was significantly depleted in pSS compared to the controls. Exploration of microbial diversity measures (Chao1, observed species and Shannon index) revealed a significant increase in the diversity in patients with renal tubular acidosis. An opposite trend was noted, with depletion of diversity in patients with steroids. CONCLUSION: Our analysis suggests that while no significant changes in the diversity of the salivary microbiome could be observed in Sjögren's syndrome compared to the controls, a set of four genera were significantly and consistently differential in the saliva of patients with pSS. Additionally, a difference in alpha diversity in patients with renal tubular acidosis and those on steroids was observed.

Can Gut Microbiota Affect Dry Eye Syndrome?

Using metagenomics, continuing evidence has elicited how intestinal microbiota trigger distant autoimmunity. Sjögren's syndrome (SS) is an autoimmune disease that affects the ocular surface, with frequently unmet therapeutic needs requiring new interventions for dry eye management. Current studies also suggest the possible relation of autoimmune dry eye with gut microbiota. Herein, we review the current knowledge of how the gut microbiota interact with the immune system in homeostasis as well as its influence on rheumatic and ocular autoimmune diseases, and compare their characteristics with SS. Both rodent and human studies regarding gut microbiota in SS and environmental dry eye are explored, and the effects of prebiotics and probiotics on dry eye are discussed. Recent clinical studies have commonly observed a correlation between gut dysbiosis and clinical manifestations of SS, while environmental dry eye portrays characteristics in between normal and autoimmune. Moreover, a decrease in both the Firmicutes/Bacteroidetes ratio and genus Faecalibacterium have most commonly been observed in SS subjects. The presumable pathways forming the "gut dysbiosis-ocular surface-lacrimal gland axis" are introduced. This review may provide perspectives into the link between the gut microbiome and dry eye, enhance our understanding of the pathogenesis in autoimmune dry eye, and be useful in the development of future interventions.

Connection between the Gut Microbiome, Systemic Inflammation, Gut Permeability and FOXP3 Expression in Patients with Primary Sjögren's Syndrome.

The aims of this study were to explore intestinal microbial composition and functionality in primary Sjögren's syndrome (pSS) and to relate these findings to inflammation, permeability and the transcription factor Forkhead box protein P3 (FOXP3) gene expression in peripheral blood. The study included 19 pSS patients and 19 healthy controls matched for age, sex, and body mass index. Fecal bacterial DNA was extracted and analyzed by 16S rRNA sequencing using an Ion S5 platform followed by a bioinformatics analysis using Quantitative Insights into Microbial Ecology (QIIME II) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Our data suggest that the gut microbiota of pSS patients differs at both the taxonomic and functional levels with respect to healthy controls. The gut microbiota profile of our pSS patients was characterized by a lower diversity and richness and with Bacteroidetes dominating at the phylum level. The pSS patients had less beneficial or commensal butyrate-producing bacteria and a higher proportion of opportunistic pathogens with proinflammatory activity, which may impair intestinal barrier function and therefore contribute to inflammatory processes associated with pSS by increasing the production of proinflammatory cytokines and decreasing the release of the anti-inflammatory cytokine IL-10 and the peripheral FOXP3 mRNA expression, implicated in the development and function of regulatory T cells (Treg) cells. Further studies are needed to better understand the real impact of dysbiosis on the course of pSS and to conceive preventive or therapeutic strategies to counteract microbiome-driven inflammation.

Protective role of commensal bacteria in Sjögren Syndrome.

CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4+IFN-γ+ cells than conventional mice. CD4+ T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4+ T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4+IFN-γ+ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4+T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.

Dysbiosis of the buccal mucosa microbiome in primary Sjögren's syndrome patients.

Objectives: Environmental factors in the aetiology of primary Sjögren's syndrome (pSS) are largely unknown. Host-microbiome interaction at mucosal surfaces is presumed to be involved in the aetiopathogenesis of pSS. Here, we assessed whether the microbiome of the buccal mucosa is specific for pSS compared with symptom-controls. Methods: The bacterial composition of buccal swab samples from 37 pSS patients, 86 non-SS sicca patients (with similar dryness symptoms to pSS patients, but not fulfilling the classification criteria) and 24 healthy controls (HCs) was determined with 16S rRNA sequencing. Multivariate Association with Linear Models was used to find associations between individual taxa and pSS, taking into account smoking and dental status. Associations were replicated in a general population cohort (n = 103). Results: The buccal mucosa microbiome of pSS and non-SS sicca patients both differed from HCs. A higher Firmicutes/Proteobacteria ratio was characteristic for both pSS and non-SS sicca patients. Disease status (pSS, non-SS sicca, HCs) and salivary secretion rate contributed almost equally to the variation in bacterial composition between individuals (3.8 and 4.3%, respectively). Two taxa were associated with pSS compared with non-SS sicca patients and 19 compared with HCs. When salivary secretion rate was taken into account, no taxon was associated with pSS compared with non-SS sicca. Twelve of the 19 pSS-associated taxa were correlated with salivary secretion. Conclusion: Dysbiosis of the buccal mucosa microbiome in pSS patients resembles that of symptom-controls. The buccal mucosa microbiome in pSS patients is determined by a combination of reduced salivary secretion and disease-specific factors.

Prevalence and significance of anti-saccharomyces cerevisiae antibodies in primary Sjögren's syndrome.

OBJECTIVES: Saccharomyces cerevisiae is a common yeast used in the food industry. IgG and IgA antibodies against the phosphopeptidomannan of the S. cerevisiae cell wall (ASCA) are a well known marker of disease severity in Crohn's disease. Moreover, a number of studies assessed ASCA in several systemic and organ-specific autoimmune diseases postulating molecular mimicry as a possible link between ASCA and autoimmunity. However, since they have never been tested in primary Sjögren's syndrome (pSS), the purpose of this study was to investigate these antibodies in a large cohort of pSS patients, compared to healthy donors (HD), and their significance as potentially helpful biomarker in a clinical setting. METHODS: ASCA IgG+IgA were assessed with ASCA screen dot for Blue Diver instrument (Alphadia sa/nv, Belgium). The comparison between the aminoacid sequence of mannan of S. cerevisiae and well characterised auto-antigens peculiar to pSS (52kD and 60kD Ro/SSA, La/SSB) was performed with the Basic Local Alignment Search Tool (BLAST). RESULTS: The prevalence of ASCA in our pSS cohort was 4.8%. We also reported that the ASCA target protein has a high similarity with Ro60/SSA protein further supporting the molecular mimicry hypothesis. Finally, we observed that ASCA positivity is associated with pSS specific clinical and serological features. ASCA+ pSS patients displayed a triple combination of circulating anti-Ro52/SSA, anti-Ro60/SSA and anti-La/SSB antibodies, associated with low complement and cutaneous involvement. CONCLUSIONS: Our data suggest a possible pathogenic/prognostic significance of ASCA in pSS.

Oral yeast colonization in patients with primary and secondary Sjögren's syndrome.

OBJECTIVE: The study aimed to investigate the pattern of oral yeast colonization of Sjögren's syndrome patients and its correlation to salivary flow rates, age, and time of the disease progression. MATERIALS AND METHODS: Saliva and swab specimens were obtained from 45 patients (primary Sjögren's syndrome = 15/ secondary Sjögren's syndrome = 15/ healthy controls = 15). Yeast species were identified using culture method through chromogenic medium followed by polymerase chain reaction and Sanger sequencing. RESULTS: Eleven species from six different genera were detected. The most prevalent species found was Candida albicans followed by Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei. Both groups of Sjögren's syndrome showed higher counts of C. albicans (Total and CFU counts) when compared to control group. In contrast, a greater variety of yeast species was identified on samples of the control group. CONCLUSIONS: This study showed that C. albicans is the most prevalent yeast, but also that a variety of other yeast species can colonize the oral cavity of Sjogren's syndrome patients. The identification of most of the colonies was not obtained by culturing-PCR methods combined.

Interferons and Dry Eye in Sjögren's Syndrome.

Various cytokines, including interferon (IFN)-γ and IL-17, are augmented, and autoreactive T cells and B cells are activated in the immune pathogenesis of Sjögren's syndrome (SS). In particular, IFNs are involved in both the early stages of innate immunity by high level of type I IFN in glandular tissue and sera and the later stages of disease progression by type I and type II IFN producing T cells and B cells through B cell activating factor in SS. Genetically modified mouse models for some of these molecules have been reported and will be discussed in this review. New findings from human SS and animal models of SS have elucidated some of the mechanisms underlying SS-related dry eye. We will discuss IFN-γ and several other molecules that represent candidate targets for treating inflammation in SS-related dry eye.

Chronic inflammation and extra-nodal marginal-zone lymphomas of MALT-type.

Extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT) is an indolent B-cell non-Hodgkin lymphoma (NHL) arising in lymphoid populations that are induced by chronic inflammation in extra nodal sites. The stomach is the most commonly affected organ, and MALT lymphoma is clearly associated with a gastroduodenitis induced by a microbial pathogen, Helicobacter pylori, thus gastric MALT lymphoma represents a paradigm for evaluating inflammatory-associated lymphomagenesis. Variable levels of evidence have indicated a possible association between other microorganisms and non-gastric MALT lymphomas. In addition to infectious etiology, chronic inflammation arising as a result of autoimmune diseases such as Sjogren's syndrome or Hashimoto thyroiditis, poses a significant risk factor for developing NHL. Recently, genetic alterations affecting the NF-κB pathway, a major signaling pathway involved in many cancers, have been identified in MALT lymphoma. This review will present MALT lymphoma as an example of the close pathogenetic link between chronic microenvironmental inflammation and tumor development, showing how these observations can be integrated into daily clinical practice, also in terms of therapeutic implications, with particular focus on the NF-κB pathway.

Characteristics of the oral microbiota in patients with primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (pSS) is an autoimmune disease with unknown etiology that is considered to be related to environmental and genetic factors. The aim of this study was to clarify the oral microflora characteristics of pSS patients and to reveal the connection between oral bacterial composition and dental caries using a high-throughput sequencing technique. METHODS: Thirty-five pSS patients and 20 healthy controls were enrolled in this study. We collected saliva and plaque samples from pSS patients and saliva samples from healthy controls. We used 16S ribosomal DNA (16S rDNA) high-throughput sequencing targeting the V3-V4 hypervariable region to determine the composition and structure of the microbiota in the three sample sets. Finally, bioinformatics analyses, including the diversity of the microbiota, species differences, and functional prediction were performed. RESULTS: In the alpha diversity and beta diversity analysis, the Chao1 (P < 0.01), observed species (P < 0.01), and PD whole tree indices (P < 0.01) were significantly lower in the saliva and plaque samples of pSS patients than in the saliva samples of healthy controls, but the Shannon (P < 0.01) and Simpson indices (P < 0.01) were significantly higher in the healthy controls, and their total diversity significantly differed. In the main flora composition at the genus level (top 10), we identified Prevotella and Veillonella as more enriched in the saliva of pSS patients and Fusobacterium, Actinomyces, and Leptotrichia as more enriched in the plaque of pSS patients. Predictive functional analysis showed that the oral microbiota of pSS patients was related to translation, metabolism of cofactors and vitamins, and nucleotide metabolism. CONCLUSIONS: The oral microbial ecology of patients with pSS is dysregulated, resulting in a decrease in overall diversity. Prevotella and Veillonella may be related to pSS, while Fusobacterium, Actinomyces, and Leptotrichia may be related to dental caries in pSS patients. Key Points • This study revealed differences in the oral microbial composition of patients with pSS compared to healthy controls. • We included a plaque group of pSS patients to identify the microbiota related to pSS and dental caries. • Prevotella and Veillonella may contribute to pSS, and Fusobacterium, Actinomyces, and Leptotrichia are associated with dental caries in pSS patients.

Fermentation of sugars and sugar alcohols by plaque Lactobacillus strains.

OBJECTIVE: The objective was to analyse the ability of Lactobacillus strains isolated from supragingival plaque of subjects with hyposalivation and from healthy controls to ferment sugars and sugar alcohols. MATERIAL AND METHODS: Fifty strains isolated from interproximal plaque from subjects with radiation-induced hyposalivation (25 strains), subjects with primary Sjögren's syndrome (16 strains) and from subjects with normal salivary secretion rate (9 strains) were tested. Growth and pH were determined after 24 and 48 h of anaerobic incubation in vials containing basal media with 1 % of glucose, fructose, sucrose, mannitol, sorbitol or xylitol. RESULTS: No differences between strains isolated from hyposalivated subjects and controls were detected. All strains lowered the pH to <5.0 from fructose and the majority of the strains from glucose and sucrose. A pH of <5.5 was seen for 52 % of the strains using mannitol, 50 % using sorbitol and 36 % using xylitol. The ability to produce acids from sugars and sugar alcohols was highest among strains of Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus paracasei and lowest among Lactobacillus fermentum strains. CONCLUSION: A large number of Lactobacillus strains are able to ferment not only sugars but also the sugar substitutes mannitol, sorbitol and xylitol to pH levels critical for enamel demineralisation. CLINICAL RELEVANCE: Our findings suggest that products containing mannitol, sorbitol and/or xylitol may contribute to the acidogenic potential of the dental plaque and especially in hyposalivated subjects with high numbers of lactobacilli.

[Detection of oral fungal flora in patients with primary Sjogren's syndrome by real-time PCR].

OBJECTIVE: To compare the quantity of the main important Candida species in the oral cavity between the patients with primary Sjogren's syndrome (pSS) and no dry mouth healthy controls,and to explore the discriminative species of fungi between the two groups. METHODS: In this study, 25 pSS patients from Department of Oral Medicine of Peking University School and Hospital of Stomatology were enrolled, with 25 residents from a community in Beijing as the control group. All the participants were examined for the oral mucosa status, and 5 Candida DNA loads were compared between the two groups by use of the FQ-PCR technique. RESULTS: Candida albicans was the dominant Candida in both of the groups. In addition, we found that the species of Candida tropicalis and Parapsilosis were more detected in the pSS group by use of the FQ-PCR technique. Quantitative of Candida tropicalis and Candida parapsilosis in pSS group (10.6 ± 1.07, 9.47 ± 4.86) was significantly higher than that in healthy controls group (8.30 ± 3.82, 5.24 ± 6.05), the difference was statistically significant (P <0.05). Quantitative of fluconazole-sensitive strains in pSS group (12.21 ± 0.82) compared with which in healthy controls group (11.53 ± 0.81) was significantly increased (P <0.01). CONCLUSION: Candida albicans was the dominant Candida in both of the groups. Candida tropicalis and Candida parapsilosis were more detected in the pSS group.

Oral microbial biomap in the drought environment: Sjogren's syndrome.

Sjogren's syndrome (SS) is an autoimmune disease that affects primarily the salivary glands, making perturbations in the oral ecosystem and potential factors of salivary flow that influence the onset and development of the disease. The oral cavity contains diverse microorganisms that inhabit various niches such as the oral microbial "biomap." It does not seem specific enough to establish a characteristic microbiome, given the diversity of clinical manifestations, variable rates of salivary secretion, and influential risk factors in patients with SS. This review discusses the biogeography of the oral microbiome in patients with SS such as saliva, tongue, tooth, mucosa, and gum. The microorganisms that were more abundant in the different oral niches were Gram-positive species, suggesting a higher survival of cell wall bacteria in this arid oral environment. Reduced salivary flow appears not to be linked to the cause of dysbiosis alone but influences host-associated risk factors. However, much work remains to be done to establish the role of the microbiome in the etiopathogenesis of autoimmune diseases such as SS. Future studies of the microbiome in autoimmunity will shed light on the role of specific microorganisms that have never been linked before with SS.

Characteristics of gut microbiota in patients with primary Sjögren's syndrome in Northern China.

This study analyzes and compares the structure and diversity of gut microbiota in patients with primary Sjögren's syndrome (pSS) in Northern China to healthy individuals to identify clinical features associated with dysbiosis. We included 60 Chinese pSS patients and 50 age- and gender-matched healthy controls. DNA was extracted from stool samples and subjected to 16S ribosomal RNA gene analysis (V3-V4) for intestinal dysbiosis. In addition, patients were examined for laboratory and serological pSS features. A Spearman's correlation analysis was performed to assess correlations between individual bacteria taxa and clinical characteristics. The alpha-diversity (Chao1 and Shannon Index) and beta-diversity (unweighted UniFrac distances) of the gut microbiota differed significantly between pSS patients and healthy controls. Further analysis showed that several gut opportunistic pathogens (Bacteroides, Megamonas, and Veillonella) were significantly more abundant in pSS patients and positively correlated with their clinical indicators. In contrast, some probiotic genera (Collinsella, unidentified_Ruminococcaceae, Romboutsia, and Dorea) were significantly decreased in pSS patients and negatively correlated with their clinical indicators. Therefore, pSS patients in Northern China showed a dysbiotic intestinal microbiome enriched for potentially pathogenic genera that might be associated with autoimmune disease.

Bacterial distribution on the ocular surface of patients with primary Sjögren's syndrome.

Many studies have shown that gut microbial dysbiosis is a major factor in the etiology of autoimmune diseases but none have suggested that the ocular surface (OS) microbiome is associated with Sjögren's syndrome (SS). In this prospective study, we analyzed bacterial distribution on the OS in patients with primary SS. Among the 120 subjects included in this study, 48 patients (group A) had primary SS, whereas 72 subjects (group B) had dry eye symptoms that were unrelated to SS. We evaluated clinical dry eye parameters such as the OS disease index, ocular staining score (OSS), Schirmer's I test, and tear break-up time (TBUT). Conjunctival swabs were used to analyze the microbial communities from the two groups. Bacterial 16S rRNA genes were sequenced using the Illumina MiSeq platform, and the data were analyzed using the QIIME 1.9.1 program. The Shannon index was significantly lower in group A than in group B microbiota (p < 0.05). An analysis of similarity using the Bray-Curtis distance method found no difference in beta-diversity between the two groups (p > 0.05). In group A, Actinobacteria at the phylum level and Corynebacteria at the genus level exhibited low abundance than group B, but the differences were not statistically significant (p > 0.05). SS apparently decreases the diversity of the OS microbial community. These observations may be related to the pathophysiology of SS and should be investigated in future studies.

Salivary dysbiosis in Sjögren's syndrome and a commensal-mediated immunomodulatory effect of salivary gland epithelial cells.

Salivary gland epithelial cells (SGECs) have been implicated in the pathogenesis of Sjögren's syndrome due to aberrant antigen-presentation function. This study examined the hypothesis that oral dysbiosis modulates the antigen-presentation function of SGECs, which regulates CD4 T cell proliferation in primary Sjögren's syndrome (pSS). Saliva samples from 8 pSS patients and 16 healthy subjects were analyzed for bacterial 16S ribosomal DNA. As a result, 39 differentially abundant taxa were identified. Among them, the phylum Proteobacteria comprised 21 taxa, and this phylum was mostly enriched in the healthy controls. The proteobacterium Haemophilus parainfluenzae was enriched in the healthy controls, with the greatest effect size at the species level. Treatment of A253 cells in vitro with H. parainfluenzae upregulated PD-L1 expression, and H. parainfluenzae-pretreated A253 cells suppressed CD4 T cell proliferation. The suppression was partially reversed by PD-L1 blockade. Among low-grade xerostomia patients, salivary abundance of H. parainfluenzae decreased in pSS patients compared to that in non-pSS sicca patients. Our findings suggest that H. parainfluenzae may be an immunomodulatory commensal bacterium in pSS.