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Microbiological and bioinformatics analysis of primary Sjogren's syndrome patients with normal salivation.

ABSTRACT: BACKGROUND:Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren's syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present. OBJECTIVE:This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation. METHODS:Bacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren's saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used. RESULTS:Saliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in pSS and 22% in controls), about a twofold increase in pSS of Streptococcus (28% vs. 17%) and Veillonella (26% vs. 12%) was detected. Prevotella melaninogenica was the major species in controls (13%) while Veillonella atypica and the Veillonella parvula groups dominated in patient samples (14 and 14%). The scarcity in bacterial species in pSS compared with controls was also demonstrated by alpha and beta diversity analyses, as well as read abundance depicted in a phylogenetic tree. CONCLUSIONS:While Firmicutes was significantly higher in pSS patients than in controls, Synergistetes and Spirochaetes were significantly lower. The number of bacterial genera and species was also lower. These data showed that microbial dysbiosis is another key characteristic of pSS whole saliva which can occur independent of hyposalivation.

SUBMITTER: Siddiqui H

PROVIDER: S-EPMC5075221 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Microbiological and bioinformatics analysis of primary Sjogren's syndrome patients with normal salivation.

Siddiqui Huma H Chen Tsute T Aliko Ardita A Mydel Piotr M PM Jonsson Roland R Olsen Ingar I

Journal of oral microbiology 20161020

<h4>Background</h4>Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren's syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present.<h4>Objective</h4>This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS ...[more]

PMID: 27770517

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Project description:BackgroundPrimary Sjögren's syndrome (pSS) is a relatively common diffuse connective tissue disease that often invades exocrine glands, such as the lacrimal and salivary glands, and manifests as dry eyes and dry mouth. At present, the molecular mechanism of pSS is not clear. This study was designed to explore the internal mechanism of pSS from the gene level and screen out the immune-related diagnostic markers of pSS.MethodsThe gene expression profiles GSE84844, GSE7451, and GSE40611 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified with R software. Then, the DEGs were intersected with the immune genes obtained from the ImmPort database to acquire differentially expressed immune-related genes (DEIRGs), and functional enrichment analyses were performed. The DEIRGs were screened through the least absolute shrinkage and selection operator (LASSO) logistic regression algorithm to obtain the optimal immune-related genes (IRGs). Expression levels of the optimal IRGs were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to obtain the key genes. Next, gene chips GSE7451 and GSE40611, from other tissues, were selected as the training sets to verify the sensitivity and specificity of the diagnosis of the key genes by receiver operating characteristic (ROC) analysis.ResultsA total of 54 DEIRGs were obtained. The functional enrichment analysis results showed that they play an important role in immune and inflammatory responses. Nine optimal IRGs were screened from the DEIRGs by the LASSO logistic regression algorithm. After qRT-PCR verification, eight out of nine optimal IRGs (IL-18, JAK2, TBK1, EED, TNFSF10, TNFSF13B, CYSLTR1, and ICOS) were significantly highly expressed in pSS patients and were defined as key genes. ROC analysis identified that TNFSF13B and CYSLTR1 had high sensitivity and specificity. Finally, the lack of previous research on EED and CYSLTR1 in pSS suggests that these IRGs may be regarded as new gateways to explore the diagnosis and pathogenesis of pSS.ConclusionsThe key DEIRGs play a decisive role during the occurrence and development of pSS.

Project description:Objective: Salivary gland ultrasound (SGUS) is emerging as a valid tool in the management of primary Sjögren's syndrome (pSS). This study aimed to investigate whether pSS patients with normal-appearing or pathological SGUS findings showed different clinical, laboratory, and pathologic pSS-related features, and to compare the results by using two different SGUS scores. Methods: Consecutive pSS patients, according to the ACR-EULAR classification criteria, were evaluated. Salivary glands were scored using the early 1992 score by De Vita et al. and the latest 2019 OMERACT score, both being semiquantitative 0-3 scoring systems focused on ultrasonographic parenchymal inhomogeneity (grades 0 and 1, normal-appearing; grades 2 and 3, pathological). The patients were then divided into two groups: "SGUS normal-appearing" if all the salivary glands had normal-appearing parenchyma (grade 0 or 1), or "SGUS pathological" if the grade was 2 or 3 in at least one salivary gland. The associations between SGUS and pSS-related clinical, laboratory, and pathological features were then investigated in the two groups. Results: One hundred pSS patients were evaluated, the mean age (±SD) was 60.9 ± 12.0 years, and mean disease duration was 11.7 ± 7.2 years. Twenty-nine out of 100 (29%) patients were in the "SGUS normal-appearing" group and 71/100 (71%) were in the "SGUS pathological" group. A normal-appearing SGUS was significantly associated with the absence of anti-La/SSB antibodies (p < 0.001) and normal unstimulated salivary flow rate (p = 0.02) by both univariate and multivariate analyses. By univariate analysis, a normal-appearing SGUS was significantly associated also with the absence of rheumatoid factor (p = 0.002) and of serum monoclonal component (p = 0.003), ESSDAI < 5 (p = 0.03), and with a negative lip biopsy (p = 0.029). No associations were found with other items, including anti-Ro/SSA (p = 0.145), Schirmer's test (p = 0.793), ESSPRI (p = 0.47), and demographic data. No differences in these results were observed by using the two SGUS scoring systems. Conclusion: The SGUS allowed the identification of different phenotypes of pSS, and different SGUS scores focused on salivary gland inhomogeneity may be effective to this end.

Project description:BackgroundPrimary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease of the exocrine glands characterized by specific pathological features. Previous studies have pointed out that salivary glands from pSS patients express a unique profile of cytokines, adhesion molecules, and chemokines compared to those from healthy controls. However, there is limited evidence supporting the utility of individual markers for different stages of pSS. This study aimed to explore potential biomarkers associated with pSS disease progression and analyze the associations between key genes and immune cells.MethodsWe combined our own RNA sequencing data with pSS datasets from the NCBI Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) via bioinformatics analysis. Salivary gland biopsies were collected from 14 pSS patients, 6 non-pSS patients, and 6 controls. Histochemical staining and scanning electron micrographs (SEM) were performed to macroscopically and microscopically characterize morphological features of labial salivary glands in different disease stages. Then, we performed quantitative PCR to validate hub genes. Finally, we analyzed correlations between selected hub genes and immune cells using the CIBERSORT algorithm.ResultsWe identified twenty-eight DEGs that were upregulated in pSS patients compared to healthy controls. These were mainly involved in immune-related pathways and infection-related pathways. According to the morphological features of minor salivary glands, severe interlobular and periductal lymphocytic infiltrates, acinar atrophy and collagen in the interstitium, nuclear shrinkage, and microscopic organelle swelling were observed with pSS disease progression. Hub genes based on above twenty-eight DEGs, including MS4A1, CD19, TCL1A, CCL19, CXCL9, CD3G, and CD3D, were selected as potential biomarkers and verified by RT-PCR. Expression of these genes was correlated with T follicular helper cells, memory B cells and M1 macrophages.ConclusionUsing transcriptome sequencing and bioinformatics analysis combined with our clinical data, we identified seven key genes that have potential value for evaluating pSS severity.

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Microbiological and bioinformatics analysis of primary Sjogren's syndrome patients with normal salivation.

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Microbiological and bioinformatics analysis of primary Sjogren's syndrome patients with normal salivation.

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