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. 2021 Jul 6:12:681941.
doi: 10.3389/fimmu.2021.681941. eCollection 2021.

The Transcriptome of Paired Major and Minor Salivary Gland Tissue in Patients With Primary Sjögren's Syndrome

Gwenny M Verstappen  1 , Lu Gao  2 , Sarah Pringle  1 , Erlin A Haacke  3 , Bert van der Vegt  3 , Silvia C Liefers  1 , Vishal Patel  2 , Yanhua Hu  2 , Sumanta Mukherjee  2 , Julie Carman  2 , Laurence C Menard  2 , Frederik K L Spijkervet  4 , Arjan Vissink  4 , Hendrika Bootsma  1 , Frans G M Kroese  1
Affiliations

The Transcriptome of Paired Major and Minor Salivary Gland Tissue in Patients With Primary Sjögren's Syndrome

Gwenny M Verstappen et al. Front Immunol. .

Abstract

Background: While all salivary glands (SGs) can be involved in primary Sjögren's syndrome (pSS), their respective role in pathogenesis remains unclear. Our objective was to assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients.

Methods: Paraffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. RNA was extracted, complementary DNA libraries were prepared and sequenced. For analysis of differentially expressed genes (DEGs), patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology.

Results: With principal component analysis, only biopsy-positive pSS could be separated from non-SS sicca patients based on SG gene expression. When comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<0.05, log2FC<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy-negative pSS and non-SS sicca patients was scarce. Overall, transcript expression levels correlated strongly between parotid and labial glands (R2 = 0.86, p-value<0.0001). Gene signatures present in both glands of biopsy-positive pSS patients included IFN-α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients.

Conclusion: Transcriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. Different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches.

Keywords: B cell abnormalities; Sjogren’s syndrome; T cell activation, salivary gland; autoimmune disease; transcriptome (RNA-seq).

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Conflict of interest statement

LG, YH, and LM are employed at BMS. VP is past employee of BMS and currently employed at Novartis. SM is past employee of BMS and currently employed at GlaxoSmithKline. JC is past employee of BMS and currently employed at Johnson&Johnson. BV is scientific advisory board member of Visiopharm. HB received unrestricted grants from BMS and Roche, is consultant for BMS, Roche, Novartis, Medimmune, Union Chimique Belge, speaker for BMS and Novartis. FK received unrestricted grants from BMS, is consultant for BMS, speaker for BMS, Roche and Janssen-Cilag. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Transcriptome analysis of paired parotid and labial gland samples. Fifty-two parotid samples and 57 labial samples were included. (A) Principal component analysis of parotid and labial gland samples. Colors indicate the patient groups and triangles indicate the 10 samples with the highest area fraction of CD45+ cells. (B) Volcano plots showing differentially expressed genes (DEGs) between biopsy-positive pSS patients (group-IV) and biopsy-negative non-SS sicca patients (group-I) for parotid and labial gland samples. (C) Venn diagram of (overlapping) DEGs between parotid and labial gland samples and correlation plot for up- and down-regulated genes (adj.p<0.05, log2FC>1) in either tissue. (D) Volcano plots showing hardly any differential gene expression between biopsy-negative pSS patients (group-III) and biopsy-negative non-SS sicca patients (group-I) for parotid and labial gland samples.
Figure 2
Figure 2
Upregulated immune pathways and immune-related genes in labial and parotid gland tissue of primary SS patients. (A) Metacore pathway analysis of differentially expressed genes (DEGs) between biopsy-positive primary SS and biopsy-negative non-SS sicca patients. A separate pathway analysis was performed for labial and parotid gland tissue. Only the top (auto)immune-related pathways that were enriched in both glands are shown (FDR<0.05). Enrichment values provided by Metacore are displayed as −Log(adj.p) for parotid gland tissue (grey bars) and labial gland tissue (white bars). (B) DEGs upregulated in salivary gland tissue of biopsy-positive pSS patients are depicted in association with their predicted cell of origin. Other DEGs with known immune function were categorized and are listed in separate boxes. Created with Biorender.com.
Figure 3
Figure 3
Gene signature analysis of paired salivary gland tissue samples. (A) Gene signature scores were calculated for all individual samples and plotted per signature per group for either the parotid or labial gland. *p < 0.005 (B) IFN-α signature scores in parotid and labial gland tissue and PBMCs were correlated with blood MxA levels. Colors represent the different patient groups as indicated in Figure 1 .
Figure 4
Figure 4
Hierarchical clustering of parotid and labial gland samples from pSS patients. Unsupervised hierarchical clustering for all pSS patients was performed based on expression of the top 50 upregulated genes in each tissue. Each column represents a patient and each row represents a gene. The following clinical parameters were categorized and marked: Diagnostic biopsy focus score, EULAR Sjögren’s syndrome disease activity index (ESSDAI), EULAR Sjögren’s syndrome patient reported index (ESSPRI), unstimulated whole saliva (UWS) flow rates, rheumatoid factor (RF) positivity, anti-SSA positivity, histopathological phenotype.
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