Perturbation of transcriptome in non-neoplastic salivary gland epithelial cell lines derived from patients with primary Sjögren's syndrome

The data presented here are related to the research article titled “Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation” (Vakrakou et al., Journal of Autoimmunity, in press, 2017). In the cited manuscript, using comparative analyses of salivary gland biopsy specimens and ductal salivary gland epithelial cell (SGEC) lines from SS patients and disease controls, we have demonstrated that the ductal epithelia of SS patients display constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function that were associated with cell-autonomously activated NF-κB and IL-1β pathways in these cells. Herein, the comparative transcriptome analysis of SGEC lines is presented. We show that the ductal epithelia of SS patients with severe lymphoepithelial lesions manifest constitutive perturbation in various inflammation- and metabolism related signaling pathways.


a b s t r a c t
The data presented here are related to the research article titled "Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation" (Vakrakou et al., Journal of Autoimmunity, in press, 2017). In the cited manuscript, using comparative analyses of salivary gland biopsy specimens and ductal salivary gland epithelial cell (SGEC) lines from SS patients and disease controls, we have demonstrated that the ductal epithelia of SS patients display constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function that were associated with cell-autonomously activated NF-κB and IL-1β pathways in these cells. Herein, the comparative transcriptome analysis of SGEC lines is presented. We show that the ductal epithelia of SS patients with severe lymphoepithelial lesions manifest constitutive perturbation in various inflammation-and metabolism related signaling pathways. Data accessibility The data will be available with this article. All microarray data are available in the GEO archive under the accession number GSE97614.

Value of the data
This is the first study to analyze the differential gene expression between non-neoplastic SGEC lines derived from SS patients and those from non-SS controls.
The results of this study provide supportive evidence for the active role of ductal epithelia in the pathogenesis of SS, as well as for the occurrence of cell-autonomous aberrations in these cells.
The identification of affected signaling pathways in the SGEC lines of SS patients sheds new light on the investigation of disease pathogenesis and for novel treatment targets.

Data
The data presented here are related to the research article entitled "Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation" [1]. To better examine the hitherto gathered evidence of epithelial deregulation in SS patients [2], we performed for the first time comparative analysis of gene expression profiles in longterm cultured non-neoplastic SGEC lines derived from SS patients (SS-SGEC; n ¼ 9) and non-SS controls (normal-SGEC lines; n ¼ 3). The SS-SGEC lines studied included two subgroups, which were selected on the basis of the histological severity of lymphoepithelial infiltrations found in the MSG biopsies from which they were obtained (SS-Group-1; n ¼ 3, focus score r 2 and SS-Group-2; n ¼ 6, focus score 4 2). Fig. 1A and Excel File 1 present the differentially expressed genes (DEG), which were obtained following the comparisons between the various groups of SGEC lines studied. Selected DEG (3 up-regulated and one down-regulated) were validated by quantitative RT-PCR (Fig. 2). Fig. 1B and Fig. 3 show the hierarchical clustering of DEG and of total genes studied, respectively. The various signaling pathways found affected in the various groups of SS-SGEC lines are presented in Excel File 2. The analysis of the subgroups of SS-SGEC lines revealed the perturbation of various inflammation-

Patients and cell lines
Long-term cultures of non-neoplastic secondary salivary gland epithelial cell (SGEC) lines were established from minor salivary gland(MSG) biopsies and maintained in serum-free keratinocyte basal medium (KBM; Clonetics), as previously described [1]. The study was approved by the Ethics Committee of the School of Medicine, National University of Athens, Greece (protocol no. 5107). The exclusive epithelial nature and ductal epithelial origin of cultured SGEC lines was routinely verified by morphology, as well as by the uniform and consistent expression of epithelial specific markers and the absence of markers indicative of other types of cells. All our SS patients were diagnosed on the basis of the American-European SS classification criteria. The SS-SGEC lines studied were selected on  the basis of the intensity of lymphoepithelial infiltrates in the respective MSG biopsies (all with sialadenitis focus score Z 1), and consisted of two subgroups; SS-Group-1 (n ¼ 3) derived from biopsies with moderate lymphocytic infiltrations (focus score o 2) and SS-Group-2 (n ¼ 6) derived from biopsies with severe infiltrations (focus score Z 2). Normal-SGEC lines, used as controls, were derived from non-SS individuals who had sicca complaints, but did not fulfill the SS classification criteria and did not have any histopathologic or serologic evidence for SS.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted with the RNeasy mini kit (Qiagen). RNA (0,25 μg) was reversed transcribed using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems®). Pro-IL1β, Versican, Pycard/ ASC and NFKBIA mRNA were analyzed by quantitative Real-time PCR using commercially available primers specific for each gene (TaqMan® Gene Expression Assays; Applied Biosystems). All samples were run in duplicate. PCR program included an initial denaturation step at 95°C for 10-min followed by 50 cycles of 95°C for 15-s and by a final extension step at 60°C for 1-min. Analysis was performed by the 2 -ΔΔCT method using HPRT1 as reference gene and HeLa cells as calibrator.

Microarray gene expression profiling
Transcriptome analyses were performed in total RNA specimens isolated from long-term cultured SGEC lines derived from non-SS controls (normal-SGEC lines; n ¼ 3) and from SS patients (SS-SGEC; n ¼ 9), using the Affymetrix microarray technology (GeneChipHuGene 1.0ST arrays with 28,869 annotated genes, following the manufacturer's workflow. Microarray analysis was performed with the R statistical environment version 2.13 using the Bioconductor package [3]. RMA normalization was performed in the microarray and identification of differentially expressed genes was conducted with Student's t-test (p-value o 0.05) [4]. Gene ontology annotation of differentially expressed genes, signal pathway analysis and network construction was performed with the use of Ingenuity Pathway Analysis (IPA) software and the Kyoto Encyclopedia of Genes and Genomes (KEGG). All microarray data are available in the GEO archive under the accession number GSE97614. Grouping of DEG into signaling pathways by ingenuity pathway analysis (IPA) in the SS-Group-1 and the SS-Group-2 of SS-SGEC lines studied is shown. Detailed presentations of genes that belong to each pathway for each of the two SS subgroups are provided in the respective Excel files 1 and 2.

Statistical analyses
Correlations were calculated using the Spearman's rank correlation coefficient. Group comparisons were performed by Mann-Whitney rank sum test. For paired comparisons, Wilcoxon signed-rank test and one-way analysis of variance were used, when appropriate. Analyses were conducted using SPSS 15.0 and Graph Pad 5.0 softwares. Clustering of selected differentially expressed genes was performed with euclidean distance metric and average linkage with MultiExperiment Viewer software [5].