Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression patterns in IgG4-RD before and after steroid treatment and healthy control groups

ABSTRACT: Transcriptome analysis in patients with IgG4-RD and healthy controls IgG4-related disease (IgG4-RD) is a new emerging disease entity characterized by elevated serum IgG4 concentrations and tissue tumefaction or infiltration by IgG4-positive plasma cells. At present, however, it is not clear whether IgG4-RD is caused by abnormalities in acquired immunity, like autoimmune diseases, or whether the excess production of IgG4 is a true cause of IgG4-RD or an epiphenomenon associated with inflammatory and/or allergic reactions. We therefore attempted to identify genes related to the pathogenesis or clinicopathology of IgG4-RD. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with IgG4-RD before and after steroid therapy and from healthy controls. Total RNA was extracted and DNA microarray analysis was performed to screen for genes showing changes in expression. To exclude any gender-related differences in gene expression, DNA microarray analysis was performed only on samples obtained from male patients and controls. Total RNA was reverse transcribed to cDNA using AmbionM-BM-. WT Expression kits (Applied Biosystems, Foster City, CA), labeled with GeneChipM-BM-. WT Terminal Labeling and Controls kits (Affymetrix, Santa Clara, CA), and hybridized to GeneChipM-BM-. Human Gene 1.0 ST Arrays (Affymetrix), which include 28869 probes. Digitalized image data were processed using GeneChipM-BM-. Operating Software (Affymetrix).

ORGANISM(S): Homo sapiens

SUBMITTER: Akio Nakajima

PROVIDER: E-GEOD-66465 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:<Objective> To compare gene expression in labial salivary glands (LSG) of IgG4-related disease (IgG4-RD) with SjM-CM-6grenM-bM-^@M-^Ys syndrome (SS). <Methods> Gene expression was analyzed by DNA microarray in LSG of IgG4-RD (n=5), SS (n=5), and healthy controls (n=3). Differentially expressed genes (DEGs) in IgG4-RD and SS were identified, and gene-annotation enrichment analysis of these DEGs was performed using Gene Ontology (GO) annotation. Validation of the result was performed by quantitative PCR using LSG from IgG4-RD (n=9), SS (n=10), and controls (n=4). <Results> Gene expression patterns in IgG4-RD, SS, and healthy controls were quite different with each other in hierarchical clustering as well as principal component analysis. In IgG4-RD, 1771 up-regulated probe sets (corresponding to 1321 up-regulated genes) and 1785 down-regulated probe sets (corresponding to 1320 down-regulated genes) were identified as DEGs (false discovery rate <0.05). GO term analysis indicated that the up-regulated set of DEGs in IgG4-RD encoded proteins that function in cell proliferation, extracellular matrix organization, and organ development. PCR validated significantly higher expression of lactotransferrin (LTF) in IgG4-RD than SS (P<0.05), and chemokine (C-C motif) ligand 18 (CCL18) in IgG4-RD than SS and controls (P<0.05). <Conclusion> The results clearly showed that the gene expression pattern in LSG of IgG4-RD is different from that of SS. LSG samples were collected from 5 Japanese patients with IgG4-RD, as well as from 5 Japanese patients with SS who had been followed up at University of Tsukuba Hospital (Ibaraki, Japan). All patients with IgG4-RD satisfied the comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD) 2011 proposed by the All Japan IgG4 team [3]. The diagnosis of IgG4-RD was based on the presence of all three items; 1) clinical examination showing characteristic diffuse/localized swelling or masses in single or multiple organs, 2) Hematological examination shows elevated serum IgG4 concentrations (135 mg/dl), 3) Histopathologic examination shows: (1) Marked lymphocyte and plasmacyte infiltration and fibrosis. (2) Infiltration of IgG4+ plasma cells: ratio of IgG4+/IgG+ cells > 40% and >10 IgG4+ plasma cells/HPF. All patients with SS satisfied the Japanese Ministry of Health criteria for the diagnosis of SS (1999) [4]. These criteria included four clinicopathological findings: lymphocytic infiltration of the salivary or lacrimal glands, dysfunction of salivary secretion, keratoconjunctivitis sicca, and presence of anti-SS-A or SS-B antibodies. The diagnosis of SS was based on the presence of two or more of the above four items. Approval for this study was obtained from the local ethics committee and a signed informed consent was obtained from each subject.

Project description:Objective: This study aimed to investigate plasma exosome profiling of IgG4-RD and its potential role in B cell differentiation and tissue damage. Methods: One hundred untreated IgG4-RD patients and 135 sex, age matched healthy controls (HCs) were enrolled in this study. A combination of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) and TMT label quantitation was used to detect exosome and B cell profiling. To validate differentially expressed proteins, western blot and ELISA were performed. RT-qPCR was used to detect gene expression after exosome stimulation. Flowcytometry was used to measure B cell activation, apoptosis, differentiation and reactive oxygen species (ROS) expression. Besides, correlation analysis between differentially expressed complement protein and laboratory parameters was also performed. Results: In total, 82 proteins were upregulated and 96 proteins were downregulated in exosome of IgG4-RD patients compared with HCs. Pathway analysis revealed that complement cascade pathway in exosome of IgG4-RD was activated, together with reduced C3 complement and C5 complement. Validation by ELISA and WB, exosome C3 and C5 was decrease in IgG4-RD compared with HCs. Stimulation of B cells with exosome, naïve B cells decreased, while memory B cells and plasmablast were increased together with increased CYCS with activation of downstream complement system. ROS was increased in B cells of IgG4-RD compared with HC. In affected submandibular glands, BCR signaling pathway was activated and exosome may potentially participate in tissue damage. Conclusion: There were 82 differentially expressed upregulated proteins and 96 downregulated proteins in IgG4-RD exosome compared with HCs. Differentially expressed proteins were enriched in exosome, complement function and immune response indicating complement activation in exosome may be related to IgG4-RD. Reduced C3 and C5 expression in exosome of IgG4-RD may indicate the disease severity. Our research suggests that IgG4-RD exosomes can participate in the differentiation B cells into plasmablast, activate the B cell auto-oxidative damage pathway and potentially participate in the pathogenesis of IgG4-RD.

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Gene expression patterns in IgG4-RD before and after steroid treatment and healthy control groups

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