Date Published: October 11, 2018
Publisher: Public Library of Science
Author(s): Manjistha Sengupta, Bi-Dar Wang, Norman H. Lee, Alexander Marx, Linda L. Kusner, Henry J. Kaminski, William D Phillips.
A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance.
MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction.
Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs were found to be involved in immune response pathways and identified in other autoimmune diseases. The cellular and molecular functions of the mRNAs showed involvement in cell death and cell survival, cellular proliferation, cytokine signaling and extra-cellular matrix reorganization. Eleven miRNA and mRNA pairs were reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-5p. MiRNA target sites were validated by dual luciferase assay. Transfection of miRNA mimics led to down regulation of RGS13 expression in Raji cells. Our study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus. We demonstrate for the first time that miR-139-5p and miR-452-5p negatively regulate RGS13 expression.
Myasthenia gravis (MG) is an autoimmune neuromuscular disorder mediated by antibodies against neuromuscular junction proteins, primarily the acetylcholine receptor (AChR) . A large proportion of AChR antibody positive early onset myasthenia gravis (EOMG) patients have thymic lymphofollicular hyperplasia with ectopic germinal centers (GC)  that are rarely observed in thymus of normal individuals. The hyperplastic thymus possesses all the components of the MG immune response with expression of the antigenic target, the AChR or AChR-like proteins [3–5], B cells producing AChR antibodies , and AChR autoreactive T cells . The GCs are sites where B cells proliferate, differentiate, undergo selection, and antibody genes undergo somatic hypermutation and class switch. Removal of the thymus improves the clinical course of EOMG patients . These observations make the thymus a likely site of disease initiation and maintenance in MG.
This study used thymus blocks from the MGTX trial , which showed benefit of thymectomy. Inclusion criteria allowed for the use of glucocorticoids prior to thymectomy. Of the seventeen thymuses, 16 blocks were assessed for miRNA and 13 blocks were designated for mRNA (Table 1). Due to the heterogeneity of the thymus, each block was assessed and categorized based on the presence or absence of GC (S1 Fig). By structuring the analysis into the two groups, the results allow for the identification of the most critical expression differences that drive thymic changes to formation and maintenance of GCs.
Our studies of miRNA and gene expression profiles provide an assessment of mechanistic pathways that could underlie germinal center development and maintenance in the MG thymus (Fig 8). We assessed samples from the MGTX trial for the presence of GCs, which allowed the use of glucocorticoids for inclusion into the study. Studies have shown that glucocorticoids reduce the presence of GCs in the thymus and return transcript levels to normal state [34–36]. We demonstrate miRNA and transcriptional profiles of thymus from MG subjects segregate based on the presence of germinal centers. Differently regulated transcripts fall into categories related to immune response, cell proliferation and cell communication/movement, while signaling pathways that are differentially identified by over-representation analysis (i.e. differentially expressed transcripts and miRNAs over-represented in specific signaling pathways) include cell death and survival as well as inflammation. The differentially regulated miRNAs are primarily involved in immune response and cell proliferation/apoptosis. Although IPA placed differentially regulated transcripts into specific categories, a clear functional relationship exists among the differentially expressed transcripts, which identified pathways involved in immune-related response, cell death/survival, and cell migration. Our profiling identified miRNA and gene transcripts known to be involved in germinal center formation and maintenance in secondary lymphatic structures. The differentially expressed miRNAs in MG thymus that we identified have also been linked to systemic lupus erythematosus, rheumatoid arthritis, and autoimmune thyroid disease. The following sections provide a more specific discussion of particular study results.
Our results suggest that development and maintenance of germinal centers involves cellular processes that are common to neoplastic pathway such as cell proliferation, reorganization of cellular matrix, cell migration and apoptosis as evidenced by profiles of gene transcription and miRNA. Gene transcripts that are responsible for cellular growth and proliferation FOS-JUN, EGR1, and EGR3 were downregulated in the mRNA profile. For organization of GC and B cell maturation, cellular movement and cell matrix reorganization is required and such pathways were identified. The GC-rich thymus was found to have a transcriptional profile consistent with state of inflammation. The results begin to provide a comprehensive picture of the pathological thymus and identify key target genes and miRNA that could be targeted for therapeutic development.