Decoding the microRNA Landscape in Systemic Sclerosis: Insights from in Silico Predictions

The article ‘In Silico Prediction of NOS2, NOS3, and Arginase 1 Genes Targeting by MicroRNAs Upregulated in Systemic Sclerosis’ published in the Indian Journal of Rheumatology explores the role of microRNAs (miRNAs) in the pathogenesis of systemic sclerosis (SSc). SSc is an autoimmune disorder marked by fibrosis of the skin and internal organs, vascular abnormalities, and immune system dysfunction. ¹ In SSc, there is initial damage to vascular endothelial cells, which leads to vascular remodelling. This process includes arteriole intimal proliferation, capillary degradation, and vascular blockage. These events are essential in the progression of SSc.^2,3 Modern research has highlighted the intricate interplay between immune cells, particularly macrophages, and miRNAs, ~22 nucleotide long RNAs in the development and progression of SSc. ⁴ The study focuses on the role of NOS3 in controlling vascular tone, leucocyte migration, and blood clotting, as well as the reduced expression of NOS3 in SSc, leading to endothelial dysfunction. The study examines the involvement of macrophages and T cells in SSc, a connective tissue disorder marked by fibrosis and endothelial dysfunction.

Macrophages are crucial for maintaining tissue homeostasis and immune surveillance. In individuals with SSc, the ability of macrophages to clear apoptotic cells is significantly reduced compared to healthy individuals. This deficiency leads to the appearance of circulating nuclear antigens and stimulates the proliferation of proinflammatory fibroblasts.^5,6 Activated macrophages in SSc generate a range of cytokines, including elevated CXCL8, CCL2, and CCL18, and low IL-10 expression. These cytokines gather in the perivascular regions of severely fibrotic SSc skin, promoting the growth of proinflammatory fibroblasts.^7,8 Furthermore, macrophages’ overproduction of CXCL13 and vascular endothelial growth factor (VEGF) contributes to fibrosis, immune system activation, and irregular vascular structure in SSc.^6,9 Macrophage plasticity to adopt a proinflammatory (M1) classically activated, anti-inflammatory, or pro-regenerative (M2) alternatively activated state is the outcome of an orchestrated process. It is imperative to understand different macrophage phenotypes, their clinical significance, and how they are altered in SSc. ¹⁰ Cytokines such as IL-4, IL-13, IL-10, and transforming growth factor-β (TGF-β), along with immune complexes, complements, and apoptotic cells, can all trigger the polarisation of M2 macrophages. ¹¹ These M2 macrophages are characterised by their high phagocytic activity and their production of TGF-β, IL-10, VEGF, and other regulatory factors. They play a crucial role in reducing inflammation, enhancing wound healing, immune response modulation and angiogenesis, and generation of the extracellular matrix (ECM). ¹² M2 macrophages facilitate wound healing, tissue repair, and structural remodelling after an inflammatory injury, mainly through factors such as TGF-β. ¹³ However, if these macrophages are abnormally activated, it could result in permanent inflammatory fibrosis in the tissue. ¹⁴ Unlike M1 macrophages, M2 macrophages release many profibrotic molecules like TGF-β and VEGF. This has led us to shift our attention towards M2 macrophages. ¹⁰ CD163, a marker gene for M2 macrophages, is present at significantly higher levels in the plasma and urine samples of SSc patients compared to healthy controls. Gene interaction networks, developed from three transcriptomic datasets of SSc skin biopsies, have revealed that the activation of M2 macrophages is the core molecular mechanism in the SSc molecular network. ¹⁵ The pathogenesis of SSc vasculopathy and the uncontrolled fibrotic process in SSc still need to be fully comprehended. Recent research into the molecular pathways involved in these processes has highlighted the crucial role of epigenetic mechanisms in the progression of the disease. In particular, these studies have identified miRNAs as significant epigenetic regulators. MiRNAs have a unique potential to elucidate the pathogenesis of SSc, improve diagnostic accuracy, and assist in the development of effective targeted therapies for the disease. ¹⁶ miRNAs can regulate multiple mRNA targets. Similarly, the translation of a single mRNA transcript into protein can be influenced by various miRNAs. ¹⁷ In patients with SSc, both upregulation and downregulation of different miRNAs have been observed in blood samples and tissue biopsies.^18,19

miRNAs play a critical role as epigenetic regulators, influencing gene expression in endothelial cells ²⁰ and fibroblasts.^21,22 miR-21 and miR-31 upregulate endothelial to mesenchymal transition (EndoMT) and are increased in SSc patients’ serum. miR-34a has been recognised as an inhibitor of canonical Wnt signalling,^23,24 whereas miR-155 acts to enhance Wnt signalling. Both miR-34a and miR-155 are overexpressed in the serum of patients with SSc.^23,24 Reduced levels of miR-193b in SSc contribute to proliferative vasculopathy through increased expression of urokinase-type plasminogen activator. ²⁵ miRNA modulates endothelin-1 production^26,27 and plays a role in connecting vasculopathy and fibroproliferation involved in SSc pathogenesis. ¹⁶

The present study emphasises the roles of M1 macrophages (expressing NOS2), M2 macrophages (expressing Arginase1), and Th2 cells in the pathogenesis of SSc, representing significant contributors to immune dysregulation. Because of their plasticity, dysregulation of the macrophage polarisation, favouring M1 over M2 phenotype, is implicated in SSc. ¹⁰ The study centres on using in silico methods to predict miRNAs that target the NOS2, NOS3, and Arginase 1 genes, which are implicated in endothelial dysfunction and macrophage dysregulation in SSc. It suggests targeting specific miRNAs may help restore the balance between M1 and M2 macrophages, reducing endothelial dysfunction and fibrosis. The study by Singh et al. provides detailed information about the extraction of target gene sequences NOS2, NOS3, and ARG1 from the NCBI database in FASTA format, collection of miRNA expression data from various published studies, extraction of miRNA and target gene sequences, and the evaluation of miRNA binding strength using RNA22. It includes the unique sequence IDs, accession numbers, and the nucleotide sequences for each gene. The document also explains the format of the FASTA definition line and the specific characters allowed in the sequence ID. Additionally, it outlines the particular nucleotide sequences extracted for each target gene to ensure accuracy in the study. Overall, it is a comprehensive guide to extracting and formatting gene sequences for research purposes. The use of Gibbs Free Energy in target prediction is explained, focusing on stable base pairing and identifying target sequences with Gibbs Free Energy ≤ –18 Kcal/mol. The computational study identified distinct upregulation of miRNAs in SSc, with a higher number targeting NOS2 and NOS3 genes compared to Arginase 1. This suggests that miRNAs likely contribute significantly to the development of SSc by modulating the expression of these genes. Singh et al. present the study’s findings, including the library of miRNAs with altered expression, the analysis of miRNA targeting strength against the NOS2, NOS3, and Arginase1 genes, and the identification of miRNAs with strong targeting potential. The authors document that alterations in L-Arginine metabolism play an important role in the pathophysiology of SSc.

The study has significant implications for understanding the molecular mechanisms underlying SSc. By identifying specific miRNAs that target NOS2 and NOS3 genes, the study provides insights into the potential involvement of epigenetic regulation in the development of SSc. Furthermore, the findings may have translational potential in developing targeted therapies for SSc by modulating the expression of these miRNAs. The article offers valuable perspectives on the role of miRNAs in SSc and details the potential of in silico prediction methods in identifying therapeutic targets for the disease. The study contributes to the growing body of research aimed at unravelling the complex pathogenesis of SSc and holds promise for developing novel treatment strategies. In conclusion, the intricate interplay between macrophages and miRNAs plays a central role in the SSc pathogenesis. Understanding these complex interactions could pave the way for novel therapeutic approaches targeting immune dysregulation and fibrosis in SSc.