Inhibition of extracellular vesicle-encapsulated miRNA produced by estrogen-mediated upregulation of cellular processing suppresses target organ inflammation in a humanized model of systemic lupus erythematosus

Abstract

ABSTRACTBackground/PurposeDistinct, disease-associated intracellular miRNA (miR) expression profiles have been identified from peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. We have previously demonstrated novel estrogenic responses in PBMCs from SLE patients and discovered that estrogen lowers the threshold of immune cell activation to a greater extent in females, including significant upregulation of toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs).ObjectiveThe objective of this study was to characterize the estrogen-mediated immunomodulatory effects of distinct EV-encapsulated miR profiles in SLE and evaluate the potential therapeutic approach of miR inhibition in a humanized mouse model.MethodsSLE patients meeting revised ACR guidelines and age/sex-matched healthy controls provided informed consent to participate in this IRB-approved study. Plasma-derived EVs were isolated by differential ultracentrifugation and quantified. PBMCs were isolated from whole blood and cultured in hormone free conditions before stimulation with 17β-estradiol (estrogen; E2). RNA was isolated following E2 stimulation or EV isolation and bulk RNA-sequencing (RNAseq) reads were analyzed. Additionally, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing complementary locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis and tissue was harvested for histopathological examination.ResultsEVs were found to be increased in the plasma of SLE patients and differentially expressed EV-derived miR profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. E2 stimulation of PBMCs identified upregulated pathways involved in miR transcription/processing. Specifically, small RNA binding proteins and synthesis enzymes demonstrated significant signaling pathway association and upregulation with E2 treatment. Human immune cell subtypes were successfully recovered from whole blood of chimeric mice at similar levels with and without miR inhibition, but levels of human IL-6, IL-1β, IL-4, and TNF-α were significantly reduced by the LNA antagonists. Moreover, miR antagonists significantly reduced histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3.ConclusionThese data suggest E2-mediated regulation of miR synthesis and demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.