Journal: Frontiers in Immunology

Article Title: Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model

doi: 10.3389/fimmu.2023.1090177

Figure Lengend Snippet: RNA expression profiles from plasma-derived extracellular vesicles (EVs) in human and murine samples. (A) EVs were isolated from healthy human urine (N = 10) and plasma (N = 25) sources. (B) Plasma-derived EVs were isolated from lupus nephritis (LN) patients (N= 5) and healthy controls (N = 25). (A, B) RNA was isolated from EVs for comprehensive, global RNA-sequencing analysis for detectable small RNA sequences. Reads were aligned and principal component analysis was performed to evaluate grouping of the datasets. (C) A volcano plot was made from the data generated in (B) . (D) Plasma was isolated from NZM2410 mice with high and low blood urea nitrogen (BUN) levels (N = 15 per group). EVs were isolated and RNA was purified for sequencing to detect miRNAs. Data from all detected miRNAs (approximately 800) is shown as a heat map and expressed as log-transformed RNAseq reads. (E) Fold changes of all log-transformed miRNA expression levels detected in RNA-sequencing of isolated EVs from human (LN/healthy) and murine (high BUN/low BUN) samples were analyzed by pathway analysis software focused on miRNAs associated with LN pathology. Relative fold changes in expression level are shown in a heat map organized by hierarchy.

Article Snippet: Murine EVs isolated from plasma were submitted directly to System Biosciences (SBI) for RNA isolation and miRNA profiling according to the Exo-NGS service, exosomal RNA sequencing protocol.

Techniques: RNA Expression, Clinical Proteomics, Derivative Assay, Isolation, RNA Sequencing, Generated, Purification, Sequencing, Transformation Assay, Expressing, Software

Journal: Frontiers in Immunology

Article Title: Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model

doi: 10.3389/fimmu.2023.1090177

Figure Lengend Snippet: Extracellular vesicles (EVs) and EV-encapsulated miRs that bind and activate TLR7 and TLR8 are upregulated in SLE patients. (A) Plasma derived EVs were isolated from healthy volunteers (N = 6) and SLE patients (N = 16; 14 with active disease and 2 inactive) by ultracentrifugation and quantified by an ELISA assay. (B) RNA was extracted from EVs isolated from healthy volunteers (N = 6) and active SLE patients (N = 6). Expression of miR-21, miR29a, miR-29b, and Let7a was measured by RT-PCR analysis. Data was normalized to RNU-44 internal control expression and shown as a relative fold-change. Values are the mean ± SEM with indicated p values calculated via paired, two-tailed, Student’s t tests. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Murine EVs isolated from plasma were submitted directly to System Biosciences (SBI) for RNA isolation and miRNA profiling according to the Exo-NGS service, exosomal RNA sequencing protocol.

Techniques: Clinical Proteomics, Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model

doi: 10.3389/fimmu.2023.1090177

Figure Lengend Snippet: Schematic of the proposed mechanism of estrogen-mediated miR production and inflammation via extracellular vesicle signaling in SLE. Estrogen (E2) enters an immune cell, dimerizes with estrogen receptor (ER)α, translocates to the nucleus, and promotes the expression of miRNA (miR) processing machinery, including RNA polymerase III, Dicer1, AGO2, and Drosha. These miRs are packaged and secreted in EVs, which are taken up by recipient immune cells. When EV-derived miR cargo is taken up, it can function to regulate gene expression in the recipient cell via two pathways. The canonical pathway involves miRs binding to target mRNAs through the RNA-induced silencing complex (RISC). In the non-canonical pathway, EV-encapsulated miRs fuse with endosomes and bind to TRL7 or TLR8 to stimulate proinflammatory gene expression and additional EV secretion, which promotes SLE pathogenesis.

Article Snippet: Murine EVs isolated from plasma were submitted directly to System Biosciences (SBI) for RNA isolation and miRNA profiling according to the Exo-NGS service, exosomal RNA sequencing protocol.

Techniques: Expressing, Derivative Assay, Gene Expression, Binding Assay