Background/Purpose: In lupus nephritis (LN), genetic and environmental factors drive the chronic activation of antiviral defenses leading to immune complex-mediated glomerular and tubular damage. Increasing evidence suggests the involvement of extracellular vesicles (EVs) in autoimmune disease. Currently a role for EVs in the pathogenesis of lupus nephritis has not been proposed. Objectives: To investigate the role of EVs in the pathogenesis of LN.

Methods: To determine the presence of EVs in kidneys, biopsies from LN patients and IgA- nephropathy and Focal Segmental Glomerulosclerosis control patients were used. Serum samples from SLE patients and from RA patients as controls were used to determine the presence of circulating EVs. Primary renal tubular epithelial cells (TEC) were cultured, and Kidney injury molecule-1 (KIM1) expression was assessed by FACS. Exosomes were analyzed by electron microscopy and western blot. mRNA analysis was performed by qPCR. TLR3 inhibition was performed with TLR3/dsRNA complex inhibitor and with hydroxychloroquine.

Results: We show that EVs deliver virus-derived small RNA and activate TEC via toll-like receptor 3 (TLR3). Highly specific stem-loop RT-PCRs revealed Epstein Barr Virus (EBV)-encoded small RNAs in LN biopsies while quantitative EBV-DNA PCR, sensitive to a single copy was negative. In situ hybridization failed to detect nuclear EBV-EBER1 (i.e. EBV-infected cells) in LN biopsies. However, we observed atypical EBER signal in the cytoplasm of TECs in LN but not in disease control biopsies, suggestive of uptake of extra-renal EBER. Consistent with this, we detected EBER1 in circulating EVs of SLE sera. The LN tissues express strongly elevated levels of TLR3, Interferon induced transmembrane-1 and -3, and TNFα. Primary TEC cultured in vitro endocytose EBER1-EVs secreted by EBV-infected B cells via phosphatidylserine receptors such as KIM-1. Importantly, EV-EBER1 uptake by TEC triggers antiviral immunity and pro-inflammatory cytokine secretion in a Toll-like receptor 3 (TLR3)-dependent manner. Treatment with hydroxychloroquine (HCQ) or a small molecule inhibitor that blocks TLR3-RNA interactions strongly reduced the pro-inflammatory effects of EBER1.

Conclusion: We propose that small RNA-loaded EVs exacerbate pre-existing autoimmunity in SLE patients by engaging tubular epithelial TLR3, supporting the rationale for TLR3-blockade as therapeutic strategy in the treatment of lupus nephritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/extracellular-vesicle-mediated-delivery-of-ebv-small-rna-eber1-activates-lupus-nephritis-related-antiviral-immunity-in-tubular-epithelial-cells-via-tlr3/