In systemic lupus erythematosus (SLE) antiviral defenses are chronically activated, resulting in over-activity of the type I interferon (IFN) pathway. Studies in lupus-prone mice suggest that immune complexes associated with endogenous nucleic acids drive renal inflammation, a major cause of SLE-morbidity. However, the origin and nature of the extracellular nucleic acids driving inflammation in human lupus nephritis (LN) are incompletely understood.
Here we provide evidence that extracellular vesicles (EVs) can deliver a pro-inflammatory small RNA cargo into renal tubular epithelial cells (TECs). In situ RNA hybridization for Epstein-Barr virus (EBV)-encoded small RNAs (EBER-ISH) on LN tissue biopsies revealed atypical EBER localization in the cytoplasm of TECs. Stem-loop RT-PCR confirmed the presence of intact 167nt EBV-EBER1 RNA in LN tissues while EBV-DNA was virtually undetectable, arguing against EBV-infected (B) cells as an explanation for EBER1 detection. We hypothesised that cell-free EBER1 in circulation enters the renal tubular epithelium in predisposed individuals. We detected extracellular vesicle (EV) associated EBER1 in SLE patient sera but not in healthy and disease controls. We determined primary TEC express phosphatidylserine (PS) receptors, most notably Kidney-injury molecue-1 (KIM1), that support endocytosis of EBER1-loaded EVs. Purified EVs from EBV-infected B cells trigger an interferon stimulated gene expression (ISG) signature in TEC that is also expressed in LN tissues, but absent in renal disease control tissues. Immunohostochemstry further reveals TLR3 and KIM-1 protein expression in the renal epithelium of LN tissues. Importantly, hydroxychloroquine and Toll-like receptor-3 (TLR3) blockade inhibit EBER1-induced pro-inflammatory cytokine (IL-6 and TNFa) production in primary TECs. We propose that PS-mediated EV uptake by TECs and TLR3-mediated recognition of EBER1 induces pro-inflammatory cytokine expression may aggravate renal inflammation in human SLE. Our findings support the rationale for therapeutic blockade of TLR signalling in LN patients. Taken together our observations provide the first evidence that EBV infection and EVs are linked to the pathogenesis of LN and confirm that EBV as a model for studying EV-mediated cell-cell. Our results support the model of exosome-mediated tubulointerstitial communication and inflammation and agree with the observation that systemic pro-inflammatory EVs target specific cell types in target organs.
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