“Endogenous Adjuvant” Activity of the RNA Components of Lupus Autoantigens Sm/RNP and Ro 60

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Abstract

Objective

Most lupus patients produce autoantibodies against small ribonucleoproteins such as Sm/RNP and Ro 60 (containing U1 and Y1–Y5 RNAs, respectively). We undertook this study to investigate whether the RNA components of these antigens, which contain extensive tracts of single- and double-stranded RNA, signatures of viral infection, activate innate immunity.

Methods

U1 and Y RNAs were affinity purified from K562 cells. Murine bone marrow–derived dendritic cells (DCs), human HEK 293 cells, and murine RAW264.7 cells were stimulated with U1 RNA and other known Toll-like receptor (TLR) ligands. Expression of the interferon (IFN)–inducible gene Mx1 and other genes was quantified using real-time polymerase chain reaction, and cytokine production was measured by enzyme-linked immunosorbent assay. DC maturation was assessed using flow cytometry.

Results

Purified U1 and Y1–Y5 RNAs and synthetic stem-loop II of U1 RNA stimulated type I IFN (IFN-I) production by cell lines and murine bone marrow–derived DCs and promoted DC maturation (CD86 expression). U1 RNA–stimulated, but not TLR-3 ligand–stimulated, IFN-I was blocked by bafilomycin A1, indicating that immunostimulation by U1 RNA requires endosomal acidification. Myeloid differentiation factor 88–deficient cells responded poorly to U1 RNA, suggesting that an endosomal TLR, probably TLR-7, mediates the stimulatory effects of U1 RNA. U1 RNA–induced IFN-I and interleukin-6 production also were protein kinase R (PKR) dependent (abrogated by 2-aminopurine and greatly reduced in PKR−/− cells).

Conclusion

We conclude that the RNA components of the Ro 60 (Y1–Y5 RNA) and Sm/RNP (U1 RNA) small ribonucleoproteins act as endogenous adjuvants that could play a role in the pathogenesis of autoimmunity by stimulating DC maturation and IFN-I production.

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