In vivo adjuvant activity of the RNA component of the Sm/RNP lupus autoantigen

Abstract

Many lupus autoantigens contain small, highly structured RNAs, and studies have shown that the RNA components of lupus autoantigens activate production of type I interferon by dendritic cells (DCs) in vitro via the Toll-like receptor (TLR)-myeloid differentiation factor 88 pathway. This study was undertaken to examine whether U1 RNA possesses adjuvant activity in vivo. U1 RNA was affinity purified from K562 cells. C57BL/6 or OT-II mice were immunized with 4-hydroxy-3-nitrophenyl acetyl (NP)-conjugated keyhole limpet hemocyanin (NP-KLH) or ovalbumin(323-337) peptide, using either U1 RNA or aluminum hydroxide (alum) as the adjuvant. Activation of DCs and lymphocytes was measured using flow cytometry. NP-specific antibody responses were measured using enzyme-linked immunosorbent assay. Antigen-specific T cell proliferation was determined using 3H-thymidine incorporation. Similar to the results with the standard adjuvant, alum, U1 RNA coadministered with NP-KLH enhanced production of NP-specific IgM and IgG (on days 8 and 16 postinjection, respectively). Moreover, proliferation of antigen-specific CD4+ T cells was enhanced to comparable levels in the mice immunized with either U1 RNA or alum. Injection of U1 RNA into the footpad of mice resulted in DC recruitment to draining lymph nodes and induction of DC maturation. U1 RNA, at 24 hours' postinjection, also increased expression of the early activation marker CD69 in both B and T lymphocytes. Pretreatment of U1 RNA with RNase or coadministration with a TLR-7 antagonist inhibited the effects of this adjuvant. A small RNA of cellular origin can drive DC maturation, B and T cell activation/proliferation, and antibody responses to exogenous antigens. These results support the idea that U1 RNA is an endogenous adjuvant, helping to explain the striking predilection of lupus autoantibodies for RNA-protein complexes such as Sm/RNP.