"Endogenous adjuvant" activity of the RNA components of lupus autoantigens Sm/RNP and Ro 60.
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).


CONCLUSIONS
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.
Kelly
Kindra M
KM
University of Florida, Gainesville, USA.
Zhuang
Haoyang
H
Nacionales
Dina C
DC
Scumpia
Philip O
PO
Lyons
Robert
R
Akaogi
Jun
J
Lee
Pui
P
Williams
Bryan
B
Yamamoto
Masahiro
M
Akira
Shizuo
S
Satoh
Minoru
M
Reeves
Westley H
WH
D000818:Animals; D001324:Autoantigens / Q000276:immunology*; D002478:Cells, Cultured; D011992:Endosomes / Q000276:immunology; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D051379:Mice; D020733:RNA, Small Cytoplasmic / Q000276:immunology*; D012261:Ribonucleoproteins / Q000276:immunology*; D017411:Ribonucleoproteins, Small Nuclear / Q000276:immunology*; D051193:Toll-Like Receptors / Q000502:physiology; D000099207:SS-A Antigen
D001324:Autoantigens; D007370:Interferon Type I; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D051193:Toll-Like Receptors; C116006:RO60 protein, human; D000099207:SS-A Antigen