Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor γRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.

Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.

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Regulatory T cells (Tregs), including natural CD4+CD25+ Tregs and inducible IL-10 producing T regulatory type 1 (TR1) cells, maintain tolerance and inhibit autoimmunity. Recently, increased percentages of Tregs have been observed in the blood of septic patients, and ex vivo-activated Tregs were shown to prevent polymicrobial sepsis mortality. Whether endogenous Tregs contribute to sepsis outcome remains unclear. Polymicrobial sepsis, induced by cecal ligation and puncture, caused an increased number of splenic Tregs compared with sham-treated mice. Splenic CD4+CD25+ T cells from septic mice expressed higher levels of Foxp3 mRNA and were more efficient suppressors of CD4+CD25− T effector cell proliferation. Isolated CD4+ T cells from septic mice displayed increased intracellular IL-10 staining following stimulation, indicating that TR1 cells may also be elevated in sepsis. Surprisingly, Ab depletion of total CD4+ or CD4+CD25+ populations did not affect mortality. Furthermore, no difference in survival outcome was found between CD25 or IL-10 null mice and wild-type littermates, indicating that Treg or TR1-generated IL-10 are not required for survival. These results demonstrate that, although sepsis causes a relative increase in Treg number and increases their suppressive function, their presence does not contribute significantly to overall survival in this model.

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Astrocyte inflammation, reactive oxygen species (ROS) formation, and dysfunction form a common denominator shared by all the major neurodegenerative disorders. Viral infections are emerging as important events in the etiology of CNS damage involving astrocytes, but molecular understanding is incomplete. Double-stranded RNA (dsRNA) is a byproduct of viral replication and serves as the signature molecule for viral infection via Toll-like receptor 3 (TLR3) largely restricted to circulating peripheral dendritic cells. However, astrocytes are strategically located at the blood-brain barrier (BBB) and throughout brain tissues, making these cells ideal candidates as innate immunity sentinels within the CNS. We hypothesized that extracellular dsRNA, mimicked by polyinosinic-polycytidylic acid (Poly(I:C); PIC), initiates signaling of the double-edged sword of antiviral plus pathophysiological events in astrocytes. Using Western blot analysis and real-time qPCR, we determined that neonatal rat astrocyte cultures constitutively express TLR3 mRNA and protein, and that PIC dsRNA induced phosphorylation of eIF2alpha, as well as mRNA type I interferon (alpha/beta IFN)-response genes Mx1, PKR, and TLR3. Astrocyte TLR3 protein was downregulated after PIC treatment, however. PIC signaled degradation of IkappaBalpha with the consequence of upregulating iNOS, TNF-alpha, and IL-1beta mRNAs and proteins. In addition to antiviral protection events, dsRNA induced astrocyte dysfunction, evidenced by inhibiting EAAT1/GLAST transporter gene expression and attenuating L-glutamate uptake via sodium-dependent transport system X(AG)-, as well as inducing cytotoxicity. Anti-TLR3 blocking antibody attenuated PIC upregulation of TNF-alpha mRNA and iNOS activity. Extracellular PIC-induced events were prevented by 2-aminopurine, implicating PKR as an important downstream player in astrocyte dsRNA sensing pathways. The effects of plasma membrane impermeable poly(I:C) were dose-dependent (0-50 microM). In concert, these data provide evidence that dsRNA/TLR3-activated astrocytes initiate a battery of rapid innate pathogen-associated molecular pattern (PAMP) immune responses that are important for mounting antiviral defense in the CNS, yet also lead to pathophysiological events associated with the glutamate neurotoxicity of neurodegenerative diseases.

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.