There has been much interest in understanding why the autoimmune response in SLE is targeted selectively at certain nucleic acid–containing macromolecules that are concentrated within apoptotic cellular blebs. A popular hypothesis has been that these autoantigens become immunogenic during apoptosis, which then may drive the autoimmune response. The present results demonstrate that the prototype autoantigen U1 snRNP, which is highly represented within apoptotic cells, is not simply a passive target of the autoimmune response, but rather is directly immune stimulatory when delivered into innate immune cells expressing either TLR7 or TLR8. Delivery of U1 snRNP can occur either via transfection or more physiologically by the uptake of snRNP-containing immune complexes formed by SLE patient sera containing anti-RNP antibody through FcγRIIa (CD32) present on pDCs. The stimulatory effects require the RNA component of the particle and appear to be mediated predominately through TLR7. A modest response is seen in TLR7−/−
mice lacking functional TLR8 (15
), suggesting an additional involvement of other immune stimulatory pathways. Immune activation could be reproduced by purified U1 snRNA or by synthetic ORNs containing certain conserved U-rich autoantigen or autoantibody binding sites from the U1 snRNAs or hY RNAs. Some of these U-rich regions are conserved across various small RNAs and different species, indicating that they may stimulate TLR7-mediated immune responses, at least when present in or reaching TLR7-containing compartments. Stimulation by synthetic ORNs was mediated by G/U- or U-rich sequences containing as little as one guanosine and was dependent on the length and position of the U-rich sequence.
Type I IFN promotes T cell responses (36
) and induces autoimmunity in up to 20% of humans treated with recombinant IFN-α (for review see reference 37
). Increased expression of type I IFN occurs in the pDCs of lupus patients, is associated with disease severity, and may contribute to disease development (38
), thereby linking pDC activation to SLE pathogenesis. Activation of the IFN-α pathway in SLE patients is significantly associated with the presence of certain autoantibody specificities, especially anti-RNP and other RNA-associated factors, but not with antiphospholipid antibodies (40
). Human pDCs express TLR7, but not the other RNA-activated TLRs, TLR3 or TLR8 (42
), that localize preferentially near phagosomes containing apoptotic particles (13
). Therefore, inappropriate or excessive activation of TLR7 by U-rich RNA sequences within snRNP could trigger the observed activation of the IFN-α pathway in SLE patients. All these TLRs are in endoplasmic reticulum or endosomal/lysosomal compartments (44
), which explains the requirement we found for transfection of the purified snRNPs or snRNA-derived ORNs. Our results suggest a possible mechanism for the observed linkage between defects in apoptotic cell clearance and SLE. We propose that under normal physiologic conditions, the small amounts of apoptotic cellular debris might be cleared too rapidly to activate the TLR pathways. However, in susceptible individuals with defective clearance, or under such abnormal conditions as during an immune response to a virus or other agent, the apoptotic debris in the form of immune complexes might be taken up by pDCs at high enough levels so that the U-rich sequences within the enclosed snRNP activate TLR7, thereby initiating type I IFN production and autoimmunity. In light of our results, the recently observed association of EBV infection with SLE could be explained by the homology and antigenic cross reactivity between the EBV nuclear antigen 1 and the snRNP and other autoantigens that are associated with apoptotic blebs (45
). Molecular mimicry between EBV nuclear antigen 1 and snRNP or other lupus autoantigens would lead to the production of autoantibodies reactive with apoptotic debris. Once an individual made autoantibodies that cross reacted with any component of the apoptotic cells or snRNP, these would form immune complexes that enhance the uptake of the nucleosome and snRNP nucleic acids through FcγRII on the pDCs. The autoimmune process would then become self-perpetuating, further enhanced by autocrine type I IFN production by pDCs and exacerbated by any defect in the clearance of apoptotic debris or immune complexes. DNA-containing immune complexes from SLE sera stimulate pDCs to secrete IFN-α through TLR9 (35
). These results demonstrate that immune complexes upon intracellular delivery via CD32 can activate pDCs not only through TLR9 as reported previously (35
), but also through TLR7, demonstrating a strong link between the pDCs and autoimmunity to both DNA- and RNA-containing autoantigens. Because the snRNAs also have dsRNA regions, we cannot completely exclude the possibility that they may also stimulate TLR3 or other pathogen-associated molecular patterns, causing additional immune stimulatory effects on cells other than pDCs (20
). But the complete inhibition of the effects by chloroquine, which fails to block activation by the TLR3 ligand poly rI:rC, suggests that any such TLR3-mediated effect would be quite small.
Apoptosis may exert additional effects on nucleosomal material that contribute to the development and direction of the subsequent autoimmune response. The U1 snRNP in contrast to other RNPs is only slightly cleaved during apoptosis, which could enable the U1 snRNA to better activate the DCs taking up these particles, resulting in the presentation of the associated 70K protein and Sm proteins on a mature DC that might drive a T cell response to the antigens through epitope spreading. Thus, the RNA component of the snRNPs would act as a built-in adjuvant to drive autoimmune responses against the associated proteins, explaining their strong and unique association with systemic autoimmunity.
All of the immune stimulatory effects of the U1 snRNP or snRNA ORNs are completely blocked by chloroquine at relatively low concentrations that occur in patients taking this class of drug for the treatment of SLE. These results suggest that the mechanism of action of chloroquine in the treatment of systemic autoimmunity, which had been unknown, could be through inhibition of one or more of TLR7/8/9. Our data support the concept that chloroquine-related compounds and suppressive ODNs, which were previously reported to block DNA-mediated TLR9 stimulation, may prevent apoptotic debris from stimulating and sustaining autoimmunity. Thus, we have identified a new potential therapeutic mechanism of action for these agents, making improved TLR7/8/9 antagonists interesting candidates for therapeutic development.