Murine B cell response to TLR7 ligands depends on an IFN-beta feedback loop.

Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR(-/-) B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1(-/-) B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-beta feedback loop and constitutively low expression of TLR7 in the IFNAR1(-/-) B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.

Autoreactive B cells discriminate CpG-rich and CpG-poor DNA and this response is modulated by IFN-alpha.

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-alpha lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-alpha can contribute to the pathogenesis of systemic lupus erythematosus.

DNA and RNA autoantigens as autoadjuvants.

AM14 B cells are a prototype for those low affinity autoreactive B cells that routinely mature as naïve cells in peripheral lymphoid tissues. These cells express a transgene-encoded receptor specific for IgG2a and can be effectively activated by immune complexes that incorporate either mammalian DNA or mammalian RNA that has been released from dead or dying cells. Activation depends on the ability of the B-cell receptor to deliver antigen to an internal vesicular compartment containing either Toll-like receptor-9 (TLR9) or TLR7. Since TLR9 and TLR7 are thought to recognize microbial DNA and RNA preferentially, it is important to determine under what conditions mammalian DNA and RNA become effective TLR ligands, and whether the determining factor is delivery or structure. This issue has been addressed by using IgG2a mAbs to deliver immune complexes preloaded with defined fragments of DNA or RNA, or by using modified ODNs/ORNs. The data demonstrate that only certain nucleic acid sequences or structures can induce autoreactive B-cell proliferation, even when delivery to the appropriate TLR compartment is facilitated by uptake through the B-cell receptor (BCR).

Comparison of CpG s-ODNs, chromatin immune complexes, and dsDNA fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells.

Synthetic single-stranded oligodeoxynucleotides (15-30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments ( approximately 600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN and small molecule inhibitors suggests that stimulatory CpG sODN and chromatin IC may either access TLR9 via different routes or depend on discrete activation parameters. These data have important implications regarding the therapeutic application of TLR9 inhibitors to the treatment of systemic autoimmune diseases.

The stimulation of Toll-like receptors by nuclear antigens: a link between apoptosis and autoimmunity.

As immunologists have long understood, effective responses to foreign antigens require adjuvants. It is now apparent that the initiation of autoimmune disease is comparably facilitated by adjuvant activity. In the case of antinuclear antibodies, it seems that DNA itself can serve as an endogenous adjuvant. Similar to many of the microbial adjuvants, mammalian DNA mediates its effect through a Toll-like receptor--in this case, TLR9.

Regulation of autoreactive B cell responses to endogenous TLR ligands.

Immune complexes containing DNA and RNA are responsible for disease manifestations found in patients with systemic lupus erythematosus (SLE). B cells contribute to SLE pathology through BCR recognition of endogenous DNA- and RNA- associated autoantigens and delivery of these self-constituents to endosomal TLR9 and TLR7, respectively. B cell activation by these pathways leads to production of class-switched DNA- and RNA-reactive autoantibodies, contributing to an inflammatory amplification loop characteristic of disease. Intriguingly, self-DNA and RNA are typically non-stimulatory for TLR9/7 due to the absence of stimulatory sequences or the presence of molecular modifications. Recent evidence from our laboratory and others suggests that B cell activation by BCR/TLR pathways is tightly regulated by surface-expressed receptors on B cells, and the outcome of activation depends on the balance of stimulatory and inhibitory signals. Either IFNalpha engagement of the type I IFN receptor or loss of IgG ligation of the inhibitory FcgammaRIIB receptor promotes B cell activation by weakly stimulatory DNA and RNA TLR ligands. In this context, autoreactive B cells can respond robustly to common autoantigens. These findings have important implications for the role of B cells in vivo in the pathology of SLE.

DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas(lpr/lpr) mice in vivo.

INTRODUCTION: B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion. METHODS: We have developed a new generation of DNA-like compounds named class R inhibitory oligonucleotides (INH-ODNs). We tested their effectiveness in autoimmune B cells and interferon-alpha-producing dendritic cells in vitro and in lupus-prone MRL-Faslpr/lpr mice in vivo. RESULTS: Class R INH-ODNs have 10- to 30-fold higher inhibitory potency when autoreactive B cells are synergistically activated through the BCR and associated TLR7 or 9 than when stimulation occurs via non-BCR-engaged TLR7/9. Inhibition of TLR9 requires the presence of both CCT and GGG triplets in an INH-ODN, whereas the inhibition of the TLR7 pathway appears to be sequence-independent but dependent on the phosphorothioate backbone. This difference was also observed in the MRL-Faslpr/lpr mice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival. CONCLUSIONS: The increased potency of class R INH-ODNs for autoreactive B cells and dendritic cells may be beneficial for lupus patients by providing pathway-specific inhibition yet allowing them to generate protective immune response when needed.

Functional outcome of B cell activation by chromatin immune complex engagement of the B cell receptor and TLR9.

We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-kappaB activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.

Toll-like receptors, endogenous ligands, and systemic autoimmune disease.

The critical role of Toll-like receptors (TLRs) as mediators of pathogen recognition by the innate immune system is now firmly established. Such recognition results in the initiation of an inflammatory immune response and subsequent instruction of the adaptive immune system, both of which are designed to rid the host of the invading pathogen. More controversial is the potential role of TLRs in the recognition of endogenous ligands and what effect this might have on the consequent development of autoimmune or other chronic sterile inflammatory disorders. An increasing number of studies implicate TLRs as being involved in the immune response to self-molecules that have in some way been altered from their native state or accumulate in non-physiologic sites or amounts, although questions have been raised about possible contaminants in certain of these studies. In this review, we discuss the evidence for endogenous ligand-TLR interactions with particular emphasis on mammalian chromatin, systemic lupus erythematosus, and atherosclerosis. Overall, the data support the general concept of a role for TLRs in the recognition of endogenous ligands. However, the precise details of the interactions and the extent to which they may contribute to the pathogenesis of human disease remain to be clarified.