Toll-like receptor driven B cell activation in the induction of systemic autoimmunity

Studies over the past decade have demonstrated a key role for pattern recognition receptors in the activation of autoreactive B cells. Self reactive B cells that manage to escape negative selection often express relatively low affinity receptors for self antigens (ignorant B cells), and can only be activated by integrating a relatively weak BCR signal with signals from additional receptors. Members of the toll-like receptor (TLR) gene family, and especially the nucleic acid binding receptors TLR 7, 8 and 9, appear to play a key role in this regard and promote the production of autoantibodies reactive with DNA- or RNA-associated autoantigens. These autoantibodies are able to form immune complexes with soluble or cell-bound ligands, and these immune complexes can in turn activate a second round of proinflammatory cells that further contribute to the autoimmune disease process. Recent data have emerged showing a pathogenic role for TLR7, with an opposing, protective role for TLR9. Targeting these disregulated pathways offers a therapeutic opportunity to treat autoimmune diseases without crippling the entire immune system. Further understanding of the role of specific receptors, cell subsets, and inhibitory signals that govern these TLR-associated pathways will enable future therapeutics to be tailored to specific categories of autoimmune disease.     

FcγRIIB regulation of BCR/TLR‐dependent autoreactive B‐cell responses

Crosslinking of Fc γ receptor II B (FcγRIIB) and the BCR by immune complexes (IC) can downregulate antigen‐specific B‐cell responses. Accordingly, FcγRIIB deficiencies have been associated with B‐cell hyperactivity in patients with systemic lupus erythematosus and mouse models of lupus. However, we have previously shown that murine IgG2a‐autoreactive AM14 B cells respond robustly to chromatin‐associated IC through a mechanism dependent on both the BCR and the endosomal TLR9, despite FcγRIIB coexpression. To further evaluate the potential contribution of FcγRIIB to the regulation of autoreactive B cells, we have now compared the IC‐triggered responses of FcγRIIB‐deficient and FcγRIIB‐sufficient AM14 B cells. We find that FcγRIIB‐deficient cells respond significantly better than FcγRIIB‐sufficient cells when stimulated with DNA IC that incorporate low‐affinity TLR9 ligand (CG‐poor dsDNA fragments). AM14 B cells also respond to RNA‐associated IC through BCR/TLR7 coengagement, but such BCR/TLR7‐dependent responses are normally highly dependent on IFN‐α costimulation. However, we now show that AM14 FcγRIIB^?/? B cells are very effectively activated by RNA IC without supplemental IFN‐α priming. These results demonstrate that FcγRIIB can effectively modulate both BCR/TLR9 and BCR/TLR7 endosomal‐dependent activation of autoreactive B cells.     

Toll-like receptor 9-independent aggravation of glomerulonephritis in a novel model of SLE

The generation of anti-DNA auto-antibodies is characteristic for the human autoimmune condition systemic lupus erythematosus (SLE) and its animal models. However, the contribution of the toll-like receptor (TLR) system of innate immunity receptors and, in particular, TLR9 to this B cell-mediated autoimmune process remains controversial. Here we report that in a novel murine model of SLE, based on hyper-reactive B cell activation mediated by mutant phospholipase Cg2, the genetic deficiency of TLR9 does not protect from spontaneous anti-DNA auto-antibody formation and glomerulonephritis. On the contrary, disease induction is aggravated and additional nucleolar antibody specificity develops in autoimmune TLR9-deficient mice. In vitro studies demonstrate that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may promote autoimmunity, while TLR9 signaling can ameliorate SLE-like immune pathology in vivo.     

RAGE-independent autoreactive B cell activation in response to chromatin and HMGB1/DNA immune complexes

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive B cell receptor (BCR) transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1–RAGE interaction was important for this response. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1–DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE⁺ and RAGE^? AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.     

Emerging roles of TLR7 and TLR9 in murine SLE

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by B cell hyperactivity leading to the production of various autoantibodies and subsequent development of glomerulonephritis, i.e. lupus nephritis. Among the principal targets of autoantibodies produced in murine SLE are nucleic acid–protein complexes, such as chromatin and ribonucleoproteins, and the envelope glycoprotein gp70 of endogenous retroviruses. The preferential production of these autoantibodies is apparently promoted by the presence of genetic abnormalities leading to defects in the elimination of apoptotic cells and to an enhanced expression of endogenous retroviruses. Moreover, recent studies revealed that the innate receptors TLR7 and TLR9 are critically involved in the activation of dendritic cells and autoreactive B cells through the recognition of endogenous DNA- or RNA-containing antigens and subsequent development of autoimmune responses against nuclear autoantigens. Furthermore, the regulation of autoimmune responses against endogenous retroviral gp70 by TLR7 suggested the implication of endogenous retroviruses in this autoimmune response. Clearly, further elucidation of the precise molecular role of TLR7 and TLR9 in the development of autoimmune responses will help to develop novel therapeutic strategies and targets for SLE.     

The role of toll-like receptors in systemic lupus erythematosus

Systemic lupus erythematosus is an autoimmune disease characterized by the production of autoantibodies against a relatively limited range of nuclear antigens. These autoantibodies result in the formation of immune complexes that deposit in tissues and induce inflammation, thereby contributing to disease pathology. Growing evidence suggests that recognition of nucleic acid motifs by Toll-like receptors may play a role in both the activation of antinuclear B cells and in the subsequent disease progression after immune complex formation. The endosomal localization of the nucleic acid-sensing Toll-like receptors (TLRs), TLR3, 7, and 9, is believed to contribute to the distinction between endogenous nucleic acids and those of foreign origin. In this article we review recent work that suggests a role for the B-cell receptor and Fcγ receptors in delivering nuclear antigens to intracellular compartments allowing TLR activation by endogenous nucleic acids. A number of in vitro studies have presented evidence supporting a role for TLRs in SLE pathology. However, recent studies that have examined the contributions of individual TLRs to SLE by using TLR-deficient mice suggest that the situation is far more complicated in vivo. These studies show that under different circumstances TLR signaling may either exacerbate or protect against SLE-associated pathology. Further understanding of the role of TLRs in pathological autoreactivity of the adaptive immune system will likely lead to important insights into the etiopathogenesis of SLE and potential targets for novel therapies.     

Activation of autoreactive B cells by CpG dsDNA

The proliferative response of autoreactive rheumatoid factor (RF) B cells to mammalian chromatin-containing immune complexes (ICs) results from the sequential engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9). We have used ICs constructed from anti-hapten antibodies and defined haptenated dsDNA fragments to determine the form of mammalian DNA that mediates this process. Despite their relatively low abundance in mammalian DNA, we found that inclusion of hypomethylated CpG motifs in these ICs was necessary for effective activation. In the absence of antibody, the same fragments could efficiently stimulate low-affinity hapten-specific and DNA-reactive 3H9 B cells, but not RF B cells. These results extend the BCR/TLR9 coengagement paradigm to a second major class of autoreactive B cells, further confirm the critical role of the BCR in chromatin ligand delivery to TLR9, and implicate hypomethylated CpG motifs as ligand elements necessary for the initiation of systemic autoimmune disease.     

B cell tolerance to epidermal ribonuclear‐associated neo‐autoantigen in vivo

Defining how self‐antigens are perceived by the immune system is pivotal to understand how tolerance is maintained under homeostatic conditions. Clinically relevant, natural autoantigens targeted by autoantibodies, in e.g. systemic lupus erythematosus (SLE), commonly have an intrinsic ability to engage not only the B cell receptor (BCR), but also a co‐stimulatory pathway in B cells, such as the Toll‐like receptor (TLR)‐7 pathway. Here we developed a novel mouse model displaying inducible expression of a fluorescent epidermal neo‐autoantigen carrying an OT‐II T cell epitope, B cell antigen and associated ribonucleic acids capable of stimulating TLR‐7. The neo‐autoantigen was expressed in skin, but did not drain in intact form into draining lymph nodes, even after ultraviolet B (UVB)‐stimulated induction of apoptosis in the basal layer. Adoptively transferred autoreactive B cells were excluded follicularly and perished at the T–B border in the spleen, preventing their recirculation and encounter with antigen peripherally. This transitional check‐point was bypassed by crossing the reporter to a BCR knock‐in line on a C4‐deficient background. Adoptively transferred OT‐II T cells homed rapidly into cutaneous lymph nodes and up‐regulated CD69. Surprisingly, however, tolerance was not broken, as the T cells subsequently down‐regulated activation markers and contracted. Our results highlight how sequestration of intracellular and peripheral antigen, the transitional B cell tolerance check‐point and T cell regulation co‐operate to maintain immunological tolerance in vivo.     

Grancalcin (GCA) modulates Toll‐like receptor 9 (TLR9) mediated signaling through its direct interaction with TLR9

Toll‐like receptors (TLRs) are playing important roles in stimulating the innate immune response and intensifying adaptive immune response against invading pathogens. Appropriate regulation of TLR activation is important to maintain a balance between preventing tumor activation and inhibiting autoimmunity. Toll‐like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells and triggers myeloid differentiation primary response gene 88 (MyD88) dependent nuclear factor kappa B (NF‐κB) pathways and type I interferon (IFN) responses. However, mechanisms of how TLR9 signals are mediated and which molecules are involved in controlling TLR9 functions remain poorly understood. Here, we report that penta EF‐hand protein grancalcin (GCA) interacts and binds with TLR9 in a yeast two‐hybrid system and an overexpression system. Using siRNA‐mediated knockdown experiments, we also revealed that GCA positively regulates type I IFN production, cytokine/chemokine production through nuclear localization of interferon regulatory factor 7 (IRF7), NF‐κB activation, and mitogen‐activated protein kinase (MAPK) activation in plasmacytoid dendritic cells. Our results indicate that heterodimerization of GCA and TLR9 is important for TLR9‐mediated downstream signaling and might serve to fine tune processes against viral infection.     

Critical role of TLR7 in the acceleration of systemic lupus erythematosus in TLR9-deficient mice

Accumulating evidence supports the idea that TLR7 and TLR9 play pathogenic and protective roles, respectively, in the development of murine systemic lupus erythematosus (SLE). However, the molecular mechanism responsible for the accelerated development of SLE resulting from the deletion of TLR9 and the respective contributions of TLR7 and TLR9 to the development of different autoimmune responses against nuclear and non-nuclear autoantigens implicated in lupus nephritis have not been well defined. In the present study, we addressed these questions by assessing the effect of the TLR9 and/or TLR7 deletion on the production of various autoantibodies and the development of lupus nephritis in C57BL/6 mice congenic for the Nba2 (NZB autoimmunity 2) locus (B6.Nba2). TLR9-deficient B6.Nba2 mice displayed increased production of autoantibodies against nuclear antigens, serum retroviral gp70 and glomerular matrix antigens, and developed a markedly accelerated form of lupus nephritis. Enhanced disease was associated with functionally upregulated expression of TLR7, as documented by an increased TLR7-dependent activation of B cells and plasmacytoid dendritic cells. Notably, disease exacerbation in TLR9-deficient mice was completely suppressed by the deletion of TLR7. Our results indicate that TLR7 has a pivotal role in a wide variety of autoimmune responses against DNA- and RNA-containing nuclear antigens, retroviral gp70 and glomerular matrix antigens implicated in murine SLE, and that enhanced TLR7 activity is critical for the accelerated development of SLE in TLR9-deficient lupus-prone mice.     

Phosphorothioate‐modified CpG oligodeoxynucleotides mimic autoantigens and reveal a potential role for Toll‐like receptor 9 in receptor revision

Re‐expression of recombinase activating genes (RAG) in mature B cells may support autoreactivity by enabling revision of the B‐cell receptor (BCR). Recent reports suggest that administration of Toll‐like receptor 9 (TLR9) ‐stimulating CpG oligodeoxynucleotides (ODN) could trigger the manifestation of autoimmune disease and that TLR are involved in the selection processes eliminating autoreactive BCR. The mechanisms involved remain to be elucidated. This prompted us to ask, whether TLR9 could be involved in receptor revision. We found that phosphorothioate‐modified CpG ODN (CpG^PTO) induced expression of Ku70 and re‐expression of RAG‐1 in human peripheral blood B lymphocytes and Igλ expression in sorted Igκ⁺ B cells. Further results revealed unselective binding specificity of CpG^PTO‐induced immunoglobulin and suggested that CpG^PTO engage and/or mimic IgM receptor signalling, an important prerequisite for the initialization of receptor editing or revision. Altogether, our data describe a potential role for TLR9 in receptor revision and suggest that CpG^PTO could mimic chromatin‐bearing autoantigens by simultaneously engaging the BCR and TLR9 on IgM⁺ B cells.     

Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk-/- mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk-/- mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk-/- mice. A transgene expressing a low level of Btk in B cells (Btk(lo)) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk(lo) B cells. anti-DNA IgG was not observed in 56R.Btk(lo) mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.     

Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.     

Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus

Antibodies (Abs) to RNA- and DNA-containing autoantigens are characteristic of systemic lupus erythematosus (SLE). We showed previously that Toll-like receptor (TLR) 9, recognizing DNA, is required for the spontaneous generation of DNA autoantibodies, but not for the development of lupus nephritis in susceptible mice. We report that lupus-prone mice deficient in TLR7, a receptor for ssRNA, failed to generate Abs to RNA-containing antigens (Ags) such as Smith (Sm) Ag. TLR9 and TLR7 also had dramatic effects on clinical disease in lupus-prone mice. In the absence of TLR9, autoimmune disease was exacerbated, lymphocytes and plasmacytoid DCs were more activated, and serum IgG and IFN-alpha were increased. In contrast, TLR7-deficient mice had ameliorated disease, decreased lymphocyte activation, and decreased serum IgG. These findings reveal opposing inflammatory and regulatory roles for TLR7 and TLR9, despite similar tissue expression and signaling pathways. These results have important implications for TLR-directed therapy of autoimmune disease.     

Nucleic acid sensing receptors in systemic lupus erythematosus: development of novel DNA- and/or RNA-like analogues for treating lupus

Double‐stranded (ds) DNA, DNA‐ or RNA‐associated nucleoproteins are the primary autoimmune targets in SLE, yet their relative inability to trigger similar autoimmune responses in experimental animals has fascinated scientists for decades. While many cellular proteins bind non‐specifically negatively charged nucleic acids, it was discovered only recently that several intracellular proteins are involved directly in innate recognition of exogenous DNA or RNA, or cytosol‐residing DNA or RNA viruses. Thus, endosomal Toll‐like receptors (TLR) mediate responses to double‐stranded RNA (TLR‐3), single‐stranded RNA (TLR‐7/8) or unmethylated bacterial cytosine (phosphodiester) guanine (CpG)‐DNA (TLR‐9), while DNA‐dependent activator of IRFs/Z‐DNA binding protein 1 (DAI/ZBP1), haematopoietic IFN‐inducible nuclear protein‐200 (p202), absent in melanoma 2 (AIM2), RNA polymerase III, retinoic acid‐inducible gene‐I (RIG‐I) and melanoma differentiation‐associated gene 5 (MDA5) mediate responses to cytosolic dsDNA or dsRNA, respectively. TLR‐induced responses are more robust than those induced by cytosolic DNA‐ or RNA‐ sensors, the later usually being limited to interferon regulatory factor 3 (IRF3)‐dependent type I interferon (IFN) induction and nuclear factor (NF)‐κB activation. Interestingly, AIM2 is not capable of inducing type I IFN, but rather plays a role in caspase I activation. DNA‐ or RNA‐like synthetic inhibitory oligonucleotides (INH‐ODN) have been developed that antagonize TLR‐7‐ and/or TLR‐9‐induced activation in autoimmune B cells and in type I IFN‐producing dendritic cells at low nanomolar concentrations. It is not known whether these INH‐ODNs have any agonistic or antagonistic effects on cytosolic DNA or RNA sensors. While this remains to be determined in the future, in vivo studies have already shown their potential for preventing spontaneous lupus in various animal models of lupus. Several groups are exploring the possibility of translating these INH‐ODNs into human therapeutics for treating SLE and bacterial DNA‐induced sepsis.     

Receptors, subcellular compartments and the regulation of peripheral B cell responses: the illuminating state of anergy

Signals through the B cell antigen receptor (BCR) are necessary but not sufficient for cellular activation. Co-stimulatory signals must be provided through other immune recognition receptor systems, such as MHC class II/CD40 and the toll-like receptor (TLR) 9 that can only productively acquire their ligands in the processive environment of specialized late endosomes (MHC class II containing compartment or MIIC). It has long been appreciated that the BCR, by effectively capturing complex antigens and delivering them to late endosomes, is the link between activation events on the cell surface and those dependent on late endosomes. However, it has become increasingly apparent that the BCR also directs the translocation of MHC class II and TLR9 into the MIIC and that the endocytic flow of these receptors coincides with that of the BCR. This likely ensures close apposition of receptor complexes within the MIIC and the efficient transfer of ligands from the BCR to MHC class II and TLR9. This complex orchestration of receptor endocytic movement is dependent upon the quality of signals elicited through the BCR. Failure to activate specific signaling pathways, such as occurs in anergic B cells, prevents the entry of the BCR and TLR9 into the MIIC and abrogates TLR9 activation. Like anergy, this block in endocytic trafficking is rapidly reversible. These findings indicate that cellular responsiveness can be determined by mechanisms that control the subcellular location of important immune recognition receptors.     

Toll-like receptor 9 in murine lupus: more friend than foe!

The immune response induced by the pathogen-associated-pattern recognition receptor toll-like receptor 9 (TLR9) upon binding of CpG motif-containing DNA has been widely accepted as an important pathway in the immune defense against microbial pathogens. In contrast, the role of TLR9 in anti-DNA antibody generation and the pathogenesis of systemic lupus erythematosus (SLE) remains controversial. Indeed, the in vivo situation might consist of a delicate balance between B-cell receptor and DNA receptor signaling. Most surprisingly, TLR9 deletion does not ameliorate but rather exacerbates pathology in murine models. Such observations warrant caution with therapeutic efforts to treat autoimmune diseases, especially SLE, via TLR modulation.     

Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies that are frequently directed against nucleic acid-associated antigens. To better understand how B cells reactive with such antigens are regulated, we generated a model system in which heavy and light chain genes encoding 564 immunoglobulin have been targeted to the heavy and light chain loci of the nonautoimmune C57BL/6 mouse strain. This antibody recognizes RNA, single-stranded DNA, and nucleosomes. We show that B cells expressing this immunoglobulin were activated, producing class-switched autoantibody in vivo despite the apparently normal induction of anergy. This autoantibody production was largely dependent on Toll-like receptor 7 (TLR7). We further show that production of these autoantibodies was sufficient to cause kidney pathology in these mice. These results demonstrate that the particular threat of nucleic acid-containing autoantigens lies in their ability to bind both antigen receptor and TLR7.