The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation.

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1ß (IL-1ß) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1ß. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1ß in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.

Cross-Reactive Antigen Expressed by B6 Splenocytes Drives Receptor Editing and Marginal Zone Differentiation of IgG2a-Reactive AM14 Vκ8 B Cells.

The AM14 BCR, derived from an autoimmune MRL/lpr mouse, binds autologous IgG2aa/j with low affinity, and as a result, AM14 B cells only proliferate in response to IgG2a immune complexes that incorporate DNA, RNA, or nucleic acid-binding proteins that serve as autoadjuvants. As such, AM14 B cells have served as a useful model for demonstrating the importance of BCR/TLR coengagement in the activation of autoreactive B cells. We now show that the same receptor recognizes an additional murine-encoded Ag, expressed by B6 splenocytes, with sufficient avidity to induce a TLR-independent proliferative response of BALB/c AM14 Vκ8 B cells both in vivo and in vitro. Moreover, detection of this cross-reactive Ag by B6 AM14 Vκ8 B cells promotes an anergic phenotype as reflected by suboptimal responses to BCR cross-linking and the absence of mature B cells in the bone marrow. The B6 Ag further impacts B cell development as shown by a dramatically expanded marginal zone compartment and extensive receptor editing in B6 AM14 Vκ8 mice but not BALB/c AM14 Vκ8 mice. Despite their anergic phenotypes, B6 AM14 Vκ8 B cells can respond robustly to autoantigen/autoadjuvant immune complexes and could therefore participate in both autoimmune responses and host defense.

Synergy between Hematopoietic and Radioresistant Stromal Cells Is Required for Autoimmune Manifestations of DNase II-/-IFNaR-/- Mice.

Detection of endogenous nucleic acids by cytosolic receptors, dependent on STING, and endosomal sensors, dependent on Unc93b1, can provoke inflammatory responses that contribute to a variety of autoimmune and autoinflammatory diseases. In DNase II-deficient mice, the excessive accrual of undegraded DNA leads to both a STING-dependent inflammatory arthritis and additional Unc93b1-dependent autoimmune manifestations, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. In this study, we use bone marrow chimeras to show that clinical and histological inflammation in the joint depends upon DNase II deficiency in both donor hematopoietic cells and host radioresistant cells. Additional features of autoimmunity in these mice, known to depend on Unc93b1 and therefore endosomal TLRs, also require DNase II deficiency in both donor and host compartments, but only require functional TLRs in the hematopoietic cells. Collectively, our data demonstrate a major role of both stromal and hematopoietic cells in all aspects of DNA-driven autoimmunity. These findings further point to the importance of cytosolic nucleic acid sensors in creating an inflammatory environment that facilitates the development of Unc93b1-dependent autoimmunity.

Overexpression of Soluble Fas Ligand following Adeno-Associated Virus Gene Therapy Prevents Retinal Ganglion Cell Death in Chronic and Acute Murine Models of Glaucoma.

Glaucoma is a multifactorial disease resulting in the death of retinal ganglion cells (RGCs) and irreversible blindness. Glaucoma-associated RGC death depends on the proapoptotic and proinflammatory activity of membrane-bound Fas ligand (mFasL). In contrast to mFasL, the natural cleavage product, soluble Fas ligand (sFasL) inhibits mFasL-mediated apoptosis and inflammation and, therefore, is an mFasL antagonist. DBA/2J mice spontaneously develop glaucoma and, predictably, RGC destruction is exacerbated by expression of a mutated membrane-only FasL gene that lacks the extracellular cleavage site. Remarkably, one-time intraocular adeno-associated virus-mediated gene delivery of sFasL provides complete and sustained neuroprotection in the chronic DBA/2J and acute microbead-induced models of glaucoma, even in the presence of elevated intraocular pressure. This protection correlated with inhibition of glial activation, reduced production of TNF-α, and decreased apoptosis of RGCs and loss of axons. These data indicate that cleavage of FasL under homeostatic conditions, and the ensuing release of sFasL, normally limits the neurodestructive activity of FasL. The data further support the notion that sFasL, and not mFasL, contributes to the immune-privileged status of the eye.

TLR9 Deficiency Leads to Accelerated Renal Disease and Myeloid Lineage Abnormalities in Pristane-Induced Murine Lupus.

Systemic lupus erythematosus (SLE) is a chronic, life-threatening autoimmune disorder, leading to multiple organ pathologies and kidney destruction. Analyses of numerous murine models of spontaneous SLE have revealed a critical role for endosomal TLRs in the production of autoantibodies and development of other clinical disease manifestations. Nevertheless, the corresponding TLR9-deficient autoimmune-prone strains consistently develop more severe disease pathology. Injection of BALB/c mice with 2,6,10,14-tetramethylpentadecane (TMPD), commonly known as pristane, also results in the development of SLE-like disease. We now show that Tlr9(-/-) BALB/c mice injected i.p. with TMPD develop more severe autoimmunity than do their TLR-sufficient cohorts. Early indications include an increased accumulation of TLR7-expressing Ly6C(hi) inflammatory monocytes at the site of injection, upregulation of IFN-regulated gene expression in the peritoneal cavity, and an increased production of myeloid lineage precursors (common myeloid progenitors and granulocyte myeloid precursors) in the bone marrow. TMPD-injected Tlr9(-/-) BALB/c mice develop higher autoantibody titers against RNA, neutrophil cytoplasmic Ags, and myeloperoxidase than do TMPD-injected wild-type BALB/c mice. The TMP-injected Tlr9(-/-) mice, and not the wild-type mice, also develop a marked increase in glomerular IgG deposition and infiltrating granulocytes, much more severe glomerulonephritis, and a reduced lifespan. Collectively, the data point to a major role for TLR7 in the response to self-antigens in this model of experimental autoimmunity. Therefore, the BALB/c pristane model recapitulates other TLR7-driven spontaneous models of SLE and is negatively regulated by TLR9.

Cutting Edge: DNase II deficiency prevents activation of autoreactive B cells by double-stranded DNA endogenous ligands.

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.

Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses.

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

Cutting edge: AIM2 and endosomal TLRs differentially regulate arthritis and autoantibody production in DNase II-deficient mice.

Innate immune pattern recognition receptors sense nucleic acids from microbes and orchestrate cytokine production to resolve infection. Inappropriate recognition of host nucleic acids also results in autoimmune disease. In this study, we use a model of inflammation resulting from accrual of self DNA (DNase II(-/-) type I IFN receptor [Ifnar](-/-)) to understand the role of pattern recognition receptor-sensing pathways in arthritis and autoantibody production. Using triple knockout (TKO) mice deficient in DNase II/IFNaR together with deficiency in either stimulator of IFN genes (STING) or absent in melanoma 2 (AIM2), we reveal central roles for the STING and AIM2 pathways in arthritis. AIM2 TKO mice show limited inflammasome activation and, similar to STING TKO mice, have reduced inflammation in joints. Surprisingly, autoantibody production is maintained in AIM2 and STING TKO mice, whereas DNase II(-/-) Ifnar(-/-) mice also deficient in Unc93b, a chaperone required for TLR7/9 endosomal localization, fail to produce autoantibodies to nucleic acids. Collectively, these data support distinct roles for cytosolic and endosomal nucleic acid-sensing pathways in disease manifestations.

An unexpected role for RNA-sensing toll-like receptors in a murine model of DNA accrual.

OBJECTIVES: The goal of this study was to determine whether endosomal Toll-like receptors (TLRs) contribute to the clinical manifestation of systemic autoimmunity exhibited by mice that lack the lysosomal nuclease DNaseII. METHODS: DNaseII/IFNaR double deficient mice were intercrossed with Unc93b13d/3d mice to generate DNaseII-/-mice with non-functional endosomal TLRs. The resulting triple deficient mice were evaluated for arthritis, autoantibody production, splenomegaly, and extramedullary haematopoiesis. B cells from both strains were evaluated for their capacity to respond to endogenous DNA by using small oligonucleotide based TLR9D ligands and a novel class of bifunctional anti-DNA antibodies. RESULTS: Mice that fail to express DNaseII, IFNaR, and Unc93b1 still develop arthritis but do not make autoantibodies, develop splenomegaly, or exhibit extramedullary haematopoiesis. DNaseII-/- IFNaR-/- B cells can respond to synthetic ODNs, but not to endogenous dsDNA. CONCLUSIONS: RNA-reactive TLRs, presumably TLR7, are required for autoantibody production, splenomegaly, and extramedullary haematopoiesis in the DNaseII-/- model of systemic autoimmunity.

Overexpression of membrane-bound fas ligand (CD95L) exacerbates autoimmune disease and renal pathology in pristane-induced lupus.

Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity.

IFN regulatory factor 5 is required for disease development in the FcgammaRIIB-/-Yaa and FcgammaRIIB-/- mouse models of systemic lupus erythematosus.

Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcgammaRIIB(-/-)Yaa and FcgammaRIIB(-/-) lupus models. In contrast to IRF5-sufficient FcgammaRIIB(-/-)Yaa mice, IRF5-deficient FcgammaRIIB(-/-)Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-alpha, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcgammaRIIB(-/-)Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcgammaRIIB(-/-)Yaa mice, type I IFN receptor subunit 1-deficient FcgammaRIIB(-/-)Yaa mice maintained a substantial level of residual disease. Furthermore, in FcgammaRIIB(-/-) mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcgammaRIIB(-/-)Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

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.

Role of Interferon-γ-Producing Th1 Cells in a Murine Model of Type I Interferon-Independent Autoinflammation Resulting From DNase II Deficiency.

OBJECTIVE: Patients with hypomorphic mutations in DNase II develop a severe and debilitating autoinflammatory disease. This study was undertaken to compare the disease parameters in these patients to those in a murine model of DNase II deficiency, and to evaluate the role of specific nucleic acid sensors and identify the cell types responsible for driving the autoinflammatory response. METHODS: To avoid embryonic death, Dnase2-/- mice were intercrossed with mice that lacked the type I interferon (IFN) receptor (Ifnar-/- ). The hematologic changes and immune status of these mice were evaluated using complete blood cell counts, flow cytometry, serum cytokine enzyme-linked immunosorbent assays, and liver histology. Effector cell activity was determined by transferring T cells from Dnase2-/- × Ifnar-/- double-knockout (DKO) mice into Rag1-/- mice, and 4 weeks after cell transfer, induced changes were assessed in the recipient mice. RESULTS: In Dnase2-/- × Ifnar-/- DKO mice, many of the disease features found in DNase II-deficient patients were recapitulated, including cytopenia, extramedullary hematopoiesis, and liver fibrosis. Dnase2+/+ × Rag1-/- mice (n > 22) developed a hematologic disorder that was attributed to the transfer of an unusual IFNγ-producing T cell subset from the spleens of donor Dnase2-/- × Ifnar-/- DKO mice. Autoinflammation in this murine model did not depend on the stimulator of IFN genes (STING) pathway but was highly dependent on the chaperone protein Unc93B1. CONCLUSION: Dnase2-/- × Ifnar-/- DKO mice may be a valid model for exploring the innate and adaptive immune mechanisms responsible for the autoinflammation similar to that seen in DNASE2-hypomorphic patients. In this murine model, IFNγ is required for T cell activation and the development of clinical manifestations. The role of IFNγ in DNASE2-deficient patient populations remains to be determined, but the ability of Dnase2-/- mouse T cells to transfer disease to Rag1-/- mice suggests that T cells may be a relevant therapeutic target in patients with IFN-related systemic autoinflammatory diseases.

Taking the STING out of TLR-driven autoimmune diseases: good, bad, or indifferent?

Both endosomal and cytosolic-nucleic acid-sensing receptors can detect endogenous ligands and promote autoimmunity and autoinflammation. These responses involve a complex interplay among and between the cytosolic and endosomal sensors involving both hematopoietic and radioresistant cells. Cytosolic sensors directly promote inflammatory responses through the production of type I IFNs and proinflammatory cytokines. Inflammation-associated tissue damage can further promote autoimmune responses indirectly, as receptor-mediated internalization of the resulting cell debris can activate endosomal Toll-like receptors (TLR). Both endosomal and cytosolic receptors can also negatively regulate inflammatory responses. A better understanding of the factors and pathways that promote and constrain autoimmune diseases will have important implications for the development of agonists and antagonists that modulate these pathways.

The role of Bruton's tyrosine kinase in the development and BCR/TLR-dependent activation of AM14 rheumatoid factor B cells.

The protein kinase Btk has been implicated in the development, differentiation, and activation of B cells through its role in the BCR and TLR signaling cascades. These receptors and in particular, the BCR and either TLR7 or TLR9 also play a critical role in the activation of autoreactive B cells by RNA- or DNA-associated autoantigens. To explore the role of Btk in the development of autoreactive B cells, as well as their responses to nucleic acid-associated autoantigens, we have now compared Btk-sufficient and Btk-deficient mice that express a prototypic RF BCR encoded by H- and L-chain sdTgs. These B cells bind autologous IgG2a with low affinity and only proliferate in response to IgG2a ICs that incorporate DNA or RNA. We found that Btk-sufficient RF(+) B cells mature into naïve FO B cells, all of which express the Tg BCR, despite circulating levels of IgG2a. By contrast, a significant proportion of Btk-deficient RF(+) B cells acquires a MZ or MZ precursor phenotype. Remarkably, despite the complete inability of RF(+) Xid/y B cells to respond to F(ab')2 anti-IgM, RF(+) Xid/y B cells could respond well to autoantigen-associated ICs. These data reveal unique features of the signaling cascades responsible for the activation of autoreactive B cells.

Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.