Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells.

Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and ß2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.

The solute carrier SLC15A4 is required for optimal trafficking of nucleic acid-sensing TLRs and ligands to endolysosomes.

A function-impairing mutation (feeble) or genomic deletion of SLC15A4 abolishes responses of nucleic acid­sensing endosomal toll-like receptors (TLRs) and significantly reduces disease in mouse models of lupus. Here, we demonstrate disease reduction in homozygous and even heterozygous Slc15a4 feeble mutant BXSB male mice with a Tlr7 gene duplication. In contrast to SLC15A4, a function-impairing mutation of SLC15A3 did not diminish type I interferon (IFN-I) production by TLR-activated plasmacytoid dendritic cells (pDCs), indicating divergence of function between these homologous SLC15 family members. Trafficking to endolysosomes and function of SLC15A4 were dependent on the Adaptor protein 3 (AP-3) complex. Importantly, SLC15A4 was required for trafficking and colocalization of nucleic acid­sensing TLRs and their ligands to endolysosomes and the formation of the LAMP2+VAMP3+ hybrid compartment in which IFN-I production is initiated. Collectively, these findings define mechanistic processes by which SLC15A4 controls endosomal TLR function and suggest that pharmacologic intervention to curtail the function of this transporter may be a means to treat lupus and other endosomal TLR-dependent diseases.

Silica exposure and chronic virus infection synergistically promote lupus-like systemic autoimmunity in mice with low genetic predisposition.

Considerable evidence indicates that autoimmune disease expression depends on both genetic and environmental factors. Among potential environmental triggers, occupational airway exposure to crystalline silica and virus infections have been linked to lupus and other autoimmune diseases in both humans and mouse models. Here, we hypothesized that combined silica and virus exposures synergize and induce autoimmune manifestations more effectively than single exposure to either of these factors, particularly in individuals with low genetic predisposition. Accordingly, infection with the model murine pathogen lymphocytic choriomenigitis virus (LCMV) in early life, followed by airway exposure to crystalline silica in adult life, induced lupus-like autoantibodies to several nuclear self-antigens including chromatin, RNP and Sm, concurrent with kidney lesions, in non-autoimmune C57BL/6 (B6) mice. In contrast, given individually, LCMV or silica were largely ineffectual in this strain. These results support a multihit model of autoimmunity, where exposure to different environmental factors acting on distinct immunostimulatory pathways complements limited genetic predisposition and increases the risk of autoimmunity above a critical threshold.

Lupus acceleration by a MAVS-activating RNA virus requires endosomal TLR signaling and host genetic predisposition.

Viruses have long been implicated in the pathogenesis of autoimmunity, yet their contribution remains circumstantial partly due to the lack of well-documented information on infections prior to autoimmune disease onset. Here, we used the lymphocytic choriomeningitis virus (LCMV) as a model to mechanistically dissect the impact of viral infection on lupus-like autoimmunity. Virus persistence strongly enhanced disease in mice with otherwise weak genetic predisposition but not in highly predisposed or non-autoimmune mice, indicating a synergistic interplay between genetic susceptibility and virus infection. Moreover, endosomal Toll-like receptors (TLRs) and plasmacytoid dendritic cells (pDCs) were both strictly required for disease acceleration, even though LCMV also induces strong TLR-independent type I interferon (IFN-I) production via RNA helicases and MAVS in conventional DCs. These results suggest that LCMV enhances systemic autoimmunity primarily by providing stimulatory nucleic acids for endosomal TLR engagement, whereas overstimulation of the MAVS-dependent cytosolic pathway in the absence of endosomal TLR signaling is insufficient for disease induction.

Murine Models for Viral Hemorrhagic Fever.

Hemorrhagic fever (HF) viruses, such as Lassa, Ebola, and dengue viruses, represent major human health risks due to their highly contagious nature, the severity of the clinical manifestations induced, the lack of vaccines, and the very limited therapeutic options currently available. Appropriate animal models are obviously critical to study disease pathogenesis and develop efficient therapies. We recently reported that the clone 13 (Cl13) variant of the lymphocytic choriomeningitis virus (LCMV-Cl13), a prototype arenavirus closely related to Lassa virus, causes in some mouse strains endothelial damage, vascular leakage, platelet loss, and death, mimicking pathological aspects typically observed in Lassa and other HF syndromes. This model has the advantage that the mice used are fully immunocompetent, allowing studies on the contribution of the immune response to disease progression. Moreover, LCMV is very well characterized and exhibits limited pathogenicity in humans, allowing handling in convenient BSL-2 facilities. In this chapter we outline protocols for the induction and analysis of arenavirus-mediated pathogenesis in the NZB/LCMV model, including mouse infection, virus titer determination, platelet counting, phenotypic analysis of virus-specific T cells, and assessment of vascular permeability.

Induction of Systemic Autoimmunity by a Xenobiotic Requires Endosomal TLR Trafficking and Signaling from the Late Endosome and Endolysosome but Not Type I IFN.

Type I IFN and nucleic acid-sensing TLRs are both strongly implicated in the pathogenesis of lupus, with most patients expressing IFN-induced genes in peripheral blood cells and with TLRs promoting type I IFNs and autoreactive B cells. About a third of systemic lupus erythematosus patients, however, lack the IFN signature, suggesting the possibility of type I IFN-independent mechanisms. In this study, we examined the role of type I IFN and TLR trafficking and signaling in xenobiotic systemic mercury-induced autoimmunity (HgIA). Strikingly, autoantibody production in HgIA was not dependent on the type I IFN receptor even in NZB mice that require type I IFN signaling for spontaneous disease, but was dependent on the endosomal TLR transporter UNC93B1 and the endosomal proton transporter, solute carrier family 15, member 4. HgIA also required the adaptor protein-3 complex, which transports TLRs from the early endosome to the late endolysosomal compartments. Examination of TLR signaling pathways implicated the canonical NF-κB pathway and the proinflammatory cytokine IL-6 in autoantibody production, but not IFN regulatory factor 7. These findings identify HgIA as a novel type I IFN-independent model of systemic autoimmunity and implicate TLR-mediated NF-κB proinflammatory signaling from the late endocytic pathway compartments in autoantibody generation.

The multiple pathways to autoimmunity.

Efforts to understand autoimmunity have been pursued relentlessly for several decades. It has become apparent that the immune system evolved multiple mechanisms for controlling self-reactivity, and defects in one or more of these mechanisms can lead to a breakdown of tolerance. Among the multitude of lesions associated with disease, the most common seem to affect peripheral tolerance rather than central tolerance. The initial trigger for both systemic autoimmune disorders and organ-specific autoimmune disorders probably involves the recognition of self or foreign molecules, especially nucleic acids, by innate sensors. Such recognition, in turn, triggers inflammatory responses and the engagement of previously quiescent autoreactive T cells and B cells. Here we summarize the most prominent autoimmune pathways and identify key issues that require resolution for full understanding of pathogenic autoimmunity.

An agonist antibody that blocks autoimmunity by inducing anti-inflammatory macrophages.

We have devised a method of using intracellular combinatorial libraries to select antibodies that control cell fates. Many agonist antibodies have been selected with this method, and the process appears to be limited only by the availability of a phenotypic selection system. We demonstrate the utility of this approach to discover agonist antibodies that engage an unanticipated target and regulate macrophage polarization by selective induction of anti-inflammatory M2 macrophages. This antibody was used therapeutically to block autoimmunity in a classic mouse model of spontaneous systemic lupus erythematosus.

Interleukin-7 is required for CD4(+) T cell activation and autoimmune neuroinflammation.

IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4(+) T cell activation and that IL-7R antagonism may be effective in treating CD4(+) T cell-mediated neuroinflammation and other autoimmune inflammatory conditions.

Type I interferon is a therapeutic target for virus-induced lethal vascular damage.

The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus--a prototype of Old World arenaviruses closely related to Lassa fever virus--elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell-mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6-8 d. These pathogenic events required type I IFN (IFN-I) signaling on nonhematopoietic cells and were completely abrogated by IFN-I receptor blockade. Thus, IFN-I may play a prominent role in hemorrhagic fevers and other acute virus infections associated with severe vascular pathology, and targeting IFN-I or downstream effector molecules may be an effective therapeutic approach.

TLRs and interferons: a central paradigm in autoimmunity.

Investigations into the pathogenesis of lupus have largely focused on abnormalities in components of the adaptive immune system. Despite important advances, however, the question about the origin of the pathogenic process, the primary disease trigger, and the dominance of autoantibodies against nuclear components, remained unanswered. Discoveries in the last decade have provided some resolution to these questions by elucidating the central role of nucleic acid-sensing TLRs and the attendant inflammatory response, particularly the production of type I interferons. These priming events are responsible for initiating the adaptive responses that ultimately mediate the pathogenic process.

Role of nucleic acid-sensing TLRs in diverse autoantibody specificities and anti-nuclear antibody-producing B cells.

Nucleic acid (NA)-sensing TLRs (NA-TLRs) promote the induction of anti-nuclear Abs in systemic lupus erythematosus. However, the extent to which other nonnuclear pathogenic autoantibody specificities that occur in lupus and independently in other autoimmune diseases depend on NA-TLRs, and which immune cells require NA-TLRs in systemic autoimmunity, remains to be determined. Using Unc93b1(3d) lupus-prone mice that lack NA-TLR signaling, we found that all pathogenic nonnuclear autoantibody specificities examined, even anti-RBC, required NA-TLRs. Furthermore, we document that NA-TLRs in B cells were required for the development of antichromatin and rheumatoid factor. These findings support a unifying NA-TLR-mediated mechanism of autoantibody production that has both pathophysiological and therapeutic implications for systemic lupus erythematosus and several other humoral-mediated autoimmune diseases. In particular, our findings suggest that targeting of NA-TLR signaling in B cells alone would be sufficient to specifically block production of a broad diversity of autoantibodies.

Discovery of peptoid ligands for anti-aquaporin 4 antibodies.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disorder of the central nervous system. In most NMO patients, autoantibodies to the water channel protein Aquaporin 4 (AQP4) are present at high levels and are thought to drive pathology by mediating complement-dependent destruction of astrocytes. Here, we apply recently developed chemical library screening technology to identify a synthetic peptoid that binds anti-AQP4 antibodies in the serum of NMO patients. This finding validates, in a well-defined human disease, that synthetic, unnatural ligands for the antigen-binding site of a disease-linked antibody can be isolated by high-throughput screening.

Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus.

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4). IRF8-deficient NZB mice, lacking pDCs, showed almost complete absence of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibodies, along with reduced kidney disease. These effects were observed despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 stimuli and intact humoral responses to conventional T-dependent and -independent antigens. Moreover, Slc15a4 mutant C57BL/6-Fas(lpr) mice, in which pDCs are present but unable to produce type I IFNs in response to endosomal TLR ligands, also showed an absence of autoantibodies, reduced lymphadenopathy and splenomegaly, and extended survival. Taken together, our results demonstrate that pDCs and the production of type I IFNs by these cells are critical contributors to the pathogenesis of lupus-like autoimmunity in these models. Thus, IRF8 and SLC15A4 may provide important targets for therapeutic intervention in human lupus.

Anti-IFN-α/ß receptor antibody treatment ameliorates disease in lupus-predisposed mice.

The demonstration in humans and mice that nucleic acid-sensing TLRs and type I IFNs are essential disease mediators is a milestone in delineating the mechanisms of lupus pathogenesis. In this study, we show that Ifnb gene deletion does not modify disease progression in NZB mice, thereby strongly implicating IFN-α subtypes as the principal pathogenic effectors. We further document that long-term treatment of male BXSB mice with an anti-IFN-α/ß receptor Ab of mouse origin reduced serologic, cellular, and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment in BXSB mice was clearly evident when applied early in the disease process, but only partial reductions in some disease characteristics were observed when treatment was initiated at later stages. A transient therapeutic effect was also noted in the MRL-Fas(lpr) model, although overall mortality was unaffected. The combined findings suggest that IFN-α/ß receptor blockade, particularly when started at early disease stages, may be a useful treatment approach for human systemic lupus erythematosus and other autoimmune syndromes.

Yip1 domain family, member 6 (Yipf6) mutation induces spontaneous intestinal inflammation in mice.

Using an environmentally sensitized genetic screen we identified mutations that cause inflammatory colitis in mice. The X-linked Klein-Zschocher (KLZ) mutation created a null allele of Yipf6, a member of a gene family believed to regulate vesicular transport in yeast, but without known functions in mammals. Yipf6 is a five transmembrane-spanning protein associated with Golgi compartments. Klein-Zschocher mutants were extremely sensitive to colitis induced by dextran sodium sulfate (DSS) and developed spontaneous ileitis and colitis after 16 mo of age in specific pathogen-free housing conditions. Electron microscopy, gene expression, and immunocytochemistry analyses provided evidence that impaired intestinal homeostasis stemmed from defective formation and secretion of large secretory granules from Paneth and goblet cells. These studies support a tissue- and organ-specific function for Yipf6 in the maintenance of intestinal homeostasis and implicate the orthologous human gene as a disease susceptibility locus.

Transmethylation in immunity and autoimmunity.

The activation of immune cells is mediated by a network of signaling proteins that can undergo post-translational modifications critical for their activity. Methylation of nucleic acids or proteins can have major effects on gene expression as well as protein repertoire diversity and function. Emerging data indicate that indeed many immunologic functions, particularly those of T cells, including thymic education, differentiation and effector function are highly dependent on methylation events. The critical role of methylation in immunocyte biology is further documented by evidence that autoimmune phenomena may be curtailed by methylation inhibitors. Additionally, epigenetic alterations imprinted by methylation can also exert effects on normal and abnormal immune responses. Further work in defining methylation effects in the immune system is likely to lead to a more detailed understanding of the immune system and may point to the development of novel therapeutic approaches.

Systemic autoimmunity and lymphoproliferation are associated with excess IL-7 and inhibited by IL-7Rα blockade.

Lupus is characterized by disturbances in lymphocyte homeostasis, as demonstrated by the marked accumulation of activated/memory T cells. Here, we provide evidence that proliferation of the CD8+ precursors for the accumulating CD4⁻CD8⁻ T cells in MRL-Fas(lpr) lupus-predisposed mice is, in part, driven by commensal antigens. The ensuing lymphadenopathy is associated with increased production of IL-7 due to expansion of fibroblastic reticular cells, the primary source of this cytokine. The excess IL-7 is not, however, consumed by CD4⁻CD8⁻ T cells due to permanent down-regulation of IL-7Rα (CD127), but instead supports proliferation of autoreactive T cells and progression of autoimmunity. Accordingly, IL-7R blockade reduced T cell activation and autoimmune manifestations even when applied at advanced disease stage. These findings indicate that an imbalance favoring production over consumption of IL-7 may contribute to systemic autoimmunity, and correction of this imbalance may be a novel therapeutic approach in lymphoproliferative and autoimmune syndromes.

Intracellular nucleic acid sensors and autoimmunity.

A collection of molecular sensors has been defined by studies in the last decade that can recognize a diverse array of pathogens and initiate protective immune and inflammatory responses. However, if the molecular signatures recognized are shared by both foreign and self-molecules, as is the case of nucleic acids, then the responses initiated by these sensors may have deleterious consequences. Notably, this adverse occurrence may be of primary importance in autoimmune disease pathogenesis. In this case, microbe-induced damage or mishandled physiologic processes could lead to the generation of microparticles containing self-nucleic acids. These particles may inappropriately gain access to the cytosol or endolysosomes and, hence, engage resident RNA and DNA sensors. Evidence, as reviewed here, strongly indicates that these sensors are primary contributors to autoimmune disease pathogenesis, spearheading efforts toward development of novel therapeutics for these disorders.

Sensors of the innate immune system: their link to rheumatic diseases.

Evidence strongly suggests that excessive or protracted signaling, or both, by cell-surface or intracellular innate immune receptors is central to the pathogenesis of most autoimmune and autoinflammatory rheumatic diseases. The initiation of aberrant innate and adaptive immune responses in autoimmune diseases can be triggered by microbes and, at times, by endogenous molecules--particularly nucleic acids and related immune complexes--under sterile conditions. By contrast, most autoinflammatory syndromes are generally dependent on germline or de novo gene mutations that cause or facilitate inflammasome assembly. The consequent production of proinflammatory cytokines, principally interferon-alpha/beta and tumor necrosis factor in autoimmune diseases, and interleukin-1beta in autoinflammatory diseases, leads to the creation of autoamplification feedback loops and chronicity of these syndromes. These findings have resulted in a critical reappraisal of pathogenetic mechanisms, and provide a basis for the development of novel diagnostic and therapeutic modalities for these diseases.