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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

TLR-dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity.

We formulate a two-phase paradigm of autoimmunity associated with systemic lupus erythematosus, the archetypal autoimmune disease. The initial Toll-like receptor (TLR)-independent phase is mediated by dendritic cell uptake of apoptotic cell debris and associated nucleic acids, whereas the subsequent TLR-dependent phase serves an amplification function and is mediated by uptake of TLR ligands derived from self-antigens (principally nucleic acids) complexed with autoantibodies. Both phases depend on elaboration of type I interferons (IFNs), and therapeutic interruption of induction or activity of these cytokines in predisposed individuals might have a substantial mitigating effect in lupus and other autoimmune diseases.

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.

Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus.

Using the Unc93b1 3d mutation that selectively abolishes nucleic acid-binding Toll-like receptor (TLR) (TLR3, -7, -9) signaling, we show these endosomal TLRs are required for optimal production of IgG autoAbs, IgM rheumatoid factor, and other clinical parameters of disease in 2 lupus strains, B6-Fas(lpr) and BXSB. Strikingly, treatment with lipid A, an autoAb-inducing TLR4 agonist, could not overcome this requirement. The 3d mutation slightly reduced complete Freund's adjuvant (CFA)-mediated antigen presentation, but did not affect T-independent type 1 or alum-mediated T-dependent humoral responses or TLR-independent IFN production induced by cytoplasmic nucleic acids. These findings suggest that nucleic acid-sensing TLRs might act as an Achilles' heel in susceptible individuals by providing a critical pathway by which relative tolerance for nucleic acid-containing antigens is breached and systemic autoimmunity ensues. Importantly, this helps provide an explanation for the high frequency of anti-nucleic acid Abs in lupus-like systemic autoimmunity.

Impaired negative regulation of homeostatically proliferating T cells.

Acute lymphopenia-induced homeostatic proliferation (HP) of T cells promotes antitumor immunity, but the mechanism is unclear. We hypothesized that this is due to a lack of inhibitory signals that allows activation of T cells with low affinity for self-antigens. Tumors resist immunity in part by expressing inhibitory molecules such as PD-1 ligand 1 (PD-L1), B7-H4, and TGF-beta. In irradiated mice undergoing HP, we found that T cells displayed a severe deficit in the activation-induced expression of inhibitory molecules PD-1 and CTLA-4, and TGF-beta1-induced expression of Foxp3. HP T cells were also less suppressed by B7-H4/Ig and, unlike control T cells, failed to produce IL-10 in response to this molecule. This deficiency in regulation was reversed as normal T-cell numbers were restored. We conclude that T cells are weakly regulated by inhibitory molecules during the acute phase of HP, which could explain their increased effectiveness in cancer immunotherapy.

Deficiency of the cyclin kinase inhibitor p21(WAF-1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity.

A characteristic feature of systemic lupus erythematosus is the accumulation of activated/memory T and B cells. These G0/G1-arrested cells express high levels of cyclin-dependent kinase inhibitors such as p21, are resistant to proliferation and apoptosis, and produce large amounts of proinflammatory cytokines. Herein, we show that ablation of p21 in lupus-prone mice allows these cells to reenter the cell cycle and undergo apoptosis, leading to autoimmune disease reduction. Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential. Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax. Proliferation and apoptosis of B cells were also increased in p21-/- lupus mice. Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice.

Indirect evidence suggests that type-I interferons (IFN-alpha/beta) play a significant role in the pathogenesis of lupus. To directly examine the contribution of these pleiotropic molecules, we created congenic NZB mice lacking the alpha-chain of IFN-alpha/betaR, the common receptor for the multiple IFN-alpha/beta species. Compared with littermate controls, homozygous IFN-alpha/betaR-deleted NZB mice had significantly reduced anti-erythrocyte autoantibodies, erythroblastosis, hemolytic anemia, anti-DNA autoantibodies, kidney disease, and mortality. These reductions were intermediate in the heterozygous-deleted mice. The disease-ameliorating effects were accompanied by reductions in splenomegaly and in several immune cell subsets, including B-1 cells, the major producers of anti-erythrocyte autoantibodies. Decreases of B and T cell proliferation in vitro and in vivo, and of dendritic cell maturation and T cell stimulatory activity in vitro were also detected. Absence of signaling through the IFN-alpha/betaR, however, did not affect increased basal levels of the IFN-responsive p202 phosphoprotein, encoded by a polymorphic variant of the Ifi202 gene associated with the Nba2 predisposing locus in NZB mice. The data indicate that type-I IFNs are important mediators in the pathogenesis of murine lupus, and that reducing their activity in the human counterpart may be beneficial.

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.

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.

T cell homeostatic proliferation elicits effective antitumor autoimmunity.

Development of tumor immunotherapies focuses on inducing autoimmune responses against tumor-associated self-antigens primarily encoded by normal, unmutated genes. We hypothesized that such responses could be elicited by T cell homeostatic proliferation in the periphery, involving expansion of T cells recognizing self-MHC/peptide ligands. Herein, we demonstrate that sublethally irradiated lymphopenic mice transfused with autologous or syngeneic T cells showed tumor growth inhibition when challenged with melanoma or colon carcinoma cells. Importantly, the antitumor response depended on homeostatic expansion of a polyclonal T cell population within lymph nodes. This response was effective even for established tumors, was characterized by CD8(+) T cell-mediated tumor-specific cytotoxicity and IFN-gamma production, and was associated with long-term memory. The results indicate that concomitant induction of the physiologic processes of homeostatic T cell proliferation and tumor antigen presentation in lymph nodes triggers a beneficial antitumor autoimmune response.