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Although numerous environmental exposures have been suggested as triggers for preclinical autoimmunity, only a few have been confidently linked to autoimmune diseases. For disease-associated exposures, the lung is a common site where chronic exposure results in cellular toxicity, tissue damage, inflammation, and fibrosis. These features are exacerbated by exposures to particulate material, which hampers clearance and degradation, thus facilitating persistent inflammation. Coincident with exposure and resulting pathological processes is the posttranslational modification of self-antigens, which, in concert with the formation of tertiary lymphoid structures containing abundant B cells, is thought to promote the generation of autoantibodies that in some instances demonstrate major histocompatibility complex restriction. Under appropriate gene-environment interactions, these responses can have diagnostic specificity. Greater insight into the molecular and cellular requirements governing this process, especially those that distinguish preclinical autoimmunity from clinical autoimmunedisease, may facilitate determination of the significance of environmental exposures in human autoimmune disease.

BackgroundEbola virus (EBOV) disease has killed thousands of West and Central Africans over the past several decades. Many who survive the acute disease later experience post-Ebola syndrome, a constellation of symptoms whose causative pathogenesis is unclear.MethodsWe investigated EBOV-specific CD8+ and CD4+ T-cell responses in 37 Sierra Leonean EBOV disease survivors with (n = 19) or without (n = 18) sequelae of arthralgia and ocular symptoms. Peripheral blood mononuclear cells were infected with recombinant vesicular stomatitis virus encoding EBOV antigens. We also studied the presence of EBOV-specific immunoglobulin G, antinuclear antibodies, anti–cyclic citrullinated peptide antibodies, rheumatoid factor, complement levels, and cytokine levels in these 2 groups.ResultsSurvivors with sequelae had a significantly higher EBOV-specific CD8+ and CD4+ T-cell response. No differences in EBOV-specific immunoglobulin G, antinuclear antibody, or anti–cyclic citrullinated peptide antibody levels were found. Survivors with sequelae showed significantly higher rheumatoid factor levels.ConclusionEBOV-specific CD8+ and CD4+ T-cell responses were significantly higher in Ebola survivors with post-Ebola syndrome. These findings suggest that pathogenesis may occur as an immune-mediated disease via virus-specific T-cell immune response or that persistent antigen exposure leads to increased and sustained T-cell responses.

The biology of how faces are built and come to differ from one another is complex. Discovering the genes that contribute to differences in facial morphology is one key to untangling this complexity, with important implications for medicine and evolutionary biology. This study maps quantitative trait loci (QTL) for skeletal facial shape using Diversity Outbred (DO) mice. The DO is a randomly outcrossed population with high heterozygosity that captures the allelic diversity of eight inbred mouse lines from three subspecies. The study uses a sample of 1147 DO animals (the largest sample yet employed for a shape QTL study in mouse), each characterized by 22 three-dimensional landmarks, 56,885 autosomal and X-chromosome markers, and sex and age classifiers. We identified 37 facial shape QTL across 20 shape principal components (PCs) using a mixed effects regression that accounts for kinship among observations. The QTL include some previously identified intervals as well as new regions that expand the list of potential targets for future experimental study. Three QTL characterized shape associations with size (allometry). Median support interval size was 3.5 Mb. Narrowing additional analysis to QTL for the five largest magnitude shape PCs, we found significant overrepresentation of genes with known roles in growth, skeletal development, and sensory organ development. For most intervals, one or more of these genes lies within 0.25 Mb of the QTL’s peak. QTL effect sizes were small, with none explaining more than 0.5% of facial shape variation. Thus, our results are consistent with a model of facial diversity that is influenced by key genes in skeletal and facial development and, simultaneously, is highly polygenic.Author SummaryThe mammalian face is a complex structure serving many functions. We studied the genetic basis for facial skeletal diversity in a large sample of mice from an experimental population designed for the study of complex traits. We quantified the contribution of genetic variation to variation in three-dimensional facial shape across more than 55,000 genetic markers spread throughout the mouse genome. We found 37 genetic regions which are very likely to contribute to differences in facial shape. We then conducted a more detailed analysis of the genetic regions associated with the most variable aspects of facial shape. For these regions, a disproportionately large number of genes are known to be important to growth and to skeletal and facial development. The magnitude of these genetic contributions to differences in facial shape are consistently small. Our results therefore support the notion that facial skeletal diversity is influenced by many genes of small effect, but that some of these small effects may be related to genes that are fundamental to skeletal and facial development.

Susceptibility to autoimmune diseases is dependent on multigenic inheritance, environmental factors, and stochastic events. Although there has been substantial progress in identifying predisposing genetic variants, a significant challenge facing autoimmune disease research is the identification of the specific events that trigger loss of tolerance, autoreactivity and ultimately autoimmune disease. Accordingly, studies have indicated that a wide range of extrinsic factors including drugs, chemicals, microbes, and other environmental factors can induce autoimmunity, particularly systemic autoimmune diseases such as lupus. This review describes a class of environmental factors, namely xenobiotics, epidemiologically linked to human autoimmunity. Mechanisms of xenobiotic autoimmune disease induction are discussed in terms of human and animal model studies with a focus on the role of inflammation and the innate immune response. We argue that localized tissue damage and chronic inflammation elicited by xenobiotic exposure leads to the release of self-antigens and damage-associated molecular patterns as well as the appearance of ectopic lymphoid structures and secondary lymphoid hypertrophy, which provide a milieu for the production of autoreactive B and T cells that contribute to the development and persistence of autoimmunity in predisposed individuals.

A single nucleotide polymorphism in PTPN22 is linked to increased disease susceptibility in a range of autoimmune diseases including systemic lupus erythematosus (SLE). PTPN22 encodes the Lyp phosphatase that dampens TCR signaling and is necessary for signaling downstream of toll-like receptors in myeloid cells. To understand these dual functions in disease, we examined the impact of deficiency in PTPN22 on a spontaneous murine model of SLE. Male PTPN22 KO mice carrying BXSB chromosome 1 and the Yaa disease accelerating factor, developed disease at a similar rate and severity as PTPN22 WT. In contrast, although female BXSB mice showed no differences in survival in the absence of PTPN22, autoantibody production was significantly increased and splenic populations associated with pathogenesis in this model were expanded in the PTPN22 KO group. These findings support the notion that when coupled with other predisposing autoimmunity genes, PTPN22 deficiency contributes to a predisposition to lupus pathogenesis.

Susceptibility and resistance to systemic autoimmunity are genetically regulated. This is particularly true for murine mercury-induced autoimmunity (mHgIA) where DBA/2J mice are considered resistant to disease including polyclonal B cell activation, autoantibody responses, and immune complex deposits. To identify possible mechanisms for the resistance to mHgIA, we exposed mHgIA sensitive B10.S and resistant DBA/2J mice to HgCl2 and assessed inflammation and pro-inflammatory responses at the site of exposure and subsequent development of markers of systemic autoimmunity. DBA/2J mice showed little evidence of induration at the site of exposure, expression of proinflammatory cytokines, T cell activation, or autoantibody production, although they did exhibit increased levels of total serum IgG and IgG1. In contrast B10.S mice developed significant inflammation together with increased expression of inflammasome component NLRP3, proinflammatory cytokines IL-1β, TNF-α, and IFN-γ, hypergammaglobulinemia, splenomegaly, CD4(+) T-cell activation, and production of autoantibodies. Inflammation in B10.S mice was associated with a selective increase in activity of cysteine cathepsin B but not cathepsins L or S. Increased cathepsin B activity was not dependent on cytokines required for mHgIA but treatment with CA-074, a cathepsin B inhibitor, led to transient reduction of local induration, expression of inflammatory cytokines, and subsequent attenuation of the systemic adaptive immune response. These findings demonstrate that sensitivity to mHgIA is linked to an early cathepsin B regulated inflammatory response which can be pharmacologically exploited to abrogate the subsequent adaptive autoimmune response which leads to disease.

Mercury (Hg), shown to induce autoimmune disease in rodents, is a ubiquitous toxicant throughout Cheyenne River Sioux Tribe (CRST) lands. CRST members may be exposed to Hg through fish consumption (FC), an important component of native culture that may supplement household subsistence. Our goals were to ascertain whether total blood Hg levels (THg) reflect Hg exposure through FC and smoking, and determine whether THg is associated with the presence of anti-nuclear antibody (ANA) and specific autoantibodies (sAuAb). We recruited 75 participants who regularly consume fish from CRST waters. Hg exposure through FC and smoking were assessed via questionnaires. Whole blood samples were collected from participants, and THg was measured using ICP-MS. ANA and sAuAb in serum were modeled using demographic and exposure information as predictors. Female gender, age, and FC were significant predictors of THg and sAuAb; self-reported smoking was not. 31% of participants tested positive for ANA ≥ 2+. Although ANA was not significantly associated with Hg, the interactions of gender with Hg and proximity to arsenic deposits were statistically significant(P<0.05). FC resulted in a detectable body burden of Hg, but THg alone did not correlate with the presence of ANA or sAuAb in this population.

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.

Autoimmunity is thought to result from a combination of genetics, environmental triggers, and stochastic events. Environmental factors, such as chemicals, drugs or infectious agents, have been implicated in the expression of autoimmune disease, yet human studies are extremely limited in their ability to test isolated exposures to demonstrate causation or to assess pathogenic mechanisms. In this review we examine the research literature on the ability of chemical, physical and biological agents to induce and/or exacerbate autoimmunity in a variety of animal models. There is no single animal model capable of mimicking the features of human autoimmune disease, particularly as related to environmental exposures. An objective, therefore, was to assess the types of information that can be gleaned from the use of animal models, and how well that information can be used to translate back to human health. Our review notes the importance of genetic background to the types and severity of the autoimmune response following exposure to environmental factors, and emphasizes literature where animal model studies have led to increased confidence about environmental factors that affect expression of autoimmunity. A high level of confidence was reached if there were multiple studies from different laboratories confirming the same findings. Examples include mercury, pristane, and infection with Streptococcus or Coxsackie B virus. A second level of consensus identified those exposures likely to influence autoimmunity but requiring further confirmation. To fit into this category, there needed to be significant supporting data, perhaps by multiple studies from a single laboratory, or repetition of some but not all findings in multiple laboratories. Examples include silica, gold, TCE, TCDD, UV radiation, and Theiler’s murine encephalomyelitis virus. With the caveat that researchers must keep in mind the limitations and appropriate applications of the various approaches, animal models are shown to be extremely valuable tools for studying the induction or exacerbation of autoimmunity by environmental conditions and exposures.

Increasing evidence supports a role for the environment in the development of autoimmune diseases, as reviewed in the accompanying three papers from the National Institute of Environmental Health Sciences Expert Panel Workshop. An important unresolved issue, however, is the development of criteria for identifying autoimmune disease phenotypes for which the environment plays a causative role, herein referred to as environmentally associated autoimmune diseases. There are several different areas in which such criteria need to be developed, including: 1) identifying the necessary and sufficient data to define environmental risk factors for autoimmune diseases meeting current classification criteria; 2) establishing the existence of and criteria for new environmentally associated autoimmune disorders that do not meet current disease classification criteria; and 3) identifying in clinical practice specific environmental agents that induce autoimmune disease in individual patients. Here we discuss approaches that could be useful for developing criteria in these three areas, as well as factors that should be considered in evaluating the evidence for criteria that can distinguish individuals with such disorders from individuals without such disorders with high sensitivity and specificity. Current studies suggest that multiple lines of complementary evidence will be important and that in many cases there will be clinical, serologic, genetic, epigenetic, and/or other laboratory features that could be incorporated as criteria for environmentally associated autoimmune diseases to improve diagnosis and treatment and possibly allow for preventative strategies in the future.

Autoimmunity is thought to result from a combination of genetics, environmental triggers, and stochastic events. Gender is also a significant risk factor with many diseases exhibiting a female bias. Although the role of environmental triggers, especially medications, in eliciting autoimmunity is well established less is known about the interplay between gender, the environment and autoimmunity. This review examines the contribution of gender in autoimmunity induced by selected chemical, physical and biological agents in humans and animal models. Epidemiological studies reveal that environmental factors can be associated with a gender bias in human autoimmunity. However many studies show that the increased risk of autoimmunity is often influenced by occupational exposure or other gender biased activities. Animal studies, although often prejudiced by the exclusive use of female animals, reveal that gender bias can be strain specific suggesting an interaction between sex chromosome complement and background genes. This observation has important implications because it argues that within a gender biased disease there may be individuals in which gender does not contribute to autoimmunity. Exposure to environmental factors, which encompasses everything around us, adds an additional layer of complexity. Understanding how the environment influences the relationship between sex chromosome complement and innate and adaptive immune responses will be essential in determining the role of gender in environmentally-induced autoimmunity.

Summary Deficiency in Daf1, a complement regulatory protein, has been shown to exacerbate development of various autoimmune diseases and recent studies have suggested that this may be explained by Daf1 acting to limit T-cell hyper-responsiveness. It has been suggested that the absence of Daf1 aggravates autoimmune disease in a complement-dependent manner, but others have shown that activation of T cells in the absence of Daf1 can be complement independent. However, the relationship between Daf1, complement components, lymphocyte activation, cytokine expression and antibody production remains to be determined in mice that are not Daf1 deficient. We have recently demonstrated, in murine mercuryinduced autoimmunity (mHgIA), that an accumulation of CD44 high Daf low CD4 + T cells is associated with the development of autoimmunity. In this study we observed that complement depletion does not affect the accumulation of activated CD4 + T cells, elevation of splenic interleukin-4 expression and autoantibody production in mHgIA. In addition, neither the accumulation of CD44 high Daf low CD4 + T cells nor the down-regulation of Daf1 expression on CD4 + T cells was influenced by a lack of

Inhalation of particulate matter is associated with a number of acute and chronic disorders including autoimmune rheumatic diseases. The strongest evidence for a link with autoimmune disease comes from epidemiological studies describing the association of occupational exposure to crystalline silica dust with the systemic autoimmune diseases SLE and RA. Very little is known regarding the mechanism by which silica exposure leads to systemic autoimmune disease. However, in the case of silicosis, there is an extensive research literature that can help identify disease processes that may precede development of autoimmunity. The pathophysiology of silicosis consists of deposition of particles into the alveoli of the lung where they cannot be cleared. Ingestion of deposited particles by alveolar macrophages initiates an inflammatory response which then stimulates fibroblasts to proliferate and produce collagen. Silica particles are enveloped by collagen leading to fibrosis and nodular lesions. These findings are consistent with an autoimmune pathogenesis that begins with activation of the innate immune system leading to proinflammatory cytokine production, inflammation of the lung leading to activation of adaptive immunity, breaking of tolerance, autoantibodies, and tissue damage. The variable frequency of these features following silica exposure suggests significant genetic involvement and gene/environment interaction in silica-induced autoimmunity.

Objective Deficiency of decay-accelerating factor 1 (termed Daf1 in mice) has been shown to exacerbate autoimmunity, and recent studies have suggested that this may be explained by Daf1 acting as a regulator of T cell immunity. The aim of this study was to determine whether Daf1 expression on T cells is modulated during development of autoimmunity in mice. Methods To test this hypothesis, we examined Daf1 levels in NZB, DBA/2, and B10.S mice before and after induction of murine mercury-induced autoimmunity (mHgIA). Daf1 was measured by real-time polymerase chain reaction and flow cytometry, and levels of Daf1 were correlated with markers of lymphocyte activation and cytokine production. Results Autoimmune-prone NZB mice had low endogenous levels of Daf1 irrespective of the induction of mHgIA. Induction of autoimmunity reduced Daf1 expression in mHgIA-sensitive B10.S mice, particularly on activated/memory (CD44high) CD4+ T cells that accumulate as a result of exposure to mercury. Murine mercury-induced autoimmunity–resistant DBA/2 mice, which fail to accumulate CD44high T cells, showed no change in Daf1 expression. Modulation of Daf1 expression was found to require CD4+ T cell costimulation, since B10.S mice deficient in CD28 were unable to down-regulate Daf1 or accumulate activated/memory CD4+ T cells. In B10.S mice exposed to mercury, the production of interleukin-4 (IL-4), but not that of IL-2 or interferon-γ, in the spleen was associated with CD44high,Daf1low,CD4+ T cells. Conclusion These findings demonstrate that reduction of Daf1 expression is closely associated with CD4+ T cell activation and the accumulation of CD44high(activated/memory),CD4+ T cells in both spontaneous and induced systemic autoimmune disease.

The complement regulatory protein decay-accelerating factor (DAF or CD55) protects host tissue from complement-mediated injury by inhibiting the classical and alternative complement pathways. Besides its role in complement regulation, DAF has also been shown to be a key player in T cell immunity. Modulation of DAF expression could therefore represent a critical regulatory mechanism in both innate and adaptive immune responses. To identify and characterize key transcriptional regulatory elements controlling mouse Daf1 expression, a 2.5-kb fragment corresponding to the 5′ flanking region of the mouse Daf1 gene was cloned. Sequence analysis showed that the mouse Daf1 promoter lacks conventional TATA and CCAAT boxes and displays a high guanine and cytosine content. RACE was used to identify one major and two minor transcription start sites 47, 20, and 17 bp upstream of the translational codon. Positive and negative regulatory regions were identified by transiently transfecting sequential 5′deletion constructs of the 5′flanking region into NIH/3T3, M12.4, and RAW264.7 cells. Mutational analyses of the promoter region combined with Sp1-specific ELISA showed that the transcription factor Sp1 is required for basal transcription and LPS-induced expression of the Daf1 gene. These findings provide new information on the regulation of the mouse Daf1 promoter and will facilitate further studies on the expression of Daf1 during immune responses.

Heavy metals in the environment originate from both human activities and natural processes. Exposure to these metals can result in important changes to immune activity. Depending on the metal and dose, these changes can result in enhanced immune function, diminished immune responses, or altered responses that produce autoimmune disease. One of the intriguing aspects of these various phenomena are the multiple points of interaction with cellular machinery at which metals elicit these changes. The individual sections of this review serve to underscore the variety of targets that can be altered by exposure to heavy metals, and provide some comparisons between the effects of specific heavy metals on the immune system. These observations may ultimately lead us to a comprehensive understanding of the mechanisms by which metals alter the immune system, and may enable the development of countermeasures to offset these effects.

Systemic lupus erythematosus is a multigenic disorder of unknown etiology. To investigate the role of specific genes in lupus, we have examined the effects of single gene deletions on mercury-induced autoimmunity. Deficiency of certain genes abrogated induction of autoimmunity, while absence of others had little effect. The most interesting observations were obtained with genes related to interferon-gamma. Genes involved in upregulation of IFN-gamma expression did not significantly influence autoimmunity whereas absence of IFN-gamma or IFN-gamma receptor led to greatly reduced autoantibody responses and immunopathology. Absence of IRF-1, a gene expressed in response to IFN-gamma, resulted in selective retention of anti-chromatin autoantibodies demonstrating that specific defects in signaling pathways and gene expression subsequent to IFN-gamma/IFN-gamma receptor interaction influence specific disease parameters. These studies show that single gene deletions can have various outcomes ranging from no effect, suppression of one or more features of disease, to suppression of all features of disease, and that all three outcomes can be observed in the IFN-gamma pathway. IFN-gamma influences the expression and function of other lupus relevant genes such as IL-6 and beta2microglobulin, therefore the effects of these gene deletions on disease expression may also reflect responses downstream of IFN-gamma function.

Systemic lupus erythematosus is a multigenic disorder of unknown etiology. To investigate the roles that specific genes play in lupus, we have examined the disease profiles in mice with single-gene deletions. In total, some 17 genes have been studied. Absence of certain genes, such as CD40L, CD28, or Igh6, abrogated induction of autoimmunity. Other genes, such as Igh5, IL-4, or ICAM-1, had little effect on the development of disease. Intermediate effects were observed in IL-6-deficient mice, while absence of beta2-microglobulin resulted in loss of hypergammaglobulinemia and IgG1 autoantibodies, but produced little change in anti-chromatin antibodies or glomerular deposits. The most interesting observations were obtained with genes related to the expression or function of interferon-gamma (IFN-gamma). Reductions in IFN-gamma levels in murine lupus are associated with reductions in both autoantibody levels and immune-complex- mediated pathology. Genes involved in up-regulation of IFN-gamma expression, such as IL-12, STAT-4, or ICE, did not significantly influence autoimmunity, whereas absence of IFN-gamma or IFN-gamma receptor led to greatly reduced autoantibody response and immunopathology. Absence of IRF-1, a gene ex-pressed in response to IFN-gamma, resulted in selective retention of anti-chromatin antibodies but little glomerular pathology. These studies suggest that the presence of a baseline level of IFN-gamma, rather than increased expression, is important for autoimmunity. Furthermore, as the IRF-1 knockout demonstrates, specific defects in signaling pathways and gene expression subsequent to IFN-gamma/IFN-gamma receptor interaction may influence only certain disease parameters. It has not escaped our attention that IFN-gamma influences the expression and function of other immunologically relevant genes, such as IL-4, IL-6, and beta2-microglobulin. Thus, these genes may be part of the downstream events following IFN-gamma/IFN-gamma receptor interaction that promote the development of autoimmunity.

Our understanding of the effect of metals (including ions and their compounds) on the immune system continues to evolve. Observed effects include immunosuppression, immune stimulation, hypersensitivity, and autoimmunity. Many metals show a paradoxical dose-response pattern comprising stimulation of immune function at low doses and suppression at higher doses, but global immune function is often preserved due to the redundancy and the reserve capacity of the immune system, and clinically relevant effects are uncommon. Clinically relevant hypersensitivity reactions due to metals are dominated by T cell-mediated allergic contact dermatitis, particularly in response to exposure to beryllium, cobalt, chromium, gold, mercury, and nickel. Immediate (type I) hypersensitivity reactions dominated by airways symptoms occur infrequently, and then most often with platinum, but rarely with nickel or chromium. The induction of metal-induced autoimmunity, including the formation of immune-complex deposits, is well documented in humans, but the number of recognized cases is few. Studies in rodents using mercury and gold have increased our knowledge of the mechanisms of metal-induced autoimmunity. Of special importance is the unraveling of genetic factors that regulate susceptibility to mercury-induced autoimmunity, including the uptake and retention of mercury, as well as the threshold metal concentrations for eliciting autoimmunity. Recently mercury, lead, and cadmium have been shown to accelerate and/or exacerbate autoimmunity in autoimmune-prone animal models. The importance of metal exposure for inducing and/or accelerating autoimmunity in humans remains to be determined.

Objective. Proteins that are phosphorylated during apoptosis are commonly precipitated by autoantibodies found in the sera of patients with systemic lupus erythematosus. We sought to determine whether scleroderma autoantigens such as small nucleolar RNPs (snoRNP) also associate with phosphoproteins in response to various cellular stressors. Methods. We screened a panel of monoclonal antibodies derived from mice exposed to mercury, a well-characterized murine model of the anti-snoRNP autoimmune response, for the ability to selectively precipitate phosphoproteins from radiolabeled lysates prepared from Jurkat T cells subjected to stressful stimuli. Results. Monoclonal antibodies reactive with snoRNPs precipitated a phosphoprotein complex (pp42, pp34, and pp23) from lysates prepared from apoptotic cells. Several novel phosphoproteins (pp62 and pp18) were also observed. The phosphorylation and/or recruitment of these proteins to the snoRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, anisomycin, or ultraviolet irradiation), an effect that is blocked by overexpression of Bcl-2. We were unable to demonstrate an association of the phosphoprotein complex with snoRNPs in cells treated with the xenobiotic agent mercury. The snoRNP-associated phosphoprotein complex is composed of serine/arginine (SR) splicing factors, including SRp40. Conclusion. The association of phosphorylated SR proteins with snoRNPs in cells undergoing apoptosis suggests that the immune response to fibrillarin that characterizes a subset of patients with scleroderma may be related to cell death induced by apoptotic stimuli (e.g., Fas ligation, irradiation, or chemical toxins), or by exposure to mercury.

Imbalances of Th1- and Th2-type responses have been postulated to be a predisposing factor for both humoral and cellular mediated autoimmune diseases. To further define their roles in systemic autoimmunity, IL-4 and IFN-γ gene knockout mice were studied for susceptibility to the prototypic Th2-mediated mercury-induced autoimmunity. A predominant Th2-type response following HgCl2 treatment of wild-type B10.S mice was confirmed by the findings of a significant increase in splenic IL-4 and hypergammaglobulinemia primarily of the IgG1 isotype, without an increase in IFN-γ levels. Paradoxically, IL-4-deficient mice developed the characteristic anti-nucleolar autoantibodies and tissue deposition of immune complexes, while IFN-γ-deficient mice had very low autoantibody levels and essentially normal immunohistology. Studies to define defects in Ab responses of IFN-γ-deficient mice, using the T-dependent Ag (4-hydroxy-3-nitrophenyl)acetyl, revealed an attenuated IgG response to low and to a lesser extent high doses of (4-hydroxy-3-nitrophenyl)acetyl-hemocyanin, but maintenance of affinity maturation. These results indicate that Th1/Th2 imbalance does not directly play a role in susceptibility to mercury-induced autoimmunity, and suggest that the dependence on Th1-type responses in certain autoimmune diseases is due to the requirement for IFN-γ for Ab production to weakly antigenic self molecules.

Decay accelerating factor (DAF), a complement-regulatory protein, protects cells from bystander complement-mediated lysis and negatively regulates T cells. Reduced expression of DAF occurs in several systemic autoimmune diseases including systemic lupus erythematosus, and DAF deficiency exacerbates disease in several autoimmune models, including murine mercury-induced autoimmunity (mHgIA).Daf1, located withinHmr1, a chromosome 1 locus associated in DBA/2 mice with resistance to mHgIA, could be a candidate. Here we show that reducedDaf1transcription in lupus-prone mice was not associated with a reduction in theDaf1transcription factor SP1. Studies of NZB mice congenic for the mHgIA-resistant DBA/2Hmr1locus suggested thatDaf1expression was controlled by the host genome and not theHmr1locus. A unique pentanucleotide repeat variant in the second intron ofDaf1in DBA/2 mice was identified and shown in F2 intercrosses to be associated with less severe disease; however, analysis ofHmr1congenics indicated that this most likely reflected the presence of autoimmunity-predisposing genetic variants within theHmr1locus or thatDaf1expression is mediated by the tandem repeat in epistasis with other genetic variants present in autoimmune-prone mice. These studies argue that the effect of DAF on autoimmunity is complex and may require multiple genetic elements.

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 Unc93b13d 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.

IFN-γ is essential for idiopathic and murine mercury-induced systemic autoimmunity (mHgIA), and heterozygous IFN-γ +/− mice also exhibit reduced disease. This suggests that blocking specific IFN-γ-related pathways that may only partially inhibit IFN-γ production or function will also suppress autoimmunity. To test this hypothesis, mice deficient in genes regulating IFN-γ expression ( Casp1 , Nlrp3 , Il12a , Il12b , Stat4 ) or function ( Ifngr1 , Irf1 ) were examined for mHgIA susceptibility. Absence of either Ifngr1 or Irf1 resulted in a striking reduction of disease, while deficiency of genes promoting IFN-γ expression had modest to no effect. Furthermore, both Irf1 − and Ifng -deficiency only modestly reduced the expansion of CD44 hi and CD44 hi CD55 lo CD4 + T cells, indicating that they are not absolutely required for T cell activation. Thus, there is substantial redundancy in genes that regulate IFN-γ expression in contrast to those that mediate later signaling events. These findings have implications for the therapeutic targeting of IFN-γ pathways in systemic autoimmunity.

The lupus-prone New Zealand Black (NZB) strain uniquely develops a genetically imposed severe spontaneous autoimmune hemolytic anemia (AIHA) that is very similar to the corresponding human disease. Previous studies have mapped anti-erythrocyte Ab (AEA)-promoting NZB loci to several chromosomal locations, including chromosome 4; however, none of these have been analyzed with interval congenics. In this study, we used NZB.NZW-Lbw2 congenic (designated Lbw2 congenic) mice containing an introgressed fragment of New Zealand White (NZW) on chromosome 4 encompassing Lbw2, a locus previously linked to survival, glomerulonephritis, and splenomegaly, to investigate its role in AIHA. Lbw2 congenic mice exhibited marked reductions in AEAs and splenomegaly but not in anti-nuclear Abs. Furthermore, Lbw2 congenics had greater numbers of marginal zone B cells and reduced expansion of peritoneal cells, particularly the B-1a cell subset at early ages, but no reduction in B cell response to LPS. Analysis of a panel of subinterval congenic mice showed that the full effect of Lbw2 on AEA production was dependent on three subloci, with splenomegaly mapping to two of the subloci and expansions of peritoneal cell populations, including B-1a cells to one. These results directly demonstrated the presence of AEA-specific promoting genes on NZB chromosome 4, documented a marked influence of background genes on autoimmune phenotypes related to Lbw2, and further refined the locations of the underlying genetic variants. Delineation of the Lbw2 genes should yield new insights into both the pathogenesis of AIHA and the nature of epistatic interactions of lupus-modifying genetic variants.

Deficiency in Daf1, a complement regulatory protein, has been shown to exacerbate development of various autoimmune diseases and recent studies have suggested that this may be explained by Daf1 acting to limit T-cell hyper-responsiveness. It has been suggested that the absence of Daf1 aggravates autoimmune disease in a complement-dependent manner, but others have shown that activation of T cells in the absence of Daf1 can be complement independent. However, the relationship between Daf1, complement components, lymphocyte activation, cytokine expression and antibody production remains to be determined in mice that are not Daf1 deficient. We have recently demonstrated, in murine mercury-induced autoimmunity (mHgIA), that an accumulation of CD44(high) Daf(low) CD4(+) T cells is associated with the development of autoimmunity. In this study we observed that complement depletion does not affect the accumulation of activated CD4(+) T cells, elevation of splenic interleukin-4 expression and autoantibody production in mHgIA. In addition, neither the accumulation of CD44(high) Daf(low) CD4(+) T cells nor the down-regulation of Daf1 expression on CD4(+) T cells was influenced by a lack of complement. In conclusion, these studies show that initiating events in xenobiotic-induced autoimmunity, including lymphocyte activation, cytokine expression and autoantibody production, are not dependent on complement.

Susceptibility to most autoimmune diseases is dependent on polygenic inheritance, environmental factors, and poorly defined stochastic events. One of the significant challenges facing autoimmune disease research is in identifying the specific events that trigger loss of tolerance and autoimmunity. Although many intrinsic factors, including age, sex, and genetics, contribute to autoimmunity, extrinsic factors such as drugs, chemicals, microbes, or other environmental factors can also act as important initiators. This review explores how certain extrinsic factors, namely, drugs and chemicals, can promote the development of autoimmunity, focusing on a few better characterized agents that, in most instances, have been shown to produce autoimmune manifestations in human populations. Mechanisms of autoimmune disease induction are discussed in terms of research obtained using specific animal models. Although a number of different pathways have been delineated for drug/chemical-induced autoimmunity, some similarities do exist, and a working model is proposed.

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.

Currently, studies have shown that genetic predisposition accounts for approximately thirty percent of all autoimmune diseases. The rest, 70 percent, are due to environmental factors, including toxic chemicals, dietary components, gut dysbiosis, and infections (Figure 1). Figure 1 Factors that contribute to autoimmune disease. Autoimmune disorder symptoms are initially nonspecific and include malaise and fatigue, low-grade fevers, aches, and pains. Due to this vagueness, patients are frequently diagnosed with an autoimmune disease after they become weak and unable to function normally, making the onset of the disease difficult to pinpoint and the possible triggers uncertain. In this issue, we present a series of papers that review, discuss, and elaborate on various environmental triggers of autoimmunity. A. Vojdani presents an extensive review of potential triggers of autoimmunity. The author discusses the loss of immune homeostasis and explains the mechanism of autoimmunity as related to infectious triggers by molecular mimicry, epitope spreading, and bystander activation. He describes the effects of dietary components, focusing particularly on recent studies with sodium chloride to explain the effects of this commonly used mineral on the immune system, in particular TH17, leading to an increased risk of autoimmunity. Milk and wheat components are reviewed, as are gluten sensitivity, celiac disease, and oral pathogens in their role in the induction of autoimmune diseases. Over the last few years, studies have amplified our previous knowledge of the gut and demonstrated its wide-ranging importance and its potentials for triggering autoimmunity when dysbiosis occurs as a result of environmental factors (Figure 2). A. W. Campbell's review brings up essential facts about some of these environmental factors affecting not only the gut but also the mucosal immunity and describes gut microbiota links to autoimmune diseases. The author discusses the importance of early detection of autoimmunity via antibody testing to bring about a better outcome for patients by removing offending triggers. Figure 2 Mechanism for the induction of autoimmunity and neuroautoimmunity by environmental triggers. A very interesting research article is presented in this issue by I. Burazor and A. Vojdani, discussing the strong link between poor dental health and cardiovascular disease due to several bacteria and then studying the potential association between these pathogens, the antibodies produced against them, and elevation of markers for inflammation in patients with acute myocardial atherothrombosis (AMA). Decay-accelerating factor 1 (DAF1) or CD55 is a 70 KDa member of proteins which regulates complement system on the cell surfaces and protects cells from complement attack. In their review, C. B. Toomey et al. discussed the relationship between DAF1 and the complement system in the regulation of environmentally induced autoimmunity. They propose a hypothesis to explain how DAF expression may impact T cell differentiation via interaction with CD97 leading to T regulatory cells, increased production of IL-10, and immune tolerance. Further understanding of this novel mechanism by which DAF can regulate mercury-induced autoimmunity may lead to new strategies for regulation of DAF in various autoimmunities induced by environmental toxicants. In a research study, D. M. Cauvi et al. show that the effect of DAF on autoimmunity is complex and may require multiple genetic elements such as a tandem repeat sequence (CTTTT)n or (TTTTC)n. This association between the absence of tandem repeats and the severity of autoimmunity may be due to linkage of tandem repeats with other predisposing variants that promote DAF1 expression. Bisphenol A (BPA) may be a potential link to autoimmune diseases. It is ubiquitous in consumer products: more than 90% of Americans were found to have detectable levels of BPA in their urine. Studies have shown that BPA is an endocrine disruptor that can affect perinatal, childhood, and adult health. D. Kharrazian's review of BPA includes a general assessment of this highly prevalent chemical in our environment. He then describes eleven different pathophysiological and immunological mechanisms where BPA exposure may lead to autoimmunity. In their article, J. Ong et al. sought to clarify the role of Hg through fish consumption and its relationship to increased autoimmune disease via testing for ANA and specific autoantibodies in blood in the Cheyenne River Sioux Tribe lands (CRST) community. The interactions of gender with blood Hg and arsenic proximity were significant, suggesting that complex interactions underlie autoimmunity. A central issue in immunology is how, at different developmental stages, the fate of B-lymphocytes is determined and how B cell receptors (BCR) distinguish between signals that induce immune response versus immune tolerance. The alteration in BCR signaling by low levels of exposure to mercury for the pathogenesis of autoimmune disease is discussed by R. F. Gill et al. Their report showed that Hg2+ has little upstream effects on BHC tyrosine kinase, but SYK tyrosine kinase and B cell scaffolding protein BLNK are augmented by low levels of mercury, suggesting that low levels of mercury may interfere with central tolerance and may be a mechanism connecting mercury intoxication to autoimmune disease. J. C. Pfau et al. review the link between asbestos exposure and autoimmunity. The authors review rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus, among others, and their association with asbestos and give a review of their hypotheses regarding the discordant and inconsistent results. They also discuss the most compelling evidence for a link between asbestos exposure and autoimmunity. Trichloroethylene (TCE) is an industrial solvent known for being neurotoxic, hepatotoxic, nephrotoxic, and immunotoxic. It is also carcinogenic. To give insight into how TCE may cause possible immune related issues, K. M. Gilbert et al. provide us with a very interesting study on exposure to TCE in autoimmune prone female mice exposed during gestation or early life. The exposures were lower than acceptable human occupational exposures, yet they still resulted in changes in peripheral CD4+ T cell in those mice exposed in early life. The review article by H. A. N. El-Fawal describes the challenges and the need for neuroantibody biomarkers in neurodegenerative diseases (ND). There is a very interesting discussion of the neurotoxicity of nanoparticles (NPs). In support of body burden, the author explains that immune response to an exposure may not adhere to the old dogma of dose response and that environmental agents may affect multiple organ systems, including the brain. The rapid rise of autoimmune disease (AD) globally has led some to label the situation as epidemic. We have presented in this special issue a sampling of the many possible environmental triggers of AD, but we hope that the readers of Autoimmune Diseases will also take away from this collection one very important fact: detection of predictive biomarkers in the early stages of autoimmune disorders can be used to identify, halt, and even reverse autoimmune disease. We sound the warning bell, but we also offer readers the hope of a solution. Aristo Vojdani K. Michael Pollard Andrew W. Campbell

Coumestrol is a naturally occurring plant estrogen. As estrogen influences cellular and humoral immunity, and has known effects on murine models of lupus, we investigated the effect of coumestrol on disease expression in the NZB/W F1 mouse. Female NZB/W F1 mice were fed a “standard” rodent diet including soy proteins, a non-soy diet, or a non-soy diet with 0.01% coumestrol. Outcome measures included survival, autoantibody expression, immunoglobulin levels, proteinuria, renal histology and B cell immunohistochemistry, and renal mRNA expression. At 24 weeks, the treatment group had decreased prevalence of autoantibodies detected by immunofluorescence and less splenomegaly. At 39 weeks, the prevalence of autoantibodies was similar but the treatment group had less proteinuria. Overall, there was little effect of treatment on renal mRNA levels as assessed by gene array analysis, but functional ontology mapping revealed that genes encoding proteins involved in the immune response were most often affected. These results suggest that treatment with coumestrol may ameliorate some aspects of disease progression in this model of systemic autoimmunity.

Objective To determine whether antifibrillarin autoantibodies in scleroderma patients are associated with autoantibodies to protein components specific for U3 small nucleolar RNP (U3 snoRNP). Methods Sera from 220 scleroderma patients were examined for antinucleolar autoantibodies (ANoA) and for antibodies to fibrillarin and the U3 snoRNP–specific proteins Mpp10 and hU3-55K. Clinical correlates were determined for the different autoantibody specificities. Results Fifty-nine of the 220 patients were positive for ANoA, and 31 of these patients were antifibrillarin positive. Anti–hU3-55K was found in 10 patients, all of whom were antifibrillarin positive. Twenty-nine patients had anti-Mpp10 antibodies; 23 of these were antifibrillarin positive and 6 were antifibrillarin negative. ANoA, including antifibrillarin, anti–hU3-55K, and anti-Mpp10, were associated with diffuse, rather than limited, systemic or localized scleroderma. Esophageal and lung involvement were more common in patients with antifibrillarin and anti-Mpp10 antibodies, and the highest frequency was in patients with anti-Mpp10 alone. Conclusion Antifibrillarin autoantibodies are associated with autoantibodies to protein components specific to U3 snoRNP, particularly Mpp10. The prevalence of anti-Mpp10 antibodies in antifibrillarin-positive patients suggests that the U3 snoRNP particle is a source of immunogenic/antigenic material for the anti-snoRNP response in scleroderma. Autoantibodies to snoRNP components were more frequent in patients with diffuse scleroderma than in those with either the limited systemic or localized forms. The increased expression of these antibodies in patients with the more severe form of scleroderma, coupled with the observations that fibrillarin expression is positively linked to collagen expression in fibroblasts and that fibrillarin is overexpressed in scleroderma fibroblasts, suggests a source of snoRNP to initiate and maintain these autoantibody responses.

Fibrillarin is a conserved nucleolar SnoRNP with a diverse N-terminal glycine- and arginine-rich (GAR) domain in most eukaryotes. This region in human fibrillarin is known to contain modified dimethylarginines. In this report we demonstrate that recombinant murine fibrillarin is a substrate for protein arginine methyltransferase, including the purified recombinant enzyme (rat PRMT1 and yeast RMT1) and the protein methyltransferases present in lymphoblastoid cell extracts. Our results of protease digestion, methylation competition reactions, and immunoblotting with a methylarginine-specific antibody all indicate that the methylation of fibrillarin is in the N-terminal GAR domain and arginyl residues are modified. Finally, amino acid analyses revealed that the modification of recombinant murine fibrillarin forms methylarginines, mostly as dimethylarginines.