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Women are more likely to develop autoimmune diseases than men. Contribution from microchimerism (Mc) has been proposed, as women naturally acquire Mc from more sources than men because of pregnancy. Women with Rheumatoid Arthritis (RA) who lack RA-associated HLA alleles have been found to harbor Mc with RA-associated HLA alleles in higher amounts than healthy women in prior work. However, an immunological impact of Mc remains to be elucidated. To test the hypothesis that Mc with RA-risk associated HLA alleles can result in the production of RA-associated autoantibodies, when host genetic risk is absent. DBA/2 mice are unable to produce RA-specific anti-citrullinated autoantibodies (ACPAs) after immunization with the enzyme peptidyl arginine deiminase (PAD) in a previously developed model. DBA/2 females were mated with C57BL/6 males humanized to express HLA-DR4, which is associated with RA-risk and production of ACPAs, to evaluate DR4+ fetal Mc contribution. Next, DBA/2 females born of heterozygous DR4+/− mothers were evaluated for DR4+ Mc of maternal or littermate origin. Finally, DBA/2 females from DR4+/− mothers were crossed with DR4+ males, to evaluate the contribution of any Mc source to ACPA production. After PAD immunization, between 20 % and 43 % of DBA/2 females (otherwise unable to produce ACPAs) had detectable ACPAs (CCP2 kit) after exposure to sources of Mc with RA-associated HLA alleles, compared to 0 % of unmated/unexposed DBA/2 females. Further the microchimeric origin of the autoantibodies was confirmed by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response. Our study demonstrates that Mc cells can produce "autoantibodies" and points to a role of Mc in the biology of autoimmune diseases, including RA. • DBA/2 mice "unable" to produce RA-specific autoantibodies in a previously described model, manage to do it when exposed to maternal, littermate and fetal microchimerism from humanized C57BL/6 mice carrying RA susceptibility genes. • The microchimeric origin of the autoantibodies was confirmed by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response. • Our results show that microchimeric cells can produce RA-specific autoantibodies. [ABSTRACT FROM AUTHOR]

Fibrosis remains a serious health concern in patients with chronic liver disease. We recently reported that chemically induced chronic murine liver injury triggers increased expression of junctional adhesion molecules (JAMs) JAM-B and JAM-C by endothelial cells and de novo synthesis of JAM-C by hepatic stellate cells (HSCs). Here, we demonstrate that biopsies of patients suffering from primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) or autoimmune hepatitis (AIH) display elevated levels of JAM-C on portal fibroblasts (PFs), HSCs, endothelial cells and cholangiocytes, whereas smooth muscle cells expressed JAM-C constitutively. Therefore, localization and function of JAM-B and JAM-C were investigated in three mouse models of autoimmune-driven liver inflammation. A PBC-like disease was induced by immunization with 2-octynoic acid-BSA conjugate, which resulted in the upregulation of both JAMs in fibrotic portal triads. Analysis of a murine model of PSC revealed a role of JAM-C in PF cell-cell adhesion and contractility. In mice suffering from AIH, endothelial cells increased JAM-B level and HSCs and capsular fibroblasts became JAM-C-positive. Most importantly, AIH-mediated liver fibrosis was reduced in JAM-B −/− mice or when JAM-C was blocked by soluble recombinant JAM-C. Interestingly, loss of JAM-B/JAM-C function had no effect on leukocyte infiltration, suggesting that the well-documented function of JAMs in leukocyte recruitment to inflamed tissue was not effective in the tested chronic models. This might be different in patients and may even be complicated by the fact that human leukocytes express JAM-C. Our findings delineate JAM-C as a mediator of myofibroblast-operated contraction of the liver capsule, intrahepatic vasoconstriction and bile duct stricture. Due to its potential to interact heterophilically with endothelial JAM-B, JAM-C supports also HSC/PF mural cell function. Together, these properties allow JAM-B and JAM-C to actively participate in vascular remodeling associated with liver/biliary fibrosis and suggest them as valuable targets for anti-fibrosis therapies. [ABSTRACT FROM AUTHOR]

Dendritic cell (DC)-mediated T cell tolerance deficiencies contribute to the pathogenesis of autoimmune diseases such as type 1 diabetes. Delivering self-antigen to dendritic-cell inhibitory receptor-2 (DCIR2) + DCs can delay but not completely block diabetes development in NOD mice. These DCIR2-targeting antibodies induce tolerance via deletion and anergy, but do not increase islet-specific Tregs. Because low-dose IL-2 (LD-IL-2) administration can preferentially expand Tregs, we tested whether delivering islet-antigen to tolerogenic DCIR2 + DCs along with LD-IL-2 would boost islet-specific Tregs and further block autoimmunity. But, surprisingly, adding LD-IL-2 did not increase efficacy of DC-targeted antigen to inhibit diabetes. Here we show the effects of LD-IL-2, with or without antigen delivery to DCIR2 + DCs, on both polyclonal and autoreactive Treg and conventional T cells (Tconv). As expected, LD-IL-2 increased total Tregs, but autoreactive Tregs required both antigen and IL-2 stimulation for optimal expansion. Also, islet-specific Tregs had lower CD25 expression and IL-2 sensitivity, while islet-specific Tconv had higher CD25 expression, compared to polyclonal populations. LD-IL-2 increased activation and expansion of Tconv, and was more pronounced for autoreactive cells after treatment with IL-2 + islet-antigen. Therefore, LD-IL-2 therapy, especially when combined with antigen stimulation, may not optimally activate and expand antigen-specific Tregs in chronic autoimmune settings. [ABSTRACT FROM AUTHOR]

Objective CD6 is emerging as a new target for treating many pathological conditions in which T cells are integrally involved, but even the latest data from studies of CD6 gene engineered mice were still contradictory. To address this issue, we studied experimental autoimmune uveitis (EAU), a model of autoimmune uveitis, in wild-type (WT) and CD6 knockout (KO) mice. Methods After EAU induction in WT and CD6 KO mice, we evaluated ocular inflammation and compared retinal antigen-specific T-cell responses using scanning laser ophthalmoscopy, spectral-domain optical coherence tomography, histopathology, and T cell recall assays. Uveitogenic T cells from WT and CD6 KO mice were adoptively transferred into WT naïve mice to confirm the impact of CD6 on T cells. In addition, we immunized CD6 KO mice with recombinant CD6 protein to develop mouse anti-mouse CD6 monoclonal antibodies (mAbs) in which functional antibodies exhibiting cross-reactivity with human CD6 were screened and identified for treatment studies. Results In CD6 KO mice with EAU, we found significantly decreased retinal inflammation and reduced autoreactive T-cell responses, and confirmed the impaired uveitogenic capacity of T cells from these mice in an adoptive transfer experiment. Notably, one of these cross-reactive mAbs significantly ameliorated retinal inflammation in EAU induced by the adoptive transfer of uveitogenic T cells. Conclusions Together, these data strongly suggest that CD6 plays a previously unknown, but pivotal role in autoimmune uveitis, and may be a promising new treatment target for this blinding disease. In addition, the newly developed mouse anti-mouse/human CD6 mAbs could be valuable tools for testing CD6-targeted therapies in other mouse models of human diseases. [ABSTRACT FROM AUTHOR]

The ultraviolet (UV) radiation contained in sunlight is a powerful immune suppressant. While exposure to UV is associated with protection from the development of autoimmune diseases, particularly multiple sclerosis, the precise mechanism by which UV achieves this protection is not currently well understood. Regulatory B cells play an important role in preventing autoimmunity and activation of B cells is a major way in which UV suppresses adaptive immune responses. Whether UV-protection from autoimmunity is mediated by the activation of regulatory B cells has never been considered before. When C57BL/6 mice were exposed to low, physiologically relevant doses of UV, a unique population of B cells was activated in the skin draining lymph nodes. As determined by flow cytometry, CD1d low CD5 – MHC-II hi B220 hi UV-activated B cells expressed significantly higher levels of CD19, CD21/35, CD25, CD210 and CD268 as well as the co-stimulatory molecules CD80, CD86, CD274 and CD275. Experimental autoimmune encephalomyelitis (EAE) in mice immunized with MOG/CFA was reduced by exposure to UV. UV significantly inhibited demyelination and infiltration of inflammatory cells into the spinal cord. Consequently, UV-exposed groups showed elevated IL-10 levels in secondary lymphoid organs, delayed EAE onset, reduced peak EAE score and significantly suppressed overall disease incidence and burden. Importantly, protection from EAE could be adoptively transferred using B cells isolated from UV-exposed, but not unirradiated hosts. Indeed, UV-protection from EAE was dependent on UV activation of lymph node B cells because UV could not protect mice from EAE who were pharmacologically depleted of B cells using antibodies. Thus, UV maintenance of a pool of unique regulatory B cells in peripheral lymph nodes appears to be essential to prevent an autoimmune attack on the central nervous system. [ABSTRACT FROM AUTHOR]

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. The successes of the Non-Obese Diabetic (NOD) mouse model have come in multiple forms including identifying key genetic and environmental risk factors e.g. Idd loci and effects of microorganisms including the gut microbiota, respectively, and how they may contribute to disease susceptibility and pathogenesis. Furthermore, the NOD model also provides insights into the roles of the innate immune cells as well as the B cells in contributing to the T cell-mediated disease. Unlike many autoimmune disease models, the NOD mouse develops spontaneous disease and has many similarities to human T1D. Through exploiting these similarities many targets have been identified for immune-intervention strategies. Although many of these immunotherapies did not have a significant impact on human T1D, they have been shown to be effective in the NOD mouse in early stage disease, which is not equivalent to trials in newly-diagnosed patients with diabetes. However, the continued development of humanized NOD mice would enable further clinical developments, bringing T1D research to a new translational level. Therefore, it is the aim of this review to discuss the importance of the NOD model in identifying the roles of the innate immune system and the interaction with the gut microbiota in modifying diabetes susceptibility. In addition, the role of the B cells will also be discussed with new insights gained through B cell depletion experiments and the impact on translational developments. Finally, this review will also discuss the future of the NOD mouse and the development of humanized NOD mice, providing novel insights into human T1D. [ABSTRACT FROM AUTHOR]

In areas where helminths infections are common, autoimmune diseases are rare. Treatment with helminths and ova from helminths, improved clinical findings of inflammatory bowel disease, multiple-sclerosis and rheumatoid-arthritis. The immunomodulatory functions of some helminths were attributed to the phosphorylcholine (PC) moiety. We aimed to decipher the tolerogenic potential of Tuftsin-PC (TPC) compound in mice genetically prone to develop lupus. Lupus prone NZBXW/F1 mice received subcutaneously TPC (5 μg/1 ml), 3 times a week starting at 14 weeks age. Autoantibodies were tested by ELISA, T-regulatory-cells by FACS, cytokines profile by RT-PCR and cytokines protein levels by DuoSet ELISA. Glomerulonephritis was addressed by detection of proteinuria, and immunoglobulin complex deposition in the mesangium of the kidneys of the mice by immunofluorescence. Our results show that TPC attenuated the development of glomerulonephritis in lupus prone mice, in particular, it ameliorated proteinuria (p < 0.02), and reduced immunoglobulin deposition in the kidney mesangium. TPC also enhanced the expression of TGFβ and IL-10 (p < 0.001), and inhibited the production of IFNγ and IL-17 (p < 0.03). TPC Significantly enhanced the expansion of CD4+CD25+FOXP3+ T-regulatory cells (Tregs) phenotype in the treated mice. These data indicate that TPC hampered lupus development in genetically lupus prone mice which was exemplified by moderate glomerulonephritis, attenuation of pro-inflammatory cytokines and enhancement of anti-inflammatory cytokines expression, as well as Tregs expansion. Our results propose harnessing novel natural therapy for lupus patients. [ABSTRACT FROM AUTHOR]

Kv1.3 channels regulate the activation/proliferation of effector memory T cells and thus play a critical role in the pathogenesis of autoimmune diseases. Using a combination of immunohistochemistry, confocal microscopy, flow cytometry and electrophysiology methods we observed a significant enrichment of activated Kv1.3 + memory T cells in psoriasis plaques and synovial fluid from patients with psoriasis/psoriatic arthritis (PsA) compared to non-lesional psoriatic skin, normal skin or peripheral blood lympho-mononuclear cells. In in vitro studies performed with lesional mononuclear cells or T cells derived from skin and joints of psoriatic disease, the small molecule Kv1.3 blocker PAP-1 dose-dependently inhibited proliferation and suppressed IL-2 and IFN-γ production. To further substantiate the pathologic role of Kv1.3 high T EM cells in psoriatic disease we tested whether PAP-1 is able to improve psoriatic disease pathology in the SCID mouse-psoriasis skin xenograft model. Following four weeks of daily treatment with 2% PAP-1 ointment we noticed about 50% reduction in the epidermal thickness (rete peg length) and the number of CD3 + lymphocytes/mm 2 of dermis decreased by 85%. Vehicle treated and untreated plaques in contrast remained unchanged and showed no reduction in epidermis thickness and infiltrating CD3 + T cells and HLA-DR + T cells. Based on these results we propose the development of Kv1.3 targeted topical immunotherapy for psoriasis and possibly for other inflammatory skin conditions, where effector memory T cells are involved in the pathogenesis. [ABSTRACT FROM AUTHOR]

Hepatitis-B vaccine (HBVv) can prevent HBV-infection and associated liver diseases. However, concerns regarding its safety, particularly among patients with autoimmune diseases (i.e. SLE) were raised. Moreover, the aluminum adjuvant in HBVv was related to immune mediated adverse events. Therefore, we examined the effects of immunization with HBVv or alum on SLE-like disease in a murine model. NZBWF1 mice were immunized with HBVv (Engerix), or aluminum hydroxide (alum) or phosphate buffered saline (PBS) at 8 and 12 weeks of age. Mice were followed for weight, autoantibodies titers, blood counts, proteinuria, kidney histology, neurocognitive functions (novel object recognition, staircase, Y-maze and the forced swimming tests) and brain histology. Immunization with HBVv induced acceleration of kidney disease manifested by high anti-dsDNA antibodies ( p < 0.01), early onset of proteinuria ( p < 0.05), histological damage and deposition of HBs antigen in the kidney. Mice immunized with HBVv and/or alum had decreased cells counts mainly of the red cell lineage ( p < 0.001), memory deficits ( p < 0.01), and increased activated microglia in different areas of the brain compare with mice immunized with PBS. Anxiety-like behavior was more pronounced among mice immunized with alum. In conclusion, herein we report that immunization with the HBVv aggravated kidney disease in an animal model of SLE. Immunization with either HBVv or alum affected blood counts, neurocognitive functions and brain gliosis. Our data support the concept that different component of vaccines may be linked with immune and autoimmune mediated adverse events. [ABSTRACT FROM AUTHOR]

A strong gender bias is seen in many autoimmune diseases including systemic lupus erythematosus (SLE). To investigate the basis for the female preponderance in SLE, we have been studying BALB/c mice in which B cells express the R4A heavy chain of an anti-DNA antibody in association with an endogenous light chain repertoire (R4Atg mice). In unmanipulated mice, approximately 5% of B cells express the R4A transgene. R4Atg mice do not spontaneously develop elevated serum titers of anti-DNA antibodies. Administration of either estradiol (E2) or prolactin (Pr) results in escape from tolerance of autoreactive B cells, expressed as an increase in transgene-expressing B cells and elevated serum titers of anti-DNA antibodies. We previously demonstrated that autoreactive B cells maturing in an estrogenic milieu develop as marginal zone (MZ) B cells; when these same B cells mature in the presence of increased prolactin, they develop as follicular (Fo) B cells. To determine the long term consequence of this differential maturation of DNA-reactive B cells, we treated R4Atg BALB/c mice with E2 or Pr for 6 weeks until serum titers of anti-DNA antibody were high, at which time hormonal exposure was discontinued. In E2-treated mice, the anti-DNA titers remained high even 3 months after discontinuation of hormone exposure. Nascent B cells underwent normal tolerance induction, but existing autoreactive MZ B cells persisted and continued to secrete autoantibody. In contrast, Pr caused only a short-term increase in anti-DNA antibody titers. By 3 months after cessation of hormone treatment, serum anti-DNA antibody titers and B cell subsets were indistinguishable from those in placebo (P) treated mice. These findings suggest that autoantibody responses are sustained for variable lengths of time depending on the B cell subset producing the autoantibodies. This observation may be relevant to understanding the heterogeneous presentation of patients with SLE and to the design of therapies targeting specific B-cell populations in autoimmune disease. [ABSTRACT FROM AUTHOR]

Abstract: Lupus is a systemic autoimmune disease characterized by anti-nuclear antibodies in humans and genetically susceptible NZB/W mice that can cause immune complex glomerulonephritis. T cells contribute to lupus pathogenesis by secreting pro-inflammatory cytokines such as IL-17, and by interacting with B cells and secreting helper factors such as IL-21 that promote production of IgG autoantibodies. In the current study, we determined whether purified NKT cells or far more numerous conventional non-NKT cells in the spleen of NZB/W female mice secrete IL-17 and/or IL-21 after TCR activation in vitro, and provide help for spontaneous IgG autoantibody production by purified splenic CD19+ B cells. Whereas invariant NKT cells secreted large amounts of IL-17 and IL-21, and helped B cells, non-NKT cells did not. The subset of IL-17 secreting NZB/W NKT cells expressed the Ly108loCD4−NK1.1− phenotype, whereas the IL-21 secreting subset expressed the Ly108hiCD4+NK1.1− phenotype and helped B cells secrete a variety of IgG anti-nuclear antibodies. α-galactocylceramide enhanced the helper activity of NZB/W and B6.Sle1b NKT cells for IgG autoantibody secretion by syngeneic B cells. In conclusion, different subsets of iNKT cells from mice with genetic susceptibility to lupus can contribute to pathogenesis by secreting pro-inflammatory cytokines and helping autoantibody production. [Copyright &y& Elsevier]

Abstract: Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. The early immunological events leading to PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-β receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFβRII CD8 T cells transfer biliary disease. Herein we report significantly increased numbers of hepatic dnTGFβRII terminally differentiated (KLRG1+) CD8 T cells, a CD8 subset previously shown to be enriched in antigen specific cells during hepatic immune response to viral infections. We performed bone marrow chimera studies to assess whether dnTGFβRII CD8 mediated disease was cell intrinsic or extrinsic. Unexpectedly, mixed (dnTGFβRII and B6) bone marrow chimeric (BMC) mice were protected from biliary disease compared to dnTGFβRII single bone marrow chimerics. To define the protective B6 cell subset, we performed adoptive transfer studies, which showed that co-transfer of B6 Tregs prevented dnTGFβRII CD8 T cell mediated cholangitis. Treg mediated disease protection was associated with significantly decreased numbers of hepatic KLRG1+ CD8 T cells. In contrast, co-transfer of dnTGFβRII Tregs offered no protection, and dnTGFβRII Treg cells were functionally defective in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In vitro cholangiocyte cytotoxicity assays demonstrated significantly increased numbers of cytotoxic hepatic dnTGFβRII KLRG1+ CD8 cells compared to B6. Protection from disease by B6 Tregs was associated with elimination of hepatic dnTGFβRII CD8 mediated cholangiocyte cytotoxicity. These results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, and that an intrinsic T cell effector defect is not sufficient to mediate autoimmune biliary disease in the setting of intact immune regulation. These results have important implications for understanding the early pathogenesis of human PBC. [Copyright &y& Elsevier]

Abstract: Autoimmunity is associated with a strong genetic component, but onset and persistence of clinically apparent autoimmune diseases often require an additional environmental trigger. The balance between immunity and tolerance is regulated by numerous molecular factors including nuclear hormone and homeostatic chemokine receptors. The nuclear hormone receptor RORγt and the chemokine receptor CCR7 are both essentially involved in functional lymphoid organogenesis and maintenance of lymphocyte homeostasis. Lack of one or the other impairs thymic T cell development and alters T cell homeostasis. Mice deficient for both, Ccr7 −/− Rorγt −/−, succumbed early to acute destructive inflammation, characterized by massive recruitment of inflammatory leukocytes, pro-inflammatory cytokine and autoantibody production, and wasting disease. Antibiotic-treatment of mice before disease onset reduced the overall gut microflora and abrogated the development of fatal mucosal inflammation. Hence, commensal bacteria and a confined tissue-specific inflammatory milieu serve as complementary trigger to initiate the lethal pathophysiologic process in Ccr7 −/− Rorγt −/− mice. [Copyright &y& Elsevier]

Abstract: Interleukin-7 (IL-7) is a critical survival factor for lymphocytes and recent studies suggest targeting the IL-7/IL-7Rα pathway holds promise for the treatment of autoimmune diseases. Several lines of evidence, genetic as well as functional, indicate an important role for this cytokine in autoimmune inflammation: polymorphisms in the IL-7Rα have been associated with increased risk for autoimmune disease and blocking IL-7/IL-7Rα with antibodies showed therapeutic efficacy in several autoimmune mouse models. Insights are starting to emerge about the mechanisms underlying IL-7's role in autoimmunity and tolerance, revealing surprising novel functions beyond its traditional activity as a T cell survival factor. In the first part of this review, the functions of IL-7 in the immune system are concisely described, providing a basis for understanding their potential role in promoting autoimmune responses. In the second part, current knowledge about the role of IL-7 in various autoimmune conditions is reviewed. [Copyright &y& Elsevier]

Abstract: Activation of complement components is crucial in the progression and severity of myasthenia gravis and experimental autoimmune myasthenia gravis (EAMG). Mice deficient in complement component C4 or treated with monoclonal antibody to C1q are resistant to EAMG. In this study, we show that inhibition of complement cascade activation by suppressing the expression of a critical low-abundant protein, C2, in the classical complement pathway, significantly improved clinical and immunopathological manifestations of EAMG. Two weeks after a second booster immunization with acetylcholine receptor, when mice exhibit muscle weakness, i.p. injection of C2 siRNA significantly suppressed C2 mRNA in the blood cells and liver of EAMG mice. Treatment of EAMG mice with C2 siRNA, once a week for 5 weeks, significantly improved muscle strength, which was further evidenced by functional AChR preservation in muscle, reduction in number of C3 and membrane-attack complexes at neuro-muscular junctions in forelimb muscle sections, and a transient decrease in serum IgG2b levels. Our study shows for the first time that siRNA-mediated suppression of C2, a component of the classical complement system, following established disease, can effectively contribute to the remission of EAMG. Therefore, C2 siRNA mediated therapy can be applied in all complement mediated autoimmune diseases. [Copyright &y& Elsevier]

Abstract: Dominant-negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-β receptor is expressed by both CD4+ and CD8+ T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8+ T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-βRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC. [Copyright &y& Elsevier]

Abstract: Cytokines are key regulators of physiological inflammatory responses, while aberrant cytokine expression contributes to pathogenesis of autoimmune diseases. We noted increased IL-6 levels in human and murine epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune bullous dermatoses (AIBD) induced by autoantibodies to type VII collagen (COL7). In contrast to rheumatoid arthritis, blockade of IL-6 led to strikingly enhanced experimental EBA, while treatment with recombinant IL-6 was protective. This was due to classical IL-6 signalling and independent of IL-6 trans-signalling, as treatment of mice with sgp130Fc had no impact on EBA manifestation. Induction of EBA in mice led to increased IL-1ra levels in skin and serum, while blockade of IL-6 completely inhibited IL-1ra expression induced by autoantibodies to COL7. In line, treatment of mice with EBA with recombinant IL-6 induced IL-1ra concentrations exceeding those of untreated animals with EBA, and IL-1ra (anakinra) administration significantly impaired experimental EBA induction. We here identified a novel anti-inflammatory pathway in an organ-specific autoimmune disease. Modulation of this IL-1ra pathway by classical IL-6 signalling demonstrates anti-inflammatory and protective activities of IL-6 in vivo. [Copyright &y& Elsevier]

Abstract: Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. [Copyright &y& Elsevier]

Abstract: Atopy and autoimmunity are usually considered opposed immunological manifestations. Lyn−/− mice develop lupus-like autoimmune disease yet have coexistent intrinsic allergic traits and are prone to severe, persistent asthma induced exogenously. Recently it has been proposed that the Th2 environment and IgE auto-Abs promotes autoimmune disease in Lyn−/− mice. To examine this apparent contradiction, we derived Lyn−/− mice with a null mutation in STAT6, a regulator of Th2 immunity that integrates signaling from the IL-4/IL-13 receptor complex. Atopy and spontaneous peritoneal eosinophilia, characteristic of Lyn−/− mice, were lost in young Lyn−/−STAT6−/− mice; however, autoimmune disease was markedly exacerbated. At a time-point where Lyn−/− mice showed only mild autoimmune disease, Lyn−/−STAT6−/− mice had maximal titres of IgG and IgA auto-Abs, impaired renal function, myeloid expansion and a highly activated T cell compartment. Remarkably, low level IgE auto-Abs but not IgG1 auto-Abs were a feature of some aged Lyn−/−STAT6−/− mice. Furthermore, aged Lyn−/−STAT6−/− mice showed dramatically increased levels of serum IgE but minimal IgG1, suggesting that class-switching to IgE can occur in the absence of an IgG1 intermediate. The results show that Lyn-deficient mice can overcome the effects of disabling Th2 immunity, highlighting the importance of Lyn in controlling Th2 responses. Our data also indicates that, under certain conditions, STAT6-independent factors can promote IgE class-switching. This work has important clinical implications as many experimental therapies designed for the treatment of asthma or atopy are based on targeting the STAT6 axis, which could potentially reveal life endangering autoimmunity or promote atopy in susceptible individuals. [Copyright &y& Elsevier]

Abstract: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110δ isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110δ did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn−/−p110δ+/KD mice. Importantly, we have shown that haploinsufficiency of p110δ has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110δ PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE. [Copyright &y& Elsevier]

Abstract: The envelope glycoprotein, gp70, of endogenous retroviruses represents one of the major nephritogenic autoantigens implicated in murine systemic lupus erythematosus. Among different endogenous retroviruses (ecotropic, xenotropic and polytropic), lupus-prone mice express remarkably high levels of modified polytropic (mPT) retroviruses, which are controlled by the Sgp3 (serum gp70 production) locus. To define the contribution of the Sgp3 locus derived from lupus-prone mice to the expression of the specific mPT proviruses, the genetic origin of different mPT viruses expressed in livers and thymi of wild-type and Sgp3 congenic C57BL/6 mice was determined through clonal analysis of their transcripts. Among 13 mPT proviruses present in the C57BL/6 genome, only 3 proviruses (Mpmv6, Mpmv10 and Mpmv13) were selectively but differentially expressed in livers and thymi. This was likely a result of co-regulated expression with host genes because of their integration in the same transcriptional direction. In contrast, Sgp3 induced the steady-state expression of an additional select group of mPT proviruses and, after stimulation of TLR7, the highly upregulated expression of a potentially replication-competent mPT virus Mpmv4. These results indicated that the expression of distinct subpopulations of mPT retroviruses was regulated by Sgp3- and TLR7-dependent mechanisms. The induction of potentially replication-competent mPT viruses and the upregulation of one such virus after stimulation with TLR7 in Sgp3 congenic mice further highlight the implication of Sgp3 in autoimmune responses against nephritogenic serum gp70 through the activation of TLR7. [Copyright &y& Elsevier]

Abstract: Previously we showed that transgenic mice expressing human HLA-DR3 gene are susceptible to PLP91–110 induced experimental autoimmune encephalomyelitis (EAE) and can serve as an animal model of multiple sclerosis (MS). HLA-DR3 mice with EAE showed increased number of CD8 T cells indicating their important role in disease pathogenesis. The role of CD8 T cells in MS, an inflammatory demyelinating disease of CNS, has been enigmatic as it has been assigned both regulatory and pathogenic roles. Therefore, to evaluate the role of CD8 T cells, we generated CD8 deficient HLA-DR3 transgenic mice (DR3.CD8−/−). Immunization with PLP91–110 led to more severe EAE in DR3.CD8−/− mice compared to HLA-DR3 mice indicating a regulatory role for CD8 T cells. Interestingly, DR3.CD8−/− mice with EAE showed decreased CNS pathology compared to DR3 mice thus suggesting a pathogenic role for CD8 T cells. We show that these two subsets of CD8 T cells can be differentiated based on the surface expression of CD122 (IL-2 Rβ chain). CD8 T cells expressing CD122 (CD8+CD122+) play a regulatory role while CD8+CD122− T cells act as a pathogenic subset. CD122 expressing CD8 T cells are the regulatory subset of CD8 T cells and regulate the encephalitogenic CD4 T cells through direct modulation of antigen presenting cells and/or through the release of immunoregulatory cytokines such as IL-10, IFNγ and TGFβ. We also showed that adoptive transfer of CD8CD122− T cells caused increased spinal cord demyelination indicating that these are pathogenic subset of CD8 T cells. Our study suggests that CD8+ T cells play both regulatory as well as pathogenic role in disease pathogenesis of EAE. A better understanding of these subsets could aid in designing novel therapy for MS patients. [Copyright &y& Elsevier]

Abstract: With an increasing number of studies demonstrating alterations in T cell microRNA expression during autoimmune disease, modulation of the T cell microRNA network is considered a potential therapeutic strategy. Due to the complex and often opposing interactions of individual microRNA, prioritization of therapeutic targets first requires dissecting the dominant effects of the T cell microRNA network. Initial results utilizing a unidirectional screen suggested that the tolerogenic functions were dominant, with spontaneous colitis resulting from T cell-specific excision of Dicer. Here we performed a bidirectional screen for microRNA function by removing Dicer from the T cells of both wildtype mice and Transforming Growth Factor β (TGFβ) receptor-deficient mice. This allowed the impact of microRNA loss on T cell activation, effector T cell differentiation and autoimmune pathology to be systematically assessed. This bidirectional screen revealed a dominant immunogenic function for T cell microRNA, with potent suppression of T cell activation, IFNγ production and autoimmune pathology in all targeted organs except the colon, where Dicer-dependent microRNA demonstrated a dominant tolerogenic function. These results reverse the original conclusions of microRNA function in T cells by revealing a systemic pro-autoimmune function. [Copyright &y& Elsevier]

Abstract: Interleukin 12 (IL-12) is a pivotal Th1-associated cytokine and a potent immunoregulatory molecule. However, the role of IL-12 in inducing immune tolerance that prevents insulitis and inhibits type 1 diabetes (T1D) remains unknown. The aim of this study was to investigate whether intermittent administration of IL-12 could prevent the development of T1D in nonobese diabetic (NOD) mice. We examined whether IL-12 treatment prevented diabetes by injecting different doses of IL-12 into NOD mice and compared the incidence of diabetes and insulitis in NOD mice with the incidence in control mice. Furthermore, we investigated the potential mechanisms of IL-12-mediated prevention of diabetes and insulitis. The expression of pro-inflammatory and immunoregulatory cytokines was measured before and following therapeutic administration of IL-12 in NOD mice. Our data demonstrated that both the absolute number and the function of DCs were impaired in NOD mice and that the levels of the Th17-associated pro-inflammatory cytokines, IL-1β, IL-6 and IL-23, were elevated in NOD mice compared with age-matched BALB/c and C57BL/6 mice. However, treatment of NOD mice with IL-12 suppressed insulitis and increased the number of healthy islets, and the levels of IL-17, IL-1β, IL-6 and IL-23 were significantly decreased. Moreover, IL-12 treatment of NOD mice induced the secretion of IFN-γ, a potent inhibitor of Th17 cells. These data indicated that intermittent administration of IL-12 prevented diabetes by inducing IFN-γ, suppressing the pathogenic IL-17-producing cells and reducing the expression of Th17-associated pro-inflammatory cytokines. Our results suggest a promising strategy for the treatment of human T1D and other Th17 cell-mediated autoimmune diseases. [Copyright &y& Elsevier]

Abstract: Antigen-specific therapies are possibly the safest approach to prevent type 1 diabetes (T1D). However their clinical translation has yielded poor results and greater efforts need to be put into the development of novel strategies to ameliorate their clinical outcome. OX40 is a costimulatory molecule expressed by T cells after antigen recognition and has been implicated in the control effector but also regulatory T cells (Tregs) function in vivo. The activity of OX40 signal on Tregs function has been controversial. In this context we investigated whether an anti-OX40 agonist antibody treatment can ameliorate antigen-specific immune intervention for the prevention of T1D. We show that treatment of non-obese diabetic (NOD) mice with an OX40 agonistic antibody (OX86) reduced type 1 diabetes (T1D) incidence by inducing both CD4+CD25+Foxp3+ Tregs and CD4+Foxp3− T cells expressing the latency-associated peptide (LAP). These OX86-induced CD4+Foxp3−LAP+ T cells also demonstrated suppressive activity in vitro. A significant increase in protection was observed when OX86 was combined with insulin B9:23 (insB9:23) peptide immunizations. Synergy resulted from an expansion of IL-10-expressing insB9:23-reactive Tregs which augmented the proportion of CD4+ T cells with in vivo suppressive activity. Consequently, CD4+ T cells purified from OX86/insB9:23 combination treatment prevented T1D development when adoptively transferred into recipient mice. These findings suggest that the requirement for OX40 signaling by antigen-induced Tregs can be dominant over its well-documented need for effector memory cell function and may have potentially important implications for improving the clinical translation of antigen-specific prevention of T1D and possibly other autoimmune disorders. [Copyright &y& Elsevier]

Abstract: Lymphnode swelling during immune responses is a transient, finely regulated tissue rearrangement, accomplished with the participation of the extracellular matrix. Here we show that murine and human reactive lymph nodes express SPARC in the germinal centres. Defective follicular dendritic cell networking in SPARC-deficient mice is accompanied by a severe delay in the arrangement of germinal centres and development of humoral autoimmunity, events that are linked to Th17 development. SPARC is required for the optimal and rapid differentiation of Th17 cells, accordingly we show delayed development of experimental autoimmune encephalomyelitis whose pathogenesis involves Th17. Not only host radioresistant cells, namely follicular dendritic cells, but also CD4+ cells are the relevant sources of SPARC, in vivo. Th17 differentiation and germinal centre formation mutually depend on SPARC for a proper functional crosstalk. Indeed, Th17 cells can enter the germinal centres in SPARC-competent, but not SPARC-deficient, mice. In summary, SPARC optimizes the changes occurring in lymphoid extracellular matrix harboring complex interactions between follicular dendritic cells, B cells and Th17 cells. [Copyright &y& Elsevier]

Abstract: Autoimmune hepatitis (AIH) is a serious chronic inflammatory disease of the liver with yet unknown etiology and largely uncertain immunopathology. The hallmark of type 2 AIH is the generation of liver kidney microsomal-1 (LKM-1) autoantibodies, which predominantly react to cytochrome P450 2D6 (CYP2D6). The identification of disease initiating factors has been hampered in the past, since antibody epitope mapping was mostly performed using serum samples collected late during disease resulting in the identification of immunodominant epitopes not necessarily representing those involved in disease initiation. In order to identify possible environmental triggers for AIH, we analyzed for the first time the spreading of the anti-CYP2D6 antibody response over a prolonged period of time in AIH patients and in the CYP2D6 mouse model, in which mice infected with Adenovirus-human CYP2D6 (Ad-h2D6) develop antibodies with a similar specificity than AIH patients. Epitope spreading was analyzed in six AIH-2-patients and in the CYP2D6 mouse model using SPOTs membranes containing peptides covering the entire CYP2D6 protein. Despite of a considerable variation, both mice and AIH patients largely focus their humoral immune response on an immunodominant epitope early after infection (mice) or diagnosis (patients). The CYP2D6 mouse model revealed that epitope spreading is initiated at the immunodominant epitope and later expands to neighboring and remote regions. Sequence homologies to human pathogens have been detected for all identified epitopes. Our study demonstrates that epitope spreading does indeed occur during the pathogenesis of AIH and supports the concept of molecular mimicry as a possible initiating mechanism for AIH. [Copyright &y& Elsevier]

Abstract: We hypothesized that regulatory T cells (Treg) effectively target diabetogenic cells, and reinforcing their killing capacity will attenuate the course of disease. For proof of concept, Fas-ligand (FasL) protein was conjugated to CD25+ Treg (killer Treg) to simulate the physiological mechanism of activation-induced cell death. Cytotoxic and suppressive activity of killer Treg was superior to naïve Treg in vitro. Administration of 3–4 × 106 Treg prevented hyperglycemia in 65% prediabetic NOD females, however only killer Treg postponed disease onset by 14 weeks. CD25+ Treg homed to the pancreas and regional lymph nodes of prediabetic NOD females, proliferated and ectopic FasL protein induced apoptosis in CD25− T cells in situ. This mechanism of pathogenic cell debulking is specific to killer Treg, as FasL-coated splenocytes have no immunomodulatory effect, and only killer Treg prevent the disease in 80% of NOD.SCID recipients of effector:suppressor T cells (10:1 ratio). All immunomodulated mice displayed increased fractional expression of FoxP3 in the pancreas and draining lymph nodes, which was accompanied by CD25 only in recipients of killer Treg. A therapeutic intervention that uses the affinity of Treg to reduce the pathogenic load has long-term consequences: arrest of destructive insulitis in mice with established disease prior to β-cell extinction. [Copyright &y& Elsevier]

Abstract: Polymorphisms in the SLAM family of leukocyte cell surface regulatory molecules have been associated with lupus-like phenotypes in both humans and mice. The murine Slamf gene cluster lies within the lupus-associated Sle1b region of mouse chromosome 1. Non-autoreactive C57BL/6 (B6) mice that have had this region replaced by syntenic segments from other mouse strains (i.e. 129, NZB and NZW) are B6 congenic strains that spontaneously produce non-nephritogenic lupus-like autoantibodies. We have recently reported that genetic ablation of the SLAM family member CD48 (Slamf2) drives full-blown autoimmune disease with severe proliferative glomerulonephritis (CD48GN) in B6 mice carrying 129 sequences of the Sle1b region (B6.129CD48−/−). We also discovered that BALB/c mice with the same 129-derived CD48-null allele (BALB.129CD48−/−) have neither nephritis nor anti-DNA autoantibodies, indicating that strain specific background genes modulate the effects of CD48 deficiency. Here we further examine this novel model of lupus nephritis in which CD48 deficiency transforms benign autoreactivity into fatal nephritis. CD48GN is characterized by glomerular hypertrophy with mesangial expansion, proliferation and leukocytic infiltration. Immune complexes deposit in mesangium and in sub-endothelial, sub-epithelial and intramembranous sites along the glomerular basement membrane. Afflicted mice have low-grade proteinuria, intermittent hematuria and their progressive renal injury manifests with elevated urine NGAL levels and with uremia. In contrast to the lupus-like B6.129CD48−/− animals, neither BALB.129CD48−/− mice nor B6 × BALB/c F1.129CD48−/− progeny have autoimmune traits, indicating that B6-specific background genes modulate the effect of CD48 on lupus nephritis in a recessive manner. [Copyright &y& Elsevier]

Abstract: Regulatory functions for B lymphocytes have been reported in murine models of autoimmune diseases in which B-cell deficient mice were shown to exhibit exacerbated disease. The B cells responsible for the immune regulations were identified as a subpopulation of interleukin 10-secreting cells. However, the mechanism of induction and the characteristics of regulatory B cells in humans have been hardly studied. This study reports that regulation of T cell responses can be induced by B cells following CD40-dependent cognate interaction. T cell proliferation and cytokine production were differentially regulated. Thus, CD40-induced regulatory B cells partially inhibited T cell proliferation following CD40 interaction without requirement of soluble factor. In contrast, modulation of Th1 differentiation resulted from CD80- and CD86-dependent interactions and from IL-10 production. The suppressive effects were mediated by CD19highIgD+CD38highCD24highCD5high B cells and appeared to be indirect, through the induction of regulatory T cells as indicated by the appearance of Foxp3+CD4+CD25+T cells. These data suggest that activation signals from T cells initiate regulatory properties in B cells that modulate T cell responses involving regulatory T cells. Finally, studies in autoimmune patients revealed that regulation of T cell proliferation was defective in systemic lupus erythematosus but efficient in other diseases. Restoration of efficient B-cell regulatory activity could provide innovative B-cell based treatment of autoimmune diseases. [Copyright &y& Elsevier]

Abstract: The absence of IFN-γ signaling leads to an increased inflammatory response in many murine models of autoimmune diseases induced by a CFA-assisted immunization schedule. We investigated the role of endogenous IFN-γ in arthritis induced by immunization with glucose-6-phosphate isomerase (G6PI) in CFA in DBA/1 mice. Surprisingly, and in contrast to our previous findings in collagen-induced arthritis (CIA), G6PI-induced arthritis was found to be reduced in IFN-γ receptor-deficient (IFN-γR KO) mice, demonstrating a proinflammatory role for IFN-γ in this model. Milder disease in IFN-γR KO mice was associated with less vigorous innate and adaptive immune responses early (day 9) after immunization: less proliferation of myeloid cells in the spleen, less osteoclast formation, less G6PI-reactive Th cells (as measured by ex vivo stimulation and flow cytometry and by in vivo skin reactivity to G6PI) and lower G6PI-specific immunoglobulin serum levels. Surprisingly, on day 21, despite continued milder disease in IFN-γR KO mice, their Th cell responses were no longer diminished but augmented as compared to wild-type mice, and their numbers of immature myeloid splenocytes were also more increased. These data reveal that IFN-γ signaling is critical for the induction of the early immune responses which trigger G6PI-induced arthritis. The strikingly different clinical consequences of absent IFN-γ signaling in G6PI-induced arthritis compared with the very similarly induced CIA emphasize that the role of a single cytokine in experimentally induced arthritis depends critically on the very nature of the inciting (auto)antigen and in particular on the kinetics of the disease manifestation elicited by the antigen. [Copyright &y& Elsevier]

Abstract: It is not fully understood how the expression level of autoantigens in beta cells impacts autoimmune diabetes (T1D) development. Earlier studies using ovalbumin and also insulin had shown that secreted antigens could enhance diabetes development through facilitated presentation by antigen presenting cells. Here we sought to determine how the expression level of a membrane bound, non-secreted or cross-presented neo-antigen, the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), would influence T1D. We found that an RIP-LCMV transgenic mouse line exhibiting higher levels of beta cell GP expression developed more severe diabetes after LCMV infection or transfer of high numbers of activated autoreactive T cells. Importantly, all beta cells were lost and a significant increase in morbidity and mortality from T1D was noted. Insulitis and accumulation of autoaggressive CD8 cells was more profound in the RIP-LCMV-GP high-expressor line. Interestingly, the additional introduction of neo-antigen-specific CD4+ helper or regulatory T cells was able to influence diabetogenesis positively or negatively. We conclude that a higher degree of autoantigen expression results in increased diabetes susceptibility. Therefore, autoantigens such as insulin that are expressed at higher levels in beta cells might have a more profound impact on diabetes pathogenesis. [Copyright &y& Elsevier]

Abstract: There is increasing evidence that both autoimmune and autoinflammatory mechanisms are involved in the development of not only type 1 diabetes mellitus (T1 DM), but also type 2 diabetes mellitus (T2 DM). Our laboratory has focused on this concept, and in earlier efforts replaced the bone marrow cells (BMCs) of leptin receptor-deficient (db/db) mice, an animal model of T2DM, with those of normal C57BL/6 (B6) mice by IBM–BMT. However, the outcome was poor due to incomplete recovery of T cell function. Therefore, we hypothesized that intra-bone marrow–bone marrow transplantation plus thymus transplantation (IBM–BMT + TT) could be used to treat T2 DM by normalizing the T cell imbalance. Hence we addressed this issue by using such dual transplantation and demonstrate herein that seven weeks later, recipient db/db mice manifested improved body weight, reduced levels of blood glucose, and a reduction of plasma IL-6 and IL-1β. More importantly, this treatment regimen showed normal CD4/CD8 ratios, and increased plasma adiponectin levels, insulin sensitivity, and the number of insulin-producing cells. Furthermore, the expression of pancreatic pAKT, pLKB1, pAMPK and HO-1 was increased in the mice treated with IBM–BMT + TT. Our data show that IBM–BMT + TT treatment normalizes T cell subsets, cytokine imbalance and insulin sensitivity in the db/db mouse, suggesting that IBM–BMT + TT is a viable therapeutic option in the treatment of T2 DM. [Copyright &y& Elsevier]

Abstract: The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögren’s syndrome (SS). M3R−/− mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1−/− (M3R−/−→Rag1−/−) mice. M3R−/−→Rag1−/− mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4+ T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R−/−→Rag1−/− mice. Moreover, transfer of only CD3+ T cells from M3R−/− immunized with M3R peptides into Rag1−/− mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3+ T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis. [Copyright &y& Elsevier]

Abstract: Systemic lupus erythematosus is a common autoimmune disease, with kidney involvement a serious complication associated with poor prognosis. Humoral immune responses constitute the hallmark of disease, however T helper cells are required for the generation of autoantibodies, as well as the induction and progression of renal injury. Administration of pristane to genetically intact mice results in the development of hypergammaglobulinaemia with the production of lupus like autoantibodies and proliferative glomerulonephritis, with similarities to human lupus nephritis. TLRs are intricately linked to the development of autoimmunity and are involved in the development of lupus nephritis. We injected wild type, TLR9−/− and TLR4−/− mice with pristane and assessed cellular and humoral autoimmunity and renal injury, 8 months later. TLR9−/− mice demonstrated a predominant decrease in Th1 cytokine production which resulted in decreased anti-RNP antibody levels, while anti-dsDNA levels remained intact. Compared to wild type mice treated with pristane, functional and histological renal injury and glomerular immunoglobulin and complement deposition was decreased in TLR9−/− mice. TLR4−/− mice demonstrated a global decrease in both Th1, IFNγ, and Th17 associated IL-17A and IL-6 cytokine production. Autoantibody levels of anti-dsDNA and anti-RNP were both decreased. Renal injury was attenuated in TLR4−/− mice which demonstrated less glomerular immunoglobulin and complement deposition. These results demonstrate that both TLR9 and TLR4 are required for ‘full-blown’ autoimmunity and organ injury in experimental lupus induced by pristane. [Copyright &y& Elsevier]

Abstract: The purpose of the present study was to develop a peptide for treatment of multiple sclerosis (MS). We have tested the effect of a novel anti-inflammatory peptide (KGHYAERVG, termed IIIM1) on experimental autoimmune encephalitis (EAE), an animal model of MS. Our findings demonstrate significant reduction in neurological score following oral administration of IIIM1. Structural studies revealed that the entire peptide is required for activity. The peptide caused significant reduction in IL17, interferon γ, IL23 and IL12 production by isolated splenocytes and concomitant elevation of anti-inflammatory cytokines. IIIM1 elevated T regulatory cells (Tregs, CD4+CD25+FoxP3+) in brain and spleen of EAE mice. Similar proliferative effect was observed in isolated human and mouse Tregs in vitro. Stimulation of Tregs by IIIM1 caused production of a new peptide termed RA1 present in Oryza Sativa Japonica group. This Japanese rice peptide ameliorated neurological symptoms in the EAE model. Similar beneficial effect was observed upon oral administration of an extract of Japanese rice. In conclusion, oral treatment with IIIM1 ameliorates EAE symptoms via stimulation of Tregs to proliferate and produce RA1 which reduces EAE symptoms. RA1 might be involved in the relatively low prevalence of MS in Japan and other Japanese rice-eating populations. [Copyright &y& Elsevier]

Abstract: Immune-based self-recognition and failure to modulate this response are believed to contribute to the debilitating autoimmune pathology observed in multiple sclerosis (MS). Studies from its murine model, experimental autoimmune encephalomyelitis (EAE), have shown that neuroantigen-specific CD4+T cells are capable of inducing disease, while their immune sibling, the CD8+T cells, have largely been ignored. To understand their role in autoimmune demyelination, we first confirmed that, similar to our observations in human MS, there is robust induction of neuroantigen-reactive CD8+T cells in several models, including MOG35–55/CFA-induced EAE. However, MOG35–55-specific CD8+T-cells, when purified, were unable to adoptively transfer disease into naïve mice (in contrast to CD4+T-cells). In fact, we observed that the transfer of these neuroantigen-specific CD8+T cells was able to suppress the induction of EAE and to inhibit ongoing EAE. These regulatory CD8+T cells produced IFN-γ and perforin and were able to kill MOG loaded CD4+T-cells as well as CD4-depleted APC, suggesting a cytotoxic/suppressor mechanism. Inhibition of EAE was associated with both the modulation of APC function as well as decreased MOG-specific CD4+T cell responses. Our studies reveal a novel and unexpected immune regulatory function for neuroantigen-specific CD8+T cells and have interesting biologic and therapeutic implications. [Copyright &y& Elsevier]

Abstract: An HLA-DR variant containing Arginine at position 74 of the DRβ1 chain confers a strong genetic susceptibility to autoimmune thyroid diseases (AITD), Graves'' disease (GD) and Hashimoto''s thyroiditis (HT), while Glutamine at position DRβ1–74 is protective. We hypothesized that the DRβ1-Arg74 variant is able to present pathogenic thyroglobulin (Tg) peptides to T-cells more efficiently, thereby triggering thyroid autoimmunity. Indeed, we have previously identified 4 human Tg (hTg) peptides that bind specifically to DRβ1-Arg74 with much weaker binding to the protective variant DRβ1-Gln74. The aim of our study was to examine in vivo whether an hTg peptide that binds strongly and specifically to DRβ1-Arg74 is capable of stimulating T-cells during the induction of thyroiditis in a “humanized” mouse expressing human DR3, and in patients positive for Tg antibodies. Sequencing of exon 2 of the DR transgene in the DR3 mice, null for endogenous MHC II molecules, confirmed that they expressed the disease-associated DRβ1-Arg74 variant, thus making them an ideal in vivo model to test the presentation of hTg peptides by DRβ1-Arg74 HLA-DR. Induction of EAT in the DR3 mice lead to T-cell stimulation and proliferation to Tg.2098, a strong and specific DRβ1-Arg74 binder, while a non-binding control peptide, Tg.2766 did not induce this response. Moreover, Tg.2098 stimulated T-cells from 4 individuals who were positive for thyroglobulin antibodies, demonstrating that Tg.2098 is an immunogenic peptide capable of being presented in vivo and activating T-cells in EAT and AITD. Energetic analysis of the complex formed by Tg.2098 and DRβ-Arg74 has shown that the origin of the affinity was determined by residues 1, 7 and 9 in the peptide, while the selectivity of the peptide for the MHC was determined by the Asp in position 4. The disease-protective substitution R74Q, leads to reduction in affinity due to changes in local interaction with D4 as well as non-local interaction with other residues. The electrostatic potential on the surface of the DRβ-Arg74–Tg.2098 complex has a unique signature which may be recognized by T-cell receptors leading to autoimmune thyroiditis. Taken together these findings suggest that Tg.2098, a strong and specific binder to the disease-associated HLA-DRβ-Arg74, is a major human T-cell epitope and participant in the pathoetiology of AITD. [Copyright &y& Elsevier]

Abstract: We developed a panel of non-obese diabetic (NOD) mice deficient in major lysosomal cysteine proteases (cathepsins S, L and B) to identify protease enzymes essential for autoimmune diabetes. Null alleles for cathepsins (Cts) S, L or B were introgressed onto the NOD genetic background with 19 Idd markers at homozygosity. Diabetes onset was determined among females aged up to 6 months. We evaluated insulitis and sialadenitis in tissues using histology and computer assisted morphology. NOD mice deficient in Ctss or Ctsb were partially protected from diabetes with incidence at 33% and 28%, respectively, versus wild-type NOD (69%; p < 0.00001). NODs lacking cathepsin L (Ctsl−/−) are completely protected from IDDM, as originally shown by others. Ctsl, Ctss, or Ctsb heterozygous mice were able to develop IDDM, although incidence levels were significantly lower for Ctsb+/− (50%) and Ctsl+/− (55%) as compared to NODs (69%; p < 0.03). Ctsl−/− mice contain functional, diabetogenic T cells and an enriched Foxp3+ regulatory T cell population, and diabetes resistance was due to the presence of an expanded population of regulatory T cells. These data provide additional information about the potency of the diabetogenic T cell population in Ctsl−/− mice which were comparable in potency to wild-type NOD mice. These data illustrate the critical contribution of each of these proteases in determining IDDM in the NOD mouse and provide a useful set of models for further studies. [Copyright &y& Elsevier]

Abstract: Dysregulated expression of Bcl-xL and Bcl-2 may initiate the development of autoimmune diseases including systemic lupus erythematosus (SLE). A tolerogenic peptide designated hCDR1 was shown to ameliorate manifestations of spontaneous and induced murine SLE. Recently, we demonstrated that Bcl-xL plays a critical role in the modulating effects of hCDR1, as manifested by reducing the state of activation of lymphocytes and by down-regulating the secretion of the pathogenic cytokines, IFN-γ and IL-10. Here we studied the role of Bcl-xL in the development and function of CD4 regulatory T-cells (Treg) from hCDR1-treated, SLE-afflicted (New-Zealand-Black × New-Zealand-White) F1 mice. We report that Bcl-xL was up-regulated in CD4 Treg of tolerized mice, where it played a role in inducing the regulatory/inhibitory molecules Foxp3, CTLA-4, and TGF-β and in repressing PD-1. Further, Bcl-xL mediated the induction of CTLA-4 and TGF-β in effector T cells (Teff) by CD4 Treg of the tolerized mice. The induction of Bcl-xL in Teff by Treg was TGF-β dependent and CTLA-4 independent, leading to inhibition of proliferation and to a decrease in activated Teff. We conclude that Bcl-xL is required for the development and function of CD4 Treg, which ameliorate lupus following treatment with a tolerogenic peptide. [Copyright &y& Elsevier]

Abstract: Most individuals have viral infections at some point in their life, however, only few develop autoreactivity to cardiac myosin following infection suggesting a genetic predisposition. Population studies have shown that among all the genetic factors linked with autoimmune disease development, MHC class II genes are the most significant genetic factors. Experimental autoimmune myocarditis resembling human Dilated cardiomyopathy can be induced in susceptible mice by infection with coxsackie virus as well as immunization with purified foreign and murine cardiac specific a-myosin. We generated transgenic mice lacking endogenous class II molecules, HLA-DR3.Abo and HLA-DQ8.Abo transgenic mice in NOD and HLA-DQ8.Abo in B10 background, to study the role of MHC in spontaneous autoimmunity. The HLA molecules in these mice are expressed on cell surface and can positively select CD4+ T cells expressing various Vb T cell receptors. NOD.DQ8 female mice spontaneously developed myocarditis and dilated cardiomyopathy. Histopathology of heart revealed mononuclear infiltrate consisting of CD4 and Mac-1+ cells and myocyte necrosis. NOD.DQ8 mice showed cellular and humoral autoreactive response to self cardiac myosin.. Depletion of CD8 and CD4 + cells suggested that CD8 T cells may act as regulatory cells while CD4 cells are required as effector cells. NOD.DR3 and B10.DQ8 mice did not develop any cardiac pathology suggesting DQ8 is required for predisposition to the spontaneous autoreactivity while NOD background influences onset and progression of disease. Thus these mice provide powerful tools to understand the role of HLA class II molecules in predisposition and onset of human diseases and to develop immunotherapy. [Copyright &y& Elsevier]

Abstract: Sjögren syndrome is an autoimmune disease characterized by hyposecretion of the lacrimal and salivary glands, resulting in dryness of the eyes and mouth. Individuals may experience primary Sjögren syndrome or a secondary form accompanying another rheumatic autoimmune disease, such as rheumatoid arthritis or systemic lupus erythematosus. The pathogenic mechanisms of Sjögren syndrome remain largely unknown, in part a consequence of the heterogeneity of the disease. Animal models have shed light on the connections between specific pathways and symptoms, but an ideal system is wanting. Improved disease models will enable a better understanding of Sjögren syndrome, including how immune tolerance is lost and potential therapeutic interventions. Most importantly, an optimal model will enable detection of disease biomarkers, since injury to the salivary glands may precede lymphocytic infiltration. This review aims to characterize available mice models of Sjögren syndrome, including advantages and disadvantages, from the researcher''s perspective. [Copyright &y& Elsevier]

Cutaneous lupus erythematosus (CLE) is a relapsing autoimmune disease, but key elements that drive disease initiation and progression remain elusive. To date, the lack of ideal murine model which resemble human cutaneous lupus makes it extremely challenging for moving mechanistic discoveries and novel therapeutics. Here, we prompt a humanized murine model to develop an inducible rapid-onset murine that performs cutaneous rather than systemic lupus, depending on the successful human immune system reconstruction from active lupus patients and UVB irradiation as for essentially pathogenic triggers. In addition, we demonstrate a newly discovered population of B cell with a unique phenotype, that of the age-associated B cell (ABC, T-bet+ CD11b+), exhibits B cell clusters in humanized cutaneous lupus. In the response of IL-21 and TLR7/9 signals, recruitment of autoreactive B cells to the position of inflammation with subsequent localized antibody production of IgG2a, IgG2b, IgG3, has the potential to exacerbate ongoing inflammation and thus driving lupus-like autoimmunity in a B-cell-dominant fashion. Overall, our model provides a relevant system for exploring the pathophysiology of cutaneous lupus, a suitable model for drug development, as well as updating a potential role of IL-21 and TLR7/9 to be targeted by biologics. The regulation network of ABC development and its pathogenic role in this newly established humanized CLE model. Firstly, we established a novel humanized CLE mouse model through the successfully engraftment of human PBMCs from active lupus patients. These mice performed lupus-like cutaneous lesions under suitable UVB irradiation. In addition, cutaneous lupus skin exhibited highly expressed human IL-21. These human-derived IL-21 cytokines play an important role in driving ABCs differentiation, including facilitating the expression of CD11b and transcription factor T-bet. On the other hand, apoptotic KCs induced by external UVB irradiation led an accumulation of endogenous nucleic acid. These nuclear autoantigens of CpG DNA and ssRNA can be recognized at the surface of B cells by the B cell receptor (BCR), and then these signals can trigger TLR7/9 in endosomes, further accelerating ABC development and proliferation, thereby prompting ABC clusters infiltration in skin lesion. These vigorous ABC responses were found to cause autoantibody production, including IgG2a, IgG2b, IgG3, but not IgG1. Antibody/antigen binding results in immune complexes that may work as positive stimulatory signals in autoimmunity, further highlighting the pathogenic role of ABC in tissue damage and ongoing inflammation. [Display omitted] • A novel humanized cutaneous lupus model for pathogenesis and medicine research. • Aberrant distribution of IL-21 and age-associated B cells in this humanized model. • Age-associated B cell differentiation and IgG secretion under IL-21 and TLR signals. [ABSTRACT FROM AUTHOR]

Abstract: The molecular mechanisms of viral infection-induced glomerulonephritis are poorly understood. Toll-like receptor (TLR)-3 and TLR7 recognize viral RNA and their exposure to TLR3 or TLR7 can trigger the exacerbation of established immune complex disease in MRLlpr mice. Because coactivation of TLR3 and TLR7 was shown to synergistically activate dendritic cells in vitro, we hypothesized that simultaneous ligation of TLR3 and TLR7 would elicit additive effects on the exacerbation of glomerulonephritis in MRLlpr mice. Saline, 50μg pI:C RNA, 25μg of the TLR7 agonist imiquimod, or a combination of both were injected every other day to MRLlpr mice from week 16–18 of age. Coinjection of pI:C RNA and imiquimod had no synergistic effect on serum levels of IL-6 and IL12p70, dsDNA autoantibody levels, and glomerulonephritis. This was consistent with a lack of synergistic effects on cytokine release of TNF- and IFNγ-prestimulated monocytes in vitro. Furthermore, in glomerular mesangial cells a synergistic effect of pI:C RNA and imiquimod was generally absent due to the lack of TLR7 expression. We conclude that a number of mechanisms protect the host from additive effects of TLR3–TLR7 coactivation on renal pathology in vivo. [Copyright &y& Elsevier]

Abstract: A major handicap in understanding the pathogenesis of autoimmune cholangitis has been the absence of an informative mouse model. Recently, autoimmune cholangitis, with several features similar to PBC, has been described in NOD.c3c4 mice, including anti-mitochondrial antibodies, lymphocytic portal tract infiltrates, biliary destruction and the adoptive transfer of disease to naïve recipients using liver-derived lymphocytes. A unique feature, and a characteristic quite distinct from human PBC, is the presence of bile cyst formation. We have addressed the issue of cysts in NOD.c3c4 mice by performing comprehensive microarray analysis using cholangiocytes from NOD.c3c4 mice compared to NOD controls. Several key differences in gene expression were noted in NOD.c3c4 cholangiocytes. First, there was consistent impairment in the expression of Fas antigen (CD95). Second, cholangiocytes were PCNA positive but TUNEL negative, suggesting an absence of apoptosis despite abnormal proliferation. In conclusion, we propose that autoimmune cholangitis develops in NOD.c3c4 mice secondary to impaired biliary cell apoptosis with exposure of mitochondrial antigens, loss of tolerance and subsequent development of multi-lineage anti-mitochondrial responses. [Copyright &y& Elsevier]

Abstract: Scurfy mice which lacks functional Foxp3 transcription factor and CD4+CD25+Foxp3+ regulatory T (Treg) cells, spontaneously develop autoimmune responses against skin, lung, liver and tail. However, many organs/tissues are spared from autoimmune attack. Here, we demonstrate that scurfy mice contain dormant autoimmune T cells that induced new diseases such as sialoadenitis, dacryoadenitis, pancreatitis, gastritis, intestinal inflammation, colitis, and myositis in RAG-1 KO mice. Inflammation in as many as 12 organs/tissues was consistently induced in individual recipients with scurfy lymph node cells containing as few as 1.25×106 CD4+ T cells. Moreover, transfer of the multiple organ autoimmune diseases could be suppressed by as little as 0.5×106 CD4+CD25+ Treg cells, mediated by inhibiting autoimmune T-cell expansion. Our study provides evidence for the presence of a large repertoire of autoimmune lymphocytes against various organs/tissues in scurfy mice as well as Treg cells in B6 mice capable of suppressing the expansion of these autoimmune lymphocytes. Various conditions that control the expression of autoimmune T cells are discussed. [Copyright &y& Elsevier]

Current treatment for patients with autoimmune disorders including rheumatoid arthritis, multiple sclerosis and type 1 diabetes, often consists of long-term drug regimens that broadly dampen immune responses. These non-specific treatments are frequently associated with severe side effects creating an urgent need for safer and more effective therapy to promote peripheral tolerance in autoimmune diseases. Cell-based immunotherapy may offer an encouraging alternative, where tolerogenic CD14+ myeloid cells are infused to inhibit autoreactive effector cells. In this review, we compared in depth three promising tolerogenic CD14+ candidates for the treatment of autoimmune disease: 1) tolerogenic dendritic cells, 2) monocytic myeloid-derived suppressor cells and 3) CD14+ type 2 conventional dendritic cells. TolDC-based therapy has entered clinical testing whereas evidence from the latter two cell types m-MDSCs and CD14+ cDC2s is predominantly coming from cancer immunology research. These three cell types have distinct cellular properties and immunosuppressive mechanisms offering unique opportunities to be explored. However, these cells differ in stage of development towards immunotherapy each facing additional hurdles. Therefore, we speculate on the potential benefits and risks of these cell types as novel cell-based immunotherapies to control autoimmune disease in patients. • Standard of care involves systemic immunosuppression causing severe side effects. • Cellular immunotherapy is emerging to specifically control autoimmunity in patients. • Phase 1 trials have proven the feasibility and safety of tolDC-based therapy. • M-MDSCs and CD14+ cDC2s are evidenced as cells with potent tolerogenic activity. • M-MDSCs-based therapy showed mixed results in mouse models of autoimmune disease. [ABSTRACT FROM AUTHOR]

Abstract: Increased susceptibility to autoimmunity in females is often viewed as the consequence of enhanced immunoreactivity providing superior protection against infections. We paradoxically observed greater mortality in female compared to male mice during systemic viral infections with three large double-stranded DNA viruses (herpes simplex virus type I [HSV], murine cytomegalovirus [MCMV], and vaccinia virus [VV]). Indeed, female mice were 27-fold more susceptible to infection with HSV than male mice. Elimination of estrogen by ovariectomy in female mice or addition of estrogen to castrated male mice only partially eliminated the observed sex differences following HSV infection. However, the differences observed in survival between female and male mice were nearly abrogated in the absence of type I interferon receptor signaling and substantially mitigated in absence of DAP12 signaling. Interestingly, the sex-specific impact of type I interferon receptor and DAP12 signaling differentially influenced survival during systemic viral infections with type I interferon receptor signaling enhancing male survival and DAP12 signaling increasing the susceptibility of female mice. These results have potential implications for the sex disparities observed in human autoimmune disorders. [Copyright &y& Elsevier]

Abstract: Pregnancy modulates autoimmune diseases through diverse and still incompletely defined mechanisms, in part operating at the decidua–placenta interface. To assess the immunological contribution of placenta, we administered mouse placental proteins to a mouse model of autoimmune hypophysitis, a disease known to be strongly associated with pregnancy. Emulsified placental proteins suppressed both the cellular and humoral aspects of hypophysitis. Suppression was specific to self antigens and not seen when two foreign antigens, tetanus toxoid or tuberculin purified protein derivative, were used. Proteomic analysis revealed high levels of soluble TNF receptor 1 in placenta, suggesting that blockade of the TNF-α pathway was a mechanism of disease suppression. Placentas derived from mice deficient in TNF receptor 1 lost the ability to suppress hypophysitis. Notably, hypophysitis suppression was seen only when the TNF-α pathway was blocked locally, at the site of immunization, and not systemically. These findings provide evidence that placenta contributes to the immune tolerance of pregnancy by locally inhibiting the TNF-α pathway. [Copyright &y& Elsevier]