DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

RGS10 deficiency ameliorates the severity of disease in experimental autoimmune encephalomyelitis.

BACKGROUND: Regulator of G-protein signaling (RGS) family proteins, which are GTPase accelerating proteins (GAPs) that negatively regulate G-protein-coupled receptors (GPCRs), are known to be important modulators of immune cell activation and function. Various single-nucleotide polymorphisms in RGS proteins highly correlate with increased risk for multiple sclerosis (MS), an autoimmune, neurodegenerative disorder. An in-depth search of the gene expression omnibus profile database revealed higher levels of RGS10 and RGS1 transcripts in peripheral blood mononuclear cells (PBMCs) in MS patients, suggesting potential functional roles for RGS proteins in MS etiology and/or progression. METHODS: To define potential roles for RGS10 in regulating autoimmune responses, we evaluated RGS10-null and wild-type (WT) mice for susceptibility to experimental autoimmune encephalomyelitis (EAE), a widely studied model of MS. Leukocyte distribution and functional responses were assessed using biochemical, immunohistological, and flow cytometry approaches. RESULTS: RGS10-null mice displayed significantly milder clinical symptoms of EAE with reduced disease incidence and severity, as well as delayed onset. We observed fewer CD3+ T lymphocytes and CD11b+ myeloid cells in the central nervous system (CNS) tissues of RGS10-null mice with myelin oligodendrocyte protein (MOG)35-55-induced EAE. Lymph node cells and splenocytes of immunized RGS10-null mice demonstrated decreased proliferative and cytokine responses in response to in vitro MOG memory recall challenge. In adoptive recipients, transferred myelin-reactive RGS10-null Th1 cells (but not Th17 cells) induced EAE that was less severe than their WT counterparts. CONCLUSIONS: These data demonstrate a critical role for RGS10 in mediating autoimmune disease through regulation of T lymphocyte function. This is the first study ever conducted to elucidate the function of RGS10 in effector lymphocytes in the context of EAE. The identification of RGS10 as an important regulator of inflammation might open possibilities for the development of more specific therapies for MS.

IRF9 and STAT1 are required for IgG autoantibody production and B cell expression of TLR7 in mice.

A hallmark of SLE is the production of high-titer, high-affinity, isotype-switched IgG autoantibodies directed against nucleic acid-associated antigens. Several studies have established a role for both type I IFN (IFN-I) and the activation of TLRs by nucleic acid-associated autoantigens in the pathogenesis of this disease. Here, we demonstrate that 2 IFN-I signaling molecules, IFN regulatory factor 9 (IRF9) and STAT1, were required for the production of IgG autoantibodies in the pristane-induced mouse model of SLE. In addition, levels of IgM autoantibodies were increased in pristane-treated Irf9 -/- mice, suggesting that IRF9 plays a role in isotype switching in response to self antigens. Upregulation of TLR7 by IFN-alpha was greatly reduced in Irf9 -/- and Stat1 -/- B cells. Irf9 -/- B cells were incapable of being activated through TLR7, and Stat1 -/- B cells were impaired in activation through both TLR7 and TLR9. These data may reveal a novel role for IFN-I signaling molecules in both TLR-specific B cell responses and production of IgG autoantibodies directed against nucleic acid-associated autoantigens. Our results suggest that IFN-I is upstream of TLR signaling in the activation of autoreactive B cells in SLE.

Chemokine-like receptor-1 expression by central nervous system-infiltrating leukocytes and involvement in a model of autoimmune demyelinating disease.

We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35-55 (MOG(35-55)), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG(35-55)-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.

High-throughput methods for measuring autoantibodies in systemic lupus erythematosus and other autoimmune diseases.

Numerous groups have now validated high-throughput approaches to autoantibody profiling in a variety of systems. Recently, we have used autoantigen microarray technology to identify distinct autoantibody profiles in H-2 congenic MRL/lpr mice (Sekine et al., manuscript in preparation), and we are expanding this platform to study human and mouse models of IDDM and RA. We are also developing protein arrays for multiplex analysis of serum antibody isotypes. Multiplexed methods for autoantibody profiling will undoubtedly continue to uncover novel aspects of autoimmunity and B cell biology. It is now time to move these technologies beyond the proof-of-concept phase, and start addressing the next series of important questions. These include, but certainly are not limited to: identifying "autoantibody signatures" associated with disease state or outcome; profiling autoantibodies during the natural course of murine and human disease; and monitoring changes in autoantibody profiles of patients in response to therapeutic intervention. However, the next set of challenges is just right around the corner. As data and statistical analysis tools become more robust, it will be possible to generate and approach new hypotheses at an unprecedented pace.

A novel CMKLR1 small molecule antagonist suppresses CNS autoimmune inflammatory disease.

Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated ß-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. α-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.

Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice.

Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

Treatment with a toll-like receptor inhibitory GpG oligonucleotide delays and attenuates lupus nephritis in NZB/W mice.

Activation of the innate immune system by DNA containing hypomethylated CpG motifs has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we examined the consequences of immunostimulatory CpG-oligodeoxynucleotide (ODN) and inhibitory GpG-ODN treatment in the NZB x NZW F1 (NZB/W) murine model of SLE. Beginning at 5 months of age, we administered CpG-ODN or GpG-ODN at regular intervals to female NZB/W animals. We also determined the effects of ODN administration on NZB/W mouse lymphocyte function, and the specificity of ODN binding to Toll-like receptors (TLRs) other than TLR-9. While CpG-ODN treatment did not appear to have a major impact on disease severity, GpG-ODN treatment significantly delayed the onset of proteinuria in NZB/W mice. Interestingly, short-term GpG-ODN treatment promoted Th2-type T and B cell responses, and inhibited B lymphocyte proliferation in vitro. On the other hand, extended GpG-ODN treatment did not result in sustained Th2 responses or significantly reduced renal disease. Moreover, the binding of CpG-ODN and GpG-ODN was not restricted to TLR-9 as both ODNs also interacted with TLR-3, TLR-7, and TLR-8. Taken together, the data indicate that the protective mechanism of GpG-ODN treatment in the NZB/W model of lupus nephritis involves modulating T cell cytokine profiles and B lymphocyte activation through the inhibition of several TLRs, including TLR-7 and TLR-9.

Type I interferon receptor controls B-cell expression of nucleic acid-sensing Toll-like receptors and autoantibody production in a murine model of lupus.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of high-titer IgG autoantibodies directed against nuclear autoantigens. Type I interferon (IFN-I) has been shown to play a pathogenic role in this disease. In the current study, we characterized the role of the IFNAR2 chain of the type I IFN (IFN-I) receptor in the targeting of nucleic acid-associated autoantigens and in B-cell expression of the nucleic acid-sensing Toll-like receptors (TLRs), TLR7 and TLR9, in the pristane model of lupus. METHODS: Wild-type (WT) and IFNAR2-/- mice were treated with pristane and monitored for proteinuria on a monthly basis. Autoantibody production was determined by autoantigen microarrays and confirmed using enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Serum immunoglobulin isotype levels, as well as B-cell cytokine production in vitro, were quantified by ELISA. B-cell proliferation was measured by thymidine incorporation assay. RESULTS: Autoantigen microarray profiling revealed that pristane-treated IFNAR2-/- mice lacked autoantibodies directed against components of the RNA-associated autoantigen complexes Smith antigen/ribonucleoprotein (Sm/RNP) and ribosomal phosphoprotein P0 (RiboP). The level of IgG anti-single-stranded DNA and anti-histone autoantibodies in pristane-treated IFNAR2-/- mice was decreased compared to pristane-treated WT mice. TLR7 expression and activation by a TLR7 agonist were dramatically reduced in B cells from IFNAR2-/- mice. IFNAR2-/- B cells failed to upregulate TLR7 as well as TLR9 expression in response to IFN-I, and effector responses to TLR7 and TLR9 agonists were significantly decreased as compared to B cells from WT mice following treatment with IFN-alpha. CONCLUSIONS: Our studies provide a critical link between the IFN-I pathway and the regulation of TLR-specific B-cell responses in a murine model of SLE.

Failure of oral atorvastatin to modulate a murine model of systemic lupus erythematosus.

OBJECTIVE: Inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme (statins) are cholesterol-lowering drugs that have shown promise as therapeutic agents in various animal models of autoimmune disease. The results of initial clinical trials with statins in multiple sclerosis and rheumatoid arthritis have also been encouraging. In this study, we attempted to treat a widely studied murine model of spontaneous systemic lupus erythematosus (SLE) with atorvastatin. METHODS: (NZB x NZW)F1 (NZB/NZW) mice received daily oral doses of atorvastatin for 20 weeks. The mice were monitored weekly for survival and proteinuria. Anti-double-stranded DNA (anti-dsDNA) antibody levels in sera were determined by enzyme-linked immunosorbent assay (ELISA). T lymphocyte cytokine production in vitro, as well as cytokine levels in vivo, were measured by ELISA. T cell proliferation was assessed by thymidine incorporation assay. Serum cholesterol levels were determined using a standard fluorometric assay. Kidney tissue was harvested and evaluated for pathologic changes. RESULTS: In NZB/NZW mice, oral atorvastatin had significant effects on T cell proliferation and cytokine production in vitro. Atorvastatin also induced significant increases in serum levels of interleukin-4. However, atorvastatin treatment in NZB/NZW mice had no significant impact on proteinuria, survival, serum anti-dsDNA antibody and cholesterol levels, or extent of renal disease. CONCLUSION: Monotherapy with oral atorvastatin has no protective effects in a murine model of spontaneous SLE. The efficacy of atorvastatin in human SLE remains to be determined.

Autoantigen arrays for multiplex analysis of antibody isotypes.

We describe here a microarray-based method for multiplexed, antigen-specific assessment of immunoglobulin (Ig) subclasses. We used 1152-feature arrays composed of 140 antigens or antigen fragments to detect isotype-specific mAb, to quantitatively monitor changes in isotype mAb concentration, and to profile antigen-specific antibody isotype production in a murine model of autoimmunity. This platform can be easily adapted to a variety of applications, and has the potential to elucidate mechanisms that govern development and evolution of antibody responses in in vivo and in vitro systems.

Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

We previously described a renal protective effect of factor B deficiency in MRL/lpr mice. Factor B is in the MHC cluster; thus, the deficient mice were H2b, the haplotype on which the knockout was derived, whereas the wild-type littermates were H2k, the H2 of MRL/lpr mice. To determine which protective effects were due to H2 vs factor B deficiency, we derived H2b congenic MRL/lpr mice from the 129/Sv (H2b) strain. Autoantibody profiling using autoantigen microarrays revealed that serum anti-Smith and anti-small nuclear ribonucleoprotein complex autoantibodies, while present in the majority of H2k/k MRL/lpr mice, were absent in the H2b/b MRL/lpr mice. Surprisingly, 70% of MRL/lpr H2b/b mice were found to be serum IgG3 deficient (with few to no IgG3-producing B cells). In addition, H2b/b IgG3-deficient MRL/lpr mice had significantly less proteinuria, decreased glomerular immune complex deposition, and absence of glomerular subepithelial deposits compared with MRL/lpr mice of any H2 type with detectable serum IgG3. Despite these differences, total histopathologic renal scores and survival were similar among the groups. These results indicate that genes encoded within or closely linked to the MHC region regulate autoantigen selection and isotype switching to IgG3 but have minimal effect on end-organ damage or survival in MRL/lpr mice.

Sources of autoantigens in systemic lupus erythematosus.

PURPOSE OF REVIEW: A hallmark of systemic lupus erythematosus is the production of autoantibodies that recognize nuclear antigens. However, the underlying events and mechanisms that lead to the selection of these molecules for the autoimmune response remain poorly understood. In this review, we will examine some of the proposed explanations for sources of systemic lupus erythematosus-specific autoantigens. We will focus on events related to apoptosis, viral infection, cytokine production, innate immune system components, and alternative splicing of pre-mRNA transcripts. RECENT FINDINGS: Definitive proof of a viral etiology for lupus remains elusive. However, recent observations have added to increasing evidence that viruses contribute to the bypass of tolerance in systemic lupus erythematosus. Also, events associated with apoptosis - most notably proteolytic autoantigen cleavage by caspases and granzyme B - have been implicated in the initiation of autoimmune responses for over a decade. Results obtained from animal models and human systems suggest complex functions for pro-apoptotic pathways in the regulation of immune responses. Inducible antigen expression and alternatively spliced transcripts may represent additional ways of generating autoantigenic material. Finally, toll-like receptor family members may play critical roles in the induction of antibody responses to nucleic acids in systemic lupus erythematosus. SUMMARY: Several factors may contribute to the generation of systemic lupus erythematosus-specific autoantigens. Determining the underlying causes of autoantibody production may provide important insight into the etiology and pathogenesis of this disease.

Granzyme B is dispensable for immunologic tolerance to self in a murine model of systemic lupus erythematosus.

OBJECTIVE: Proteolytic autoantigen cleavage by the serine protease granzyme B has been implicated in the development of systemic autoimmune disease; however, there has been no conclusive demonstration of a pathogenic role for granzyme B in autoimmunity. In this study, we evaluated the role of granzyme B in a murine model of autoimmunity. METHODS: To identify potential novel granzyme B substrates, complementary DNAs encoding nuclear factor 45 (NF45) and NF90 were used to generate (35)S-methionine-labeled proteins by coupled in vitro transcription/translation. Radiolabeled proteins were then incubated with purified recombinant granzyme B or caspases, and the cleavage products were analyzed by autoradiography. We also immunized granzyme B-deficient and granzyme B-intact mice with the mineral oil pristane. Production of autoantibodies directed against granzyme B substrates in response to pristane was evaluated by Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay. RESULTS: The double-stranded RNA-binding protein NF90 was identified as a novel substrate for caspases and granzyme B, both in vitro and in vivo. NF90 is uniquely cleaved by granzyme B in vitro; however, pristane immunization still induced anti-NF90 antibodies in granzyme B-deficient mice. Pristane-treated granzyme B-deficient mice also produced antibodies directed against the U1-70-kd antigen, a previously identified granzyme B substrate. Last, antibodies directed against U1-70 kd arose spontaneously in granzyme B-deficient mice. CONCLUSION: These results demonstrate that granzyme B is not required for the production of autoantibodies directed against antigens that are granzyme B substrates in vitro. The data also suggest a protective role for this proapoptotic protease in systemic autoimmunity.

Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.