Increased adipose catecholamine levels and protection from obesity with loss of Allograft Inflammatory Factor-1.

Recent studies implicate macrophages in regulation of thermogenic, sympathetic neuron-mediated norepinephrine (NE) signaling in adipose tissues, but understanding of such non-classical macrophage activities is incomplete. Here we show that male mice lacking the allograft inflammatory factor-1 (AIF1) protein resist high fat diet (HFD)-induced obesity and hyperglycemia. We link this phenotype to higher adipose NE levels that stem from decreased monoamine oxidase A (MAOA) expression and NE clearance by AIF1-deficient macrophages, and find through reciprocal bone marrow transplantation that donor Aif1-/- vs WT genotype confers the obesity phenotype in mice. Interestingly, human sequence variants near the AIF1 locus associate with obesity and diabetes; in adipose samples from participants with obesity, we observe direct correlation of AIF1 and MAOA transcript levels. These findings identify AIF1 as a regulator of MAOA expression in macrophages and catecholamine activity in adipose tissues - limiting energy expenditure and promoting energy storage - and suggest how it might contribute to human obesity.

Rare variants and HLA haplotypes associated in patients with neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) are rare, debilitating autoimmune diseases of the central nervous system. Many NMOSD patients have antibodies to Aquaporin-4 (AQP4). Prior studies show associations of NMOSD with individual Human Leukocyte Antigen (HLA) alleles and with mutations in the complement pathway and potassium channels. HLA allele associations with NMOSD are inconsistent between populations, suggesting complex relationships between the identified alleles and risk of disease. We used a retrospective case-control approach to identify contributing genetic variants in patients who met the diagnostic criteria for NMOSD and their unaffected family members. Potentially deleterious variants identified in NMOSD patients were compared to members of their families who do not have the disease and to existing databases of human genetic variation. HLA sequences from patients from Belgrade, Serbia, were compared to the frequency of HLA haplotypes in the general population in Belgrade. We analyzed exome sequencing on 40 NMOSD patients and identified rare inherited variants in the complement pathway and potassium channel genes. Haplotype analysis further detected two haplotypes, HLA-A*01, B*08, DRB1*03 and HLA-A*01, B*08, C*07, DRB1*03, DQB1*02, which were more prevalent in NMOSD patients than in unaffected individuals. In silico modeling indicates that HLA molecules within these haplotypes are predicted to bind AQP4 at several sites, potentially contributing to the development of autoimmunity. Our results point to possible autoimmune and neurodegenerative mechanisms that cause NMOSD, and can be used to investigate potential NMOSD drug targets.

HIV-1 Tat and cocaine impact mitochondrial epigenetics: effects on DNA methylation.

Human immunodeficiency virus (HIV) infection and the psychostimulant drug cocaine are known to induce epigenetic changes in DNA methylation that are linked with the severity of viral replication and disease progression, which impair neuronal functions. Increasing evidence suggests that changes in DNA methylation and hydroxymethylation occur in mitochondrial DNA (mtDNA) and represent mitochondrial genome epigenetic modifications (mitoepigenetic modifications). These modifications likely regulate both mtDNA replication and gene expression. However, mtDNA methylation has not been studied extensively in the contexts of cocaine abuse and HIV-1 infection. In the present study, epigenetic factors changed the levels of the DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b, the Ten-eleven translocation (TET) enzymes 1, 2, and 3, and mitochondrial DNMTs (mtDNMTs) both in vitro and in vivo. These changes resulted in alterations in mtDNA methylation levels at CpG and non-CpG sites in human primary astrocytes as measured using targeted next-generation bisulphite sequencing (TNGBS). Moreover, mitochondrial methylation levels in the MT-RNR1, MT-ND5, MT-ND1, D-loop and MT-CYB regions of mtDNA were lower in the HIV-1 Tat and cocaine treatment groups than in the control group. In summary, the present findings suggest that mitoepigenetic modification in the human brain causes the mitochondrial dysfunction that gives rise to neuro-AIDS.

Clinical significance of anti-DNA/N-methyl-D-aspartate receptor 2 antibodies in de novo and post-steroid cases with neuropsychiatric systemic lupus erythematosus.

BACKGROUND: Anti-DNA/N-methyl-D-aspartate receptor 2 (NR2) antibodies (anti-DNA/NR2 antibodies) are a subset of anti-DNA autoantibodies that cross-react with the extracellular domain of the GluN2A/GluN2B subunits of NR2. These antibodies induce apoptosis of hippocampus neurons and psychiatric disorder in mice and humans. Neuropsychiatric system lupus erythematosus (NPSLE) can develop after initiation of corticosteroids (post-steroid neuropsychiatric manifestation: PSNP) or before treatment (de novo NPSLE); however, pathophysiological differences between these subtypes remain unclear. The objective of this study was to clarify the prevalence of anti-DNA/NR2 antibodies in patients with NPSLE. METHODS: This study involved a cohort of patients with NPSLE admitted to our hospital. NPSLE patients were classified into two groups, de novo NPSLE and PSNP-SLE. Serum anti-DNA antibodies and anti-DNA/NR2 antibodies were measured by enzyme-linked immunosorbent assays. RESULTS: Serum samples were obtained from 24 patients with de novo NPSLE, 25 with PSNP-SLE and 76 healthy controls (HC). The level of anti-DNA/NR2 antibodies in patients with de novo NPSLE and PSNP-SLE were also higher than those in HC. Positive correlation between anti-DNA antibodies and anti-DNA/NR2 antibodies were found in PSNP-SLE, but was not significant in de novo NPSLE. CONCLUSION: The levels of anti-DNA/NR2 antibodies in PSNP-SLE were similar to those in de novo NPSLE. Anti-DNA/NR2 antibodies in PSNP-SLE were suggested as a dominant subset of anti-DNA antibodies, indicating that anti-DNA/NR2 antibodies may be a predictive factor in PSNP-SLE.

Understanding the Antibody Repertoire in Neuropsychiatric Systemic Lupus Erythematosus and Neuromyelitis Optica Spectrum Disorder: Do They Share Common Targets?

OBJECTIVE: IgG anti-DWEYS antibodies cross-reactive with DNA and the N-methyl-d-aspartate receptor subunits GluN2A and GluN2B are known to be associated with neuropsychiatric systemic lupus erythematosus (NPSLE). IgG anti-DWEYS have not been investigated in demyelinating NPSLE or in another demyelinating disorder, neuromyelitis optica spectrum disorder (NMOSD), which is a disease also found mainly in young women and associated with aquaporin 4 (AQP-4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. This study was undertaken to investigate the frequency of all of these brain-reactive antibodies in patients with NPSLE, those with demyelinating NPSLE, and those with NMOSD. METHODS: Serum samples from patients with NPSLE (n = 108), patients with SLE without neuropsychiatric manifestations (n = 38), patients with NMOSD (n = 33), and healthy controls (n = 106) were assessed for the frequency of IgG anti-brain antibodies as well as IgG antibodies to AQP-4, MOG, GluN2A/GluN2B, and double-stranded DNA (dsDNA). RESULTS: Sera were positive for IgG anti-AQP-4 antibodies in 27 (82%) of 33 patients with NMOSD and 3 (27%) of 11 patients with demyelinating NPSLE, whereas all sera from patients with non-demyelinating NPSLE, patients with SLE, and healthy controls were negative for IgG anti-AQP-4. IgG anti-MOG were detected at high titers in 3 (50%) of 6 patients with NMOSD who were negative for IgG anti-AQP-4, and at low titers in 2 (18%) of 11 patients with demyelinating NPSLE and 1 (1%) of 97 patients with non-demyelinating NPSLE. IgG antibodies to dsDNA were present in 11 (33%) of 33 patients with NMOSD. Only 4 (12%) of 33 patients with NMOSD were positive for IgG anti-DWEYS, compared to 11 (29%) of 38 patients with SLE and 59 (55%) of 108 patients with NPSLE. IgG anti-DWEYS antibodies were present in 56 (58%) of 97 patients with non-demyelinating NPSLE and 3 (27%) of 11 patients with demyelinating NPSLE. Serum IgG brain-reactive antibodies were present at a similar frequency in patients with non-demyelinating NPSLE (72 [75%] of 96), those with demyelinating NPSLE (9 [82%] of 11), and those with SLE (32 [84%] of 38), but were less frequent in patients with NMOSD (20 [61%] of 33). CONCLUSION: Patients with demyelinating NPSLE should be tested for IgG antibodies to AQP-4, MOG, and DWEYS. IgG anti-AQP-4 can be considered diagnostic for NMOSD, whereas none of these antibodies appear to be diagnostic for demyelinating NPSLE. Moreover, IgG anti-dsDNA are present in patients with NMOSD but are not cross-reactive with IgG anti-DWEYS, indicating that the antigenic stimulus and mechanisms of tissue damage are potentially different between demyelinating NPSLE and NMOSD.

Amending HIV Drugs: A Novel Small-Molecule Approach To Target Lupus Anti-DNA Antibodies.

Systemic lupus erythematosus is an autoimmune disease that can affect numerous tissues and is characterized by the production of nuclear antigen-directed autoantibodies (e.g., anti-dsDNA). Using a combination of virtual and ELISA-based screens, we made the intriguing discovery that several HIV-protease inhibitors can function as decoy antigens to specifically inhibit the binding of anti-dsDNA antibodies to target antigens such as dsDNA and pentapeptide DWEYS. Computational modeling revealed that HIV-protease inhibitors comprised structural features present in DWEYS and predicted that analogues containing more flexible backbones would possess preferred binding characteristics. To address this, we reduced the internal amide backbone to improve flexibility, producing new small-molecule decoy antigens, which neutralize anti-dsDNA antibodies in vitro, in situ, and in vivo. Pharmacokinetic and SLE model studies demonstrated that peptidomimetic FISLE-412,1 a reduced HIV protease inhibitor analogue, was well-tolerated, altered serum reactivity to DWEYS, reduced glomeruli IgG deposition, preserved kidney histology, and delayed SLE onset in NZB/W F1 mice.

Checkpoints for Autoreactive B Cells in the Peripheral Blood of Lupus Patients Assessed by Flow Cytometry.

OBJECTIVE: Antinuclear antibodies (ANAs) are diagnostic in several autoimmune disorders, yet the failure to achieve B cell tolerance in these diseases is still poorly understood. Although secreted ANAs detected by an indirect immunofluorescence assay are the gold standard for autoreactivity, there has been no convenient assay with which to measure the frequency of circulating B cells that recognize nuclear antigens (ANA+ B cells) in patients. The aim of this study was to generate an assay to easily identify these B cells and to examine its utility in a study of autoreactive B cells in systemic lupus erythematosus (SLE). METHODS: We developed and validated a novel flow cytometry-based assay that identifies ANA+ B cells using biotinylated nuclear extracts, and utilized it to examine B cell tolerance checkpoints in peripheral blood mononuclear cells obtained from SLE patients and healthy controls. RESULTS: We observed progressive selection against ANA+ B cells as they matured from transitional to naive to CD27+IgD- and CD27+IgD+ memory cells in both healthy subjects and SLE patients; however, ANA+ naive B cells in SLE patients were not anergized to the same extent as in healthy individuals. We also showed that anergy induction is restored in SLE patients treated with belimumab, an inhibitor of BAFF. CONCLUSION: This assay will enable studies of large populations to identify potential genetic or environmental factors affecting B cell tolerance checkpoints in healthy subjects and patients with autoimmune disease and permit monitoring of the B cell response to therapeutic interventions.

Advancing drug delivery systems for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. It is characterized by demyelination of neurons and loss of neuronal axons and oligodendrocytes. In MS, auto-reactive T cells and B cells cross the blood-brain barrier (BBB), causing perivenous demyelinating lesions that form multiple discrete inflammatory demyelinated plaques located primarily in the white matter. In chronic MS, cortical demyelination and progressive axonal transections develop. Treatment for MS can be stratified into disease-modifying therapies (DMTs) and symptomatic therapy. DMTs aim to decrease circulating immune cells or to prevent these cells from crossing the BBB and reduce the inflammatory response. There are currently 10 DMTs approved for the relapsing forms of MS; these vary with regard to their efficacy, route and frequency of administration, adverse effects, and toxicity profile. Better drug delivery systems are being developed in order to decrease adverse effects, increase drug efficacy, and increase patient compliance through the direct targeting of pathologic cells. Here, we address the uses and benefits of advanced drug delivery systems, including nanoparticles, microparticles, fusion antibodies, and liposomal formulations. By altering the properties of therapeutic particles and enhancing targeting, breakthrough drug delivery technologies potentially applicable to multiple disease treatments may rapidly emerge.

Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion.

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is mediated by myelin-specific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. The molecular bases for expansion and activation of these cells, plus trafficking to the CNS for peripheral cells, are not fully understood. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca(2+) binding adapter-1 [Iba-1]) is induced in leukocytes in MS and EAE; here we provide the first assessment of Aif-1 function in this setting. After myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization, Aif-1-deficient mice were less likely than controls to develop EAE and had less CNS leukocyte infiltration and demyelination; their spinal cords contained fewer CD4 T cells and microglia and more CD8 T cells. These mice also showed significantly less splenic CD4 T-cell expansion and activation, plus decreased proinflammatory cytokine expression. These findings identify Aif-1 as a potent molecule that promotes expansion and activation of CD4 T cells, plus elaboration of a proinflammatory cytokine milieu, in MOG35-55-induced EAE and as a potential therapeutic target in MS.

Hormonal milieu at time of B cell activation controls duration of autoantibody response.

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.

Differential roles of estrogen receptors α and ß in control of B-cell maturation and selection.

It is clear that estrogen can accelerate and exacerbate disease in some lupus-prone mouse strains. It also appears that estrogen can contribute to disease onset or flare in a subset of patients with lupus. We have previously shown estrogen alters B-cell development to decrease lymphopoiesis and increase the frequency of marginal zone B cells. Furthermore, estrogen diminishes B-cell receptor signaling and allows for the increased survival of high-affinity DNA-reactive B cells. Here, we analyze the contribution of estrogen receptor α or ß engagement to the altered B-cell maturation and selection mediated by increased exposure to estrogen. We demonstrate that engagement of either estrogen receptor α or ß can alter B-cell maturation, but only engagement of estrogen receptor α is a trigger for autoimmunity. Thus, maturation and selection are regulated differentially by estrogen. These observations have therapeutic implications.

Hormonal regulation of B cell development: 17 beta-estradiol impairs negative selection of high-affinity DNA-reactive B cells at more than one developmental checkpoint.

There are increasing data suggesting that sex hormones, such as estrogen, have immunomodulatory effects and play a role in disease progression and pathogenesis in patients with the autoimmune disorder systemic lupus erythematosus. We have shown previously that treatment with 17beta-estradiol (E2) induces a lupus phenotype in BALB/c mice that express a transgene-encoded H chain of an anti-DNA Ab. Because E2 treatment interferes with normal tolerance of naive DNA-reactive B cells, we elected to study the effects of hormonal modulation on the regulation of autoreactive B cells at early developmental checkpoints. Single-cell PCR was performed to study the repertoire of DNA-reactive B cell subsets. High-affinity DNA-reactive B cells were rescued at both the immature and transitional B cell stage in E2-treated mice. Interestingly, although low-affinity DNA-reactive B cells survive negative selection in control mice, the frequency of these cells was significantly reduced in the mature pool of E2-treated mice, suggesting that the high-affinity DNA-reactive cells that mature to immunocompetence out-compete the low-affinity population for survival as mature B cells. These data provide evidence that an elevation in serum levels of E2 facilitates the maturation of a pathogenic naive autoreactive B cell repertoire and hampers the maturation of a potentially protective autoreactive B cell repertoire. Furthermore, these data show that both positive and negative selection occur within the transitional B cell stage.