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High mobility group box protein 1 (HMGB1) is an evolutionary ancient nuclear protein that exerts divergent biological tasks inside and outside of cells. The functions of HMGB1 depend on location, binding partners and redox states of the molecule. In the nucleus, HMGB1 organizes DNA and nucleosomes and regulates gene transcription. Upon cell activation or injury, nuclear HMGB1 can translocate to the cytoplasm, where it is involved in inflammasome activation and pyroptosis, as well as regulation of the autophagy/apoptosis balance. When actively secreted or passively released into the extracellular milieu, HMGB1 has cytokine, chemokine, neuroimmune and metabolic activities. Thus, HMGB1 plays multiple roles in the pathogenesis of inflammatory diseases and mediates immune responses that range from inflammation and bacterial killing to tissue repair. HMGB1 has been associated with divergent clinical conditions such as sepsis, rheumatoid arthritis and atherosclerosis. HMGB1 initiates and perpetuates immune responses during infectious and sterile inflammation, as the archetypical alarmin and damage-associated molecular pattern (DAMP) molecule. We here describe advances in the understanding of HMGB1 biology with focus on recent findings of its mission as a DAMP in danger sensing and as a therapeutic target in inflammatory diseases.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antinuclear antibodies (ANAs) that form immune complexes that mediate pathogenesis by tissue deposition or cytokine induction. Some ANAs bind DNA or associated nucleosome proteins, whereas other ANAs bind protein components of complexes of RNA and RNA-binding proteins (RBPs). Levels of anti-DNA antibodies can fluctuate widely, unlike those of anti-RBP antibodies, which tend to be stable. Because anti-DNA antibody levels can reflect disease activity, repeat testing is common; by contrast, a single anti-RBP antibody determination is thought to suffice for clinical purposes. Experience from clinical trials of novel therapies has provided a new perspective on ANA expression during disease, as many patients with SLE are ANA negative at screening despite previously testing positive. Because trial results suggest that patients who are ANA negative might not respond to certain agents, screening strategies now involve ANA and anti-DNA antibody testing to identify patients with so-called ‘active, autoantibody-positive SLE’. Evidence suggests that ANA responses can decrease over time because of the natural history of disease or the effects of therapy. Together, these findings suggest that, during established disease, more regular serological testing could illuminate changes relevant to pathogenesis and disease status.Antinuclear antibodies (ANAs), characteristic features of systemic lupus erythematosus (SLE), are a requirement for disease classification and trial enrolment. In this Review, the authors re-examine the role of ANAs in SLE and discuss changing attitudes towards using ANAs as biomarkers.

Key Points Signal transducer and activator of transcription (STAT) proteins have crucial immunoregulatory functions, and are particularly important for T helper cell differentiation. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP–seq) analysis has mapped the DNA binding sites of transcription factors such as STATs on a genome-wide scale. ChIP–seq technology also allows researchers to gain a genomic view of the histone epigenetic modifications that constitute the 'epigenome'. Genomic approaches enable us to link transcription factor binding with epigenomic modifications and gene expression to comprehensively evaluate the regulation of these activities. Genome-wide association studies have linked STAT genes and STAT-mediated cytokine signalling pathways to multiple immune deficiency and autoimmune disorders. Further applications of next-generation sequencing technologies include mapping of the DNA methylome, nucleosome positioning and DNase I hypersensitive sites, as well as profiling of the transcriptome (using RNA-seq) and microRNAs, underscoring the versatility of this powerful tool. This Review describes how next-generation sequencing has enriched our knowledge of how STAT proteins regulate cytokine-mediated T helper cell differentiation through direct DNA binding and by affecting epigenetic modifications. Signal transducer and activator of transcription (STAT) proteins are well known for their essential roles in transmitting cytokine-mediated signals and specifying T helper (T H ) cell differentiation. Recent technological advances have revealed that STAT proteins have broad and complex roles in gene regulation and epigenetic control, including important roles as functional repressors. However, the challenge of how to link signal transduction, nucleosome biology and gene regulation remains. The relevance of tackling this problem is highlighted by genome-wide association studies that link cytokine signalling and STATs to various autoimmune or immune deficiency disorders. Defining exactly how extrinsic signals control the specification and plasticity of T H cells will provide important insights and perhaps therapeutic opportunities in these diseases.

The nucleosome is the basic structural repeating unit of chromatin. DNA damage and cell apoptosis release nucleosomes into the blood circulatory system, and increased levels of circulating nucleosomes have been observed to be related to inflammation and autoimmune diseases. However, how circulating nucleosomes trigger immune responses has not been fully elucidated. cGAS (cGMP-AMP synthase) is a recently discovered pattern recognition receptor that senses cytoplasmic double-stranded DNA (dsDNA). In this study, we employed in vitro reconstituted nucleosomes to examine whether extracellular nucleosomes can gain access to the cytoplasm of mammalian cells to induce immune responses by activating cGAS. We showed that nucleosomes can be taken up by various mammalian cells. Additionally, we found that in vitro reconstituted mononucleosomes and oligonucleosomes can be recognized by cGAS. Compared to dsDNA, nucleosomes exhibit higher binding affinities to cGAS but considerably lower potency in cGAS activation. Incubation of monocytic cells with reconstituted nucleosomes leads to limited production of type I interferons and proinflammatory cytokines via a cGAS-dependent mechanism. This proof-of-concept study reveals the cGAS-dependent immunogenicity of nucleosomes and highlights the potential roles of circulating nucleosomes in autoimmune diseases, inflammation, and antitumour immunity.

Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.

GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4-nucleosome remodeling histone deacetylase repression complex at the Tb×21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.

Systemic lupus erythematosus (SLE) is an autoimmune disease with variable clinical expression. It is a potentially devastating condition affecting mostly women and leading to clinically unpredictable outcomes. Remission and flares may, in fact, alternate over time and a mild involvement limited to few articular sites may be followed by severe and widespread organ damage. SLE is the prototype of any autoimmune condition and has, for this reason, attracted the interest of basic immunologists. Therapies have evolved over time and clinical prognosis has, in parallel, been improved. What clinicians still lack is the possibility to use biomarkers of the disease as predictors of outcome and, in this area, several studies are trying to find solutions. Circulating autoantibodies are clearly a milestone of clinical research and the concrete possibility is to integrate, in the future, classical markers of activation (like C3) with target organ autoantibodies. Anti-dsDNA antibodies represent a basic point in any predictive attempt in SLE and should be considered the benchmark for any innovative proposal in the wide field of target organ pathologies related to SLE. DNA is part of the nucleosome that is the basic unit of chromatin. It consists of DNA wrapped around a histone octamer made of 2 copies each of Histone 2A, 2B, 3, and 4. The nucleosome has a plastic organization that varies over time and has the potential to stimulate the formation of antibodies directed to the whole structure (anti-nucleosome) or its parts (anti-dsDNA and anti-Histones). Here, we present an updated review of the literature on antibodies directed to the nucleosome and the nucleosome constituents, i.e., DNA and Histones. Wetriedto merge the data first published more than twenty years ago with more recent results to create a balanced bridge between old dogma and more recent research that could serve as a stimulus to reconsider mechanisms for SLE. The formation of large networks would provide the chance of studying large cohorts of patients and confirm what already presented in small sample size during the last years.

To characterize the significance of correlated autoantibodies in systemic lupus erythematosus (SLE) and its complication lupus nephritis (LN) in a large cohort of patients. Clinical data were statistically analyzed in 1699 SLE patients with or without nephritis who were diagnosed and treated during 2002-2013 in the northeast region of China. Reactivity to a list of 16 autoantibodies was detected by the serum test Euroline ANA profile (IgG). Serum titers of the anti-nucleosome autoantibodies were measured by ELISA assays. Kidney biopsies were examined by pathologists. Immune complex deposition was identified by immunohistochemistry stain. Simultaneous positivity of anti-dsDNA, -nucleosome and -histone antibodies (3-pos) was prevalent in SLE patients with LN compared to Non-renal SLE patients (41% vs 11%, p< 0.001). Significant correlations were found between any two of the above three anti-nucleosome antibodies in LN patients. In comparison to non-3-pos cohorts, 3-pos patients with LN had significantly higher serum levels of the three antibodies and more active disease; was associated with type IV disease; suffered from more severe renal damages; received more intensive treatment and had worse disease outcome. The serum levels of these three autoantibodies in 3-pos LN patients were significantly decreased when they underwent clinical recovery. Simultaneous reactivity to anti-dsDNA, -nucleosome and -histone antibodies by Euroline ANA profile (IgG) may indicate severe nephropathy in patients with SLE.

Schizophrenia is known to be accompanied not only with an imbalance in the neurotransmitter systems but also with immune system dysregulation and chronic low-grade inflammation. Extracellular histones and nucleosomes as damage-associated molecular patterns (DAMPs) trigger systemic inflammatory and toxic reactions by activating Toll-like receptors. In this work, we obtained the first evidence that polyclonal IgGs of patients with schizophrenia effectively hydrolyze five histones (H1, H2a, H2b, H3, and H4). Several strict criteria were used to demonstrate that histone-hydrolyzing activity is a property of the analyzed IgGs. The IgGs histone-hydrolyzing activity level, depending on the type of histone (H1–H4), was statistically significantly 6.1–20.2 times higher than that of conditionally healthy donors. The investigated biochemical properties (pH and metal ion dependences, kinetic characteristics) of these natural catalytic IgGs differed markedly from canonical proteases. It was previously established that the generation of natural catalytic antibodies is an early and clear sign of impaired humoral immunity. One cannot, however, exclude that histone-hydrolyzing antibodies may play a positive role in schizophrenia pathogenesis because histone removal from circulation or the inflamed area minimizes the inflammatory responses. Thus, it can be assumed that histone-hydrolyzing antibodies are a link between humoral immunity and inflammatory responses in schizophrenia.

Antibodies to double-stranded (dsDNA) are associated with systemic lupus erythematosus (SLE) and directly involved in human lupus nephritis. Information about their glomerular target antigens is inconsistent, and whether availability of target antigens, antibody specificity or avidity are nephritogenic parameters, is not determined. In this study, we analyzed renal tissue from anti-dsDNA antibody-positive lupus patients with nephritis by morphological and immunological assays, including immune electron microscopy (IEM) and colocalization IEM, an EM-based confocal microscopy assay. IEM demonstrated that antibody deposits were confined to electron dense structures (EDS) in glomerular membranes. These autoantibodies colocalized with nucleosome-binding anti-dsDNA/-histone/-transcription factor antibodies. To confirm the colocalization IEM-data, we developed a colocalization terminal deoxynucleotidyl-transferase (TdT) biotin-dUTP nicked end-labeled (TUNEL) IEM assay where extracellular DNA was traced by TdT-mediated introduction of biotinylated nucleotides and autoantibodies by IEM. Results consistently demonstrated that DNA colocalized with autoantibodies in glomerular membrane-associated EDS. The colocalization IEM and colocalization TUNEL IEM assays thus demonstrate that intra-glomerular membrane-associated nucleosomes are targeted by anti-dsDNA autoantibodies in human lupus nephritis. The data provide a new approach to understand basic molecular and immunological processes accounting for antibody-mediated nephritis in human SLE.