Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE

Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1–nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1β, IL-6, IL-10, and tumor necrosis factor (TNF) α and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2–dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1–nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA

High mobility group box 1 Protein in der Immunopathogenese des systemischen Lupus erythematodes

High mobility group box protein 1 (HMGB1) is a nuclear DNA binding protein and a potent pro-inflammatory mediator when released from cells. Recently, it has become obvious that HMGB1 plays a role in the pathogenesis of both human and experimental diseases such as arthritis and sepsis. Investigations of HMGB1 in diverse pathological conditions, however, have been precluded due to the absence of a sensitive and reliable analytical system for the detection of HMGB1 in biological fluids. In the first part of this thesis sensitive ELISAs for the detection of HMGB1 in cell culture medium and cell lysates were established. However, these assays failed to reliably quantitate HMGB1 in human serum and plasma. We found that serum/plasma components, including autoantibodies to HMGB1, bind to HMGB1 and interfere with its detection by ELISA systems. In addition to complex formation and interference with HMGB1 detection by ELISA, these HMGB1-interacting proteins may also modulate cytokine activity of HMGB1. Pathogenic autoantibodies against double stranded (ds) DNA and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the factors involved in breaking the immunological tolerance against these nuclear components are not fully understood. The “endogenous adjuvant” HMGB1 gets tightly attached to hypoacetylated chromatin during apoptotic cell death. In conditions of clearance deficiency, as it is the case in many patients with SLE, non-ingested apoptotic cells may undergo secondary necrosis, thereby releasing nucleosomes containing HMGB1. In the second part of this work, we investigated whether HMGB1 nucleosome complexes are in fact detectable in vitro and in vivo and may contribute to the breakdown of immunological tolerance against dsDNA and nucleosmes. We found that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. Moreover, HMGB1 nucleosome complexes were detected in sera and plasma from SLE patients. HMGB1 containing nucleosomes from apoptotic cells were potent inducers of cytokines from macrophages and induced cytokine release and maturation of DC, which was crucially dependent on the presence of HMGB1. Cytokine release from macrophages was dependent on the presence of MyD88 and Toll-like receptor (TLR) 2. In vivo, immunizations with nucleosomes from apoptotic cells induced anti-dsDNA and anti-histone IgG responses in non autoimmune mice, whereas nucleosomes from viable cells did not. In conclusion, HMGB1 nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the immunopathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA and could represent a target for immunomodulatory therapy.HMGB1 (high mobility group box 1) ist nicht nur intrazellulär als strukturelles Kernprotein für die Chromatinarchitektur und Transkriptionskontrolle, sondern auch extrazellulär als proinflammatorisches Zytokin bedeutend. Es wird vermutet, dass HMGB1 eine wichtige Rolle bei der Pathogenesse von Sepsis und rheumatoider Arthritis spielt und somit einen potentiellen therapeutischen Angriffspunkt darstellt. Aufgrund der mangelhaften Nachweismethoden für HMGB1 in biologischen Flüssigkeiten gibt es kaum klinische Untersuchungen insbesondere an größeren Kollektiven. Im ersten Teil dieser Arbeit sollte daher ein sensitives ELISA-System zum Nachweis von HMGB1 in Zellkultur-Überständen und Zelllysaten entwickelt werden. Mit diesem ELISA konnte jedoch kein zuverlässiger Nachweis von HMGB1 in Serum/Plasma Proben erfolgen, da Särumkomponenten, einschließlich IgG- und IgM-Autoantikörper gegen HMGB1, an zirkulierendes HMGB1 binden und dadurch die Detektion im ELISA beeinträchtigen. Außerdem können HMGB1 interagierende Serumproteine die Zytokinaktivität des HMGB1 modulieren. Antikörper gegen doppelsträngige-DNS (dsDNS) und Nukleosomen sind charakteristische serologische Marker für den systemischen Lupus erythematodes (SLE). Die Faktoren, die verantwortlich sind für den Verlust der immunologischen Toleranz gegen die nukleären Autoantigene, sind jedoch noch nicht vollständig charakterisiert. Während HMGB1 bei nekrotischem Zelltod rasch freigesetzt wird, bleibt es beim apoptotischen Zelltod intrazellulär fest mit dem Chromatin assoziiert. Da bei zahlreichen SLE-Patienten ein Defekt in der Phagozytose apoptotischer Zellen besteht, können diese sekundär nekrotisch werden und dabei Nukleosomen freisetzen, die mit dem „endogenen Adjuvans“ HMGB1 assoziiert sind. Dieser Prozess könnte somit die Autoimmunreaktion gegen das Nukleosom, einem zentralen Autoantigen beim SLE, fördern. Daher wurde im zweiten Teil dieser Arbeit untersucht, ob HMGB1-Nukleosomen Komplexe tatsächlich in vitro und in vivo freigesetzt werden und ob sie eine inflammatorische und immunogene Wirkung besitzen. Es wurde gezeigt, dass Nukleosomen, die von spät-apoptotischen Zellen freigesetzt werden, mit HMGB1 assoziiert sind. Auch im Blut von SLE-Patienten wurden HMGB1 Nukleosomen Komplexe nachgewiesen. Ferner wurde demonstriert, dass die HMGB1 beinhaltenden Nukleosomen Induktoren für die Sekretion von Zytokinen durch Makrophagen und für die Reifung dendritischer Zellen (DC) sind, wofür eindeutig das Nukleosomenassoziierte HMGB1 verantwortlich gemacht werden konnte. Die Zytokinfreisetzung von Makrophagen war dabei abhängig vom Toll-like Rezeptor 2 vermittelten Signalweg. Schließlich konnte durch die Immunisierung mit Nukleosomen, isoliert aus apoptotischen Zellen, die Bildung von Antikörpern gegen dsDNS and Histone in nichtautoimmunen Mausstämmen induziert werden. Zusammenfassend konnte gezeigt werden, dass Nukleosomen aus apoptotischen Zellen, die mit dem „endogenen Adjuvans“ HMGB1 assoziiert sind, Antigen-präsentierende Zellen aktivieren und dadurch die Entstehung der Autoimmunreaktion gegen Nukleosomen beim SLE entscheidend fördern können

Low Production of Reactive Oxygen Species Drives Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a common autoimmune disease. Recent findings have shown that a major single nucleotide variant predisposing to SLE is associated with low production of reactive oxygen species (ROS). A variant amino acid in a frequent NCF1 allele causing deficient ROS production leads to an exaggerated type I interferon (IFN) response, earlier disease onset, and higher susceptibility to SLE. It is the so far strongest identified single nucleotide variant, with an odds ratio (OR) of >3 and an allele frequency of >10%. Its functional role is in sharp contrast to the earlier belief that excessive ROS production is exclusively pathogenic rather than protective. It opens new possibilities to understand the pathogenesis of SLE and to develop novel diagnostics and treatment strategies

Association of High Mobility Group Box Chromosomal Protein 1 and Receptor for Advanced Glycation End Products Serum Concentrations With Extraglandular Involvement and Disease Activity in Sjögren's Syndrome.

OBJECTIVE To assess serum levels of high mobility group box chromosomal protein 1 (HMGB-1) and the soluble receptor for advanced glycation end products (sRAGE) in patients with Sjögren's syndrome (SS) and explore correlations with disease activity. METHODS Thirty-nine patients with SS and 21 healthy controls were included in this cross-sectional study. Clinical and laboratory values were obtained from all patients. Disease activity was assessed using the European League Against Rheumatism SS Disease Activity Index (ESSDAI). Serum samples were collected and HMGB-1 and sRAGE levels were measured using enzyme-linked immunosorbent assay (ELISA), and HMGB-1 concentrations were semiquantified by Western blotting. RESULTS In ELISA, HMGB-1 serum levels did not differ between healthy controls and patients with SS (P = 0.783). When measured by semiquantitative Western blotting, HMGB-1 levels were increased in patients with SS compared to healthy controls (P = 0.012). HMGB-1 serum levels detected by Western blotting were higher in patients with extraglandular manifestations (P = 0.003) and were correlated with ESSDAI disease activity (r = 0.544, P < 0.0001). Furthermore, sRAGE was elevated in the sera of patients with SS (P = 0.003) compared to healthy controls and was also correlated with the ESSDAI (r = 0.545, P = 0.002). CONCLUSION Serum levels of total HMGB-1 and sRAGE were elevated in patients with SS compared to healthy controls and correlated with disease activity as measured by the ESSDAI. Patients with extraglandular involvement had high serum levels of HMGB-1

Standardization of Antigen-Emulsion Preparations for the Induction of Autoimmune Disease Models

Autoimmune murine disease models are vital tools for identifying novel targets and finding better treatments for human diseases. Complete Freund’s adjuvant is commonly used to induce disease in autoimmune models, and the quality of the adjuvant/autoantigen emulsion is of critical importance in determining reproducibility. We have established an emulsification method using a standard homogenizer and specially designed receptacle. Emulsions are easy to prepare, form stable and uniform water-in-oil particles, are faster to make than the traditional syringe method, use less material and are designed to fill syringes with ease. In the present study, we have validated the emulsions for induction of experimental autoimmune encephalitis, collagen II induced arthritis, antigen induced arthritis, and delayed type hypersensitivity models. These models were induced consistently and reproducibly and, in some cases, the new method outperformed the traditional method. The method described herein is simple, cost-effective and will reduce variability, thereby requiring fewer animals for in vivo research involving animal models of autoimmune disease and in vaccine development

T cells specific for post-translational modifications escape intrathymic tolerance induction

Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism

A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis

Although anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis and generally considered pathogenic, their functional relevance is incompletely understood. In this study, the authors describe an ACPA with a protective effect against antibody-induced arthritis in mice