Luis E. Munoz

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.

The role of defective clearance of apoptotic cells in systemic autoimmunity

The inefficient clearance of dying cells can result in the accumulation of apoptotic cell remnants. This occurrence is considered an intrinsic defect that can cause the permanent presence of cellular debris responsible for the initiation of systemic autoimmunity in diseases such as systemic lupus erythematosus (SLE). If postapoptotic debris accumulates in germinal centers, activates complement and functions as a survival signal for B cells that have become autoreactive by somatic hypermutation, autoimmunity could arise (etiology). The accumulation of postapoptotic remnants and fragments derived from secondary necrotic cells in the presence of autoantibodies against apoptotic cells or adaptor molecules obliges their pathological elimination and maintains autoinflammation. The autoimmunity that occurs in patients with SLE involves complex antigens that contain nucleic acids, which can function as virus mimetics. Complexes of autoantibodies, proteins and nucleic acids are likely to be mistaken by the immune system for opsonized viruses, resulting in the production of type I interferons, a hallmark of SLE (pathogenesis). The pathogenicity of autoantibodies is thought to strongly increase if autoantigens are accessible for immune-complex formation. The immune complex could be considered a binary pyrogen formed from less proinflammatory components. The accessibility of cognate autoantigens, in turn, is likely to be related to impaired or delayed clearance of apoptotic cells.

Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines

Gout is characterized by an acute inflammatory reaction and the accumulation of neutrophils in response to monosodium urate (MSU) crystals. Inflammation resolves spontaneously within a few days, although MSU crystals can still be detected in the synovial fluid and affected tissues. Here we report that neutrophils recruited to sites of inflammation undergo oxidative burst and form neutrophil extracellular traps (NETs). Under high neutrophil densities, these NETs aggregate and degrade cytokines and chemokines via serine proteases. Tophi, the pathognomonic structures of chronic gout, share characteristics with aggregated NETs, and MSU crystals can induce NETosis and aggregation of NETs. In individuals with impaired NETosis, MSU crystals induce uncontrolled production of inflammatory mediators from neutrophils and persistent inflammation. Furthermore, in models of neutrophilic inflammation, NETosis-deficient mice develop exacerbated and chronic disease that can be reduced by adoptive transfer of aggregated NETs. These findings suggest that aggregated NETs promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation.

Apoptosis in the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a prototype inflammatory autoimmune disease resulting from autoimmune responses against nuclear autoantigens. During apoptosis many lupus autoantigens congregate inside the cells and are susceptible to modifications. Modified nuclear constituents are considered foreign and dangerous. Therefore, apoptotic cells have to has to be efficiently removed to avoid the accumulation of apoptotic debris and the subsequently development of autoimmune responses. Hence, apoptosis and clearance of apoptotic cells/material are considered key processes in the aetiology of SLE. Clearance deficiencies may account for the development of autoimmunity by inducing a loss of tolerance in lymphoid tissues. Furthermore, phagocytosis of apoptotic cells may lead to a pro-inflammatory response in the presence of autoantibodies. This may sustain inflammatory conditions and the pathology found in overt lupus.

Autophagy regulates TNFα-mediated joint destruction in experimental arthritis

Objectives Autophagy is a homeostatic process to recycle dispensable and damaged cell organelles. Dysregulation of autophagic pathways has recently been implicated in the pathogenesis of various diseases. Here, we investigated the role of autophagy during joint destruction in arthritis. Methods Autophagy in osteoclasts was analysed in vitro and ex vivo by transmission electron microscopy, Western blotting and immunohistochemistry for Beclin1 and Atg7. Small molecule inhibitors, LysMCre-mediated knockout of Atg7 and lentiviral overexpression of Beclin1 were used to modulate autophagy in vitro and in vivo. Osteoclast differentiation markers were quantified by real-time PCR. The extent of bone and cartilage destruction was analysed in human tumour necrosis factor α transgenic (hTNFα tg) mice after adoptive transfer with myeloid specific Atg7-deficient bone marrow. Results Autophagy was activated in osteoclasts of human rheumatoid arthritis (RA) showing increased expression of Beclin1 and Atg7. TNFα potently induced the expression of autophagy-related genes and activated autophagy in vitro and in vivo. Activation of autophagy by overexpression of Beclin1-induced osteoclastogenesis and enhanced the resorptive capacity of cultured osteoclasts, whereas pharmacologic or genetic inactivation of autophagy prevented osteoclast differentiation. Arthritic hTNFα tg mice transplanted with Atg7fl/fl×LysMCre+ bone marrow cells (BMC) showed reduced numbers of osteoclasts and were protected from TNFα-induced bone erosion, proteoglycan loss and chondrocyte death. Conclusions These findings demonstrate that autophagy is activated in RA in a TNFα-dependent manner and regulates osteoclast differentiation and bone resorption. We thus provide evidence for a central role of autophagy in joint destruction in RA.

Autoimmunity and chronic inflammation — Two clearance-related steps in the etiopathogenesis of SLE

Systemic lupus erythematosus (SLE) is an autoimmune disease with very prominent chronic inflammatory aspects that render into multiple symptoms and clinical signs. The precise etiology of SLE remains elusive; however, it is known that its etiopathogenesis is of multifactorial nature. The production of autoantibodies (AAb) targeting double stranded DNA (dsDNA) and other nuclear autoantigens is the main characteristic of this disease. These target antigens are often modified and/or translocated when apoptotic cells undergo secondary necrosis as a consequence of the clearance deficiency in patients with SLE. In healthy individuals, dead and dying cells are rapidly removed by macrophages in an anti-inflammatory context; this does not elicit immune responses. In SLE, apoptotic cells are often not properly cleared; autoantigens leak out, and are subsequently presented to B cells by follicular dendritic cells (FDC) in secondary lymphoid tissues. This defect challenges the peripheral self-tolerance. Autoreactive B cell activation and production of anti-nuclear AAb result as the first step in the etiopathogenesis of SLE. The second step is the formation of immune complexes (IC) with apoptotic cell-derived nuclear remnants either in situ or deposited in various tissues. Nucleic acid-containing IC may also be ingested by phagocytes, which subsequently produce pro-inflammatory cytokines. Both processes result in chronic organ and tissue damage, development and maintenance of the systemic autoimmune disease. In conclusion, clearance deficiency may contribute to SLE in two ways: first, in germinal centres it enables the affinity maturation of autoreactive B cells and second, in peripheral tissues it leads to the accumulation of accessible nuclear autoantigens. Chronic inflammation in SLE is consequently promoted by the persistently binding of AAb with their cognate autoantigens forming a binary weapon: the nucleic acid-containing IC.

Remnants of secondarily necrotic cells fuel inflammation in systemic lupus erythematosus.

OBJECTIVE Patients with systemic lupus erythematosus (SLE) are often characterized by cellular as well as humoral deficiencies in the recognition and phagocytosis of dead and dying cells. The aim of this study was to investigate whether the remnants of apoptotic cells are involved in the induction of inflammatory cytokines in blood-borne phagocytes. METHODS We used ex vivo phagocytosis assays comprising cellular and humoral components and phagocytosis assays with isolated granulocytes and monocytes to study the phagocytosis of secondarily necrotic cell-derived material (SNEC). Cytokines were measured by multiplex bead array technology. RESULTS We confirmed the impaired uptake of various particulate targets, including immunoglobulin-opsonized beads, by granulocytes and monocytes from patients with SLE compared with healthy control subjects. Surprisingly, blood-borne phagocytes from two-thirds of the patients with SLE took up SNEC, which was rarely phagocytosed by phagocytes from healthy control subjects or patients with rheumatoid arthritis. Supplementation of healthy donor blood with IgG fractions derived from patients with SLE transferred the capability to take up SNEC to the phagocytes of healthy donors. Phagocytosis-promoting immune globulins also induced secretion of huge amounts of cytokines by blood-borne phagocytes following uptake of SNEC. CONCLUSION Opsonization of SNEC by autoantibodies from patients with SLE fosters its uptake by blood-borne monocytes and granulocytes. Autoantibody-mediated phagocytosis of SNEC is accompanied by secretion of inflammatory cytokines, fueling the inflammation that contributes to the perpetuation of autoimmunity in SLE.

Phospholipids: Key Players in Apoptosis and Immune Regulation

Phosphatidylserine (PS), a phospholipid predominantly found in the inner leaflet of eukaryotic cellular membranes, plays important roles in many biological processes. During apoptosis, the asymmetric distribution of phospholipids of the plasma membrane gets lost and PS is translocated to the outer leaflet of the plasma membrane. There, PS acts as one major “eat me” signal that ensures efficient recognition and uptake of apoptotic cells by phagocytes. PS recognition of activated phagocytes induces the secretion of anti-inflammatory cytokines like interleukin-10 and transforming grow factor-beta. Deficiencies in the clearance of apoptotic cells result in the occurrence of secondarily necrotic cells. The latter have lost the membrane integrity and release immune activating danger signals, which may induce inflammatory responses. Accumulation of dead cells containing nuclear autoantigens in sites of immune selection may provide survival signals for autoreactive B-cells. The production of antibodies against nuclear structures determines the initiation of chronic autoimmunity in systemic lupus erythematosus. Since PS on apoptotic cells is an important modulator of the immune response, natural occurring ligands for PS like annexinA5 have profound effects on immune responses against dead and dying cells, including tumour cells. In this review we will focus on the role of PS exposure in the clearance process of dead cells and its implications in clinical situations where apoptosis plays a relevant role, like in cancer, chronic autoimmunity, and infections. Relevance of other phospholipids during the apoptosis process is also discussed.

Sodium Overload and Water Influx Activate the NALP3 Inflammasome

The NALP3 inflammasome is activated by low intracellular potassium concentrations [K+]i, leading to the secretion of the proinflammatory cytokine IL-1β. However, the mechanism of [K+]i lowering after phagocytosis of monosodium urate crystals is still elusive. Here, we propose that endosomes containing monosodium urate crystals fuse with acidic lysosomes. The low pH in the phagolysosome causes a massive release of sodium and raises the intracellular osmolarity. This process is balanced by passive water influx through aquaporins leading to cell swelling. This process dilutes [K+]i to values below the threshold of 90 mm known to activate NALP3 inflammasomes without net loss of cytoplasmic potassium ions. In vitro, the inhibitors of lysosomal acidification (ammonium chloride, chloroquine) and of aquaporins (mercury chloride, phloretin) all significantly decreased the production of IL-1β. In vivo, only the pharmacological inhibitor of lysosome acidification chloroquine could be used which again significantly reduced the IL-1β production. As a translational aspect one may consider the use of chloroquine for the anti-inflammatory treatment of refractory gout.

Inefficient Clearance of Dying Cells and Autoreactivity

Dying cells were basically unnoticed by scientists for a long time and only came back into the spotlight roughly 10 years ago. The process of recognition and uptake of apoptotic and necrotic cells is complex and failures in this process can contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Here, we discuss the recognition and uptake molecules which are involved in an efficient clearance of dying cells in early and late phases of cell death. The exposure of phosphatidylserine (PS) is an early surface change of apoptosing cells recognized by several receptors and adaptor molecules. We demonstrated that dying cells have cell membranes with high lateral mobility of PS, which contribute to their efficient clearance. Changes of the glycoprotein composition of apoptotic cells occur later than the exposure of PS. We further observed that complement binding is an early event in necrosis and a rather late event in apoptosis. Complement, C-reactive protein (CRP), and serum DNase I act as back-up molecules in the clearance process. Finally, we discuss how the accumulation of secondary necrotic cells and cellular debris in the germinal centers of secondary lymph organs can lead to autoimmunity. It is reasonable to argue that clearance defects are major players in the development of autoimmune diseases such as SLE.