Christina Janko

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.

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.

Monosodium urate crystals induce extracellular DNA traps in neutrophils, eosinophils, and basophils but not in mononuclear cells

Neutrophil extracellular traps (NETs) are fibers of extracellular DNA released from neutrophils due to overwhelming phagocytic stimuli. The function of NETs is to trap and kill microbes to avoid spreading of potential pathogens. NETs are formed after encounter with various gram-positive and -negative bacteria but also in response to mediators causing sterile inflammation like interleukin-8 (IL-8), tumor necrosis factor (TNF), and phorbol myristate acetate (PMA). Here we show the formation of NETs (NETting) in response to monosodium urate (MSU) crystals as further model for sterile inflammation. We identified monocytes, neutrophils, and eosinophils as MSU phagocytosing cells. Basophils did not take up the crystals, instead they upregulated their activation marker CD203c after contact with MSU. Nevertheless, MSU crystals induced extracellular trap formation also in basophils, like in eosinophils and neutrophils, which phagocytose the crystals. In contrast, monocytes do not form NETs despite uptake of the MSU crystals. In contrast to the canonical stimuli like bacteria and PMA, MSU-induced NETosis was not abrogated by plasma. Our data show that MSU crystals induce extracellular DNA trap formation in all three granulocytes lineages (NETs, EETs, and BETs) but not in monocytes, and DNA externalization does not necessitate the uptake of the crystals.

Inflammatory clearance of apoptotic remnants in systemic lupus erythematosus (SLE)

Deficiencies in the recognition and phagocytosis of dead and dying cells have been shown to be one of the main alterations in patients with systemic lupus erythematosus (SLE). Cellular as well as humoral elements play an important role in the clearance of apoptotic and necrotic cells. Non-ingested nuclear material may provide survival signals for autoreactive B-cells and consequently antibodies directed against nuclear structures will be produced. In healthy individuals, nuclear fragments are not phagocytosed in whole blood. Instead, they are mainly degraded by the action of plasma DNases and complement factors. In contrast, the uptake of nuclear fragments by blood-borne phagocytes is increased in most patients with SLE. The phagocytosis of this kind of prey, which might be opsonised by autoantibodies, may contribute to the maintenance of inflammatory responses in SLE.

Macrophages Discriminate Glycosylation Patterns of Apoptotic Cell-derived Microparticles

Background: Apoptotic cells release vesicles, which expose “eat-me” signals. Results: Vesicles originated from endoplasmic reticulum expose immature glycoepitopes and are preferentially phagocytosed by macrophages. Conclusion: Immature surface glycoepitopes serve as “eat-me” signals for the clearance of apoptotic vesicles originated from endoplasmic reticulum. Significance: Understanding the distinction by macrophages of apoptotic blebs may provide new insights into clearance-related diseases. Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Development of a lauric acid/albumin hybrid iron oxide nanoparticle system with improved biocompatibility

The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantum interference device. Using flow cytometry, we further investigated the effects of the different types of nanoparticle coating on morphology, viability, and DNA integrity of Jurkat cells. We showed that by addition of bovine serum albumin, the toxicity of nanoparticles is greatly reduced. We also investigated the effect of the particles on the growth of primary human endothelial cells to further demonstrate the biocompatibility of the particles. As proof of principle, we showed that the hybrid-coated particles are able to carry payloads of up to 800 μg/mL of the cytostatic drug mitoxantrone while still staying colloidally stable. The drug-loaded system exhibited excellent therapeutic potential in vitro, exceeding that of free mitoxantrone. In conclusion, we have synthesized a biocompatible ferrofluid that shows great potential for clinical application. The synthesis is straightforward and reproducible and thus easily translatable into a good manufacturing practice environment.

Development and characterization of magnetic iron oxide nanoparticles with a cisplatin-bearing polymer coating for targeted drug delivery

A highly selective and efficient cancer therapy can be achieved using magnetically directed superparamagnetic iron oxide nanoparticles (SPIONs) bearing a sufficient amount of the therapeutic agent. In this project, SPIONs with a dextran and cisplatin-bearing hyaluronic acid coating were successfully synthesized as a novel cisplatin drug delivery system. Transmission electron microscopy images as well as X-ray diffraction analysis showed that the individual magnetite particles were around 4.5 nm in size and monocrystalline. The small crystallite sizes led to the superparamagnetic behavior of the particles, which was exemplified in their magnetization curves, acquired using superconducting quantum interference device measurements. Hyaluronic acid was bound to the initially dextran-coated SPIONs by esterification. The resulting amide bond linkage was verified using Fourier transform infrared spectroscopy. The additional polymer layer increased the vehicle size from 22 nm to 56 nm, with a hyaluronic acid to dextran to magnetite weight ratio of 51:29:20. A maximum payload of 330 μg cisplatin/mL nanoparticle suspension was achieved, thus the particle size was further increased to around 77 nm with a zeta potential of −45 mV. No signs of particle precipitation were observed over a period of at least 8 weeks. Analysis of drug-release kinetics using the dialysis tube method revealed that these were driven by inverse ligand substitution and diffusion through the polymer shell as well as enzymatic degradation of hyaluronic acid. The biological activity of the particles was investigated in a nonadherent Jurkat cell line using flow cytometry. Further, cell viability and proliferation was examined in an adherent PC-3 cell line using xCELLigence analysis. Both tests demonstrated that particles without cisplatin were biocompatible with these cells, whereas particles with the drug induced apoptosis in a dose-dependent manner, with secondary necrosis after prolonged incubation. In conclusion, combination of dextran-coated SPIONs with hyaluronic acid and cisplatin represents a promising approach for magnetic drug targeting in the treatment of cancer.