Polystyrene microparticle distribution after ingestion by murine macrophages.

The impact of microplastic particles on organisms is currently intensely researched. Although it is well established that macrophages ingest polystyrene (PS) microparticles, little is known about the subsequent fate of the particles, such as entrapment in organelles, distribution during cell division, as well as possible mechanisms of excretion. Here, submicrometer (0.2 and 0.5 µm) and micron-sized (3 µm) particles were used to analyze particle fate upon ingestion of murine macrophages (J774A.1 and ImKC). Distribution and excretion of PS particles was investigated over cycles of cellular division. The distribution during cell division seems cell-specific upon comparing two different macrophage cell lines, and no apparent active excretion of microplastic particles could be observed. Using polarized cells, M1 polarized macrophages show higher phagocytic activity and particle uptake than M2 polarized ones or M0 cells. While particles with all tested diameters were found in the cytoplasm, submicron particles were additionally co-localized with the endoplasmic reticulum. Further, 0.5 µm particles were occasionally found in endosomes. Our results indicate that a possible reason for the previously described low cytotoxicity upon uptake of pristine PS microparticles by macrophages may be due to the preferential localization in the cytoplasm.

Stunning of neutrophils accounts for the anti-inflammatory effects of clodronate liposomes.

Clodronate liposomes (Clo-Lip) have been widely used to deplete mononuclear phagocytes (MoPh) to study the function of these cells in vivo. Here, we revisited the effects of Clo-Lip together with genetic models of MoPh deficiency, revealing that Clo-Lip exert their anti-inflammatory effects independent of MoPh. Notably, not only MoPh but also polymorphonuclear neutrophils (PMN) ingested Clo-Lip in vivo, which resulted in their functional arrest. Adoptive transfer of PMN, but not of MoPh, reversed the anti-inflammatory effects of Clo-Lip treatment, indicating that stunning of PMN rather than depletion of MoPh accounts for the anti-inflammatory effects of Clo-Lip in vivo. Our data highlight the need for a critical revision of the current literature on the role of MoPh in inflammation.

Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases.

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human disease-related stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner.

Cardiac Arrest and Successful Extracorporeal Cardiopulmonary Resuscitation as a Result of a Refeeding Syndrome in a Young Female with Anorexia Nervosa.

Anorexia nervosa is a potentially life-threatening eating disorder, characterized by an abnormally low body weight. This case report illustrates a 22-year old female with cardiac arrest due to a refeeding syndrome in a patient with anorexia nervosa. It features the successful use of extracorporeal cardiopulmonary resuscitation in a case of severe left ventricular dysfunction resulting in a favorable outcome. Conclusion. We present the first case of a cardiac arrest due to a refeeding syndrome in anorexia nervosa featuring the successful use of an extracorporeal cardiopulmonary resuscitation approach as a bridge to full recovery.

Myeloperoxidase Modulates Inflammation in Generalized Pustular Psoriasis and Additional Rare Pustular Skin Diseases.

Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19%-41% of affected individuals harbor bi-allelic mutations in IL36RN, the genetic cause is not known in most cases. To identify and characterize new pathways involved in the pathogenesis of GPP, we performed whole-exome sequencing in 31 individuals with GPP and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO). We discovered eight MPO mutations resulting in MPO -deficiency in neutrophils and monocytes. MPO mutations, primarily those resulting in complete MPO deficiency, cumulatively associated with GPP (p = 1.85E-08; OR = 6.47). The number of mutant MPO alleles significantly differed between 82 affected individuals and >4,900 control subjects (p = 1.04E-09); this effect was stronger when including IL36RN mutations (1.48E-13) and correlated with a younger age of onset (p = 0.0018). The activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO deficiency. Phorbol-myristate-acetate-induced formation of neutrophil extracellular traps (NETs) was reduced in affected cells (p = 0.015), and phagocytosis assays in MPO-deficient mice and human cells revealed altered neutrophil function and impaired clearance of neutrophils by monocytes (efferocytosis) allowing prolonged neutrophil persistence in inflammatory skin. MPO mutations contribute significantly to GPP's pathogenesis. We implicate MPO as an inflammatory modulator in humans that regulates protease activity and NET formation and modifies efferocytosis. Our findings indicate possible implications for the application of MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases.

IgA subclasses have different effector functions associated with distinct glycosylation profiles.

Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease.

Live cell imaging of macrophage/bacterium interaction demonstrates cell lysis induced by Corynebacterium diphtheriae and Corynebacterium ulcerans.

OBJECTIVES: In frame of a study to characterize the interaction of human macrophage-like cells with pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, live cell imaging experiments were carried out and time lapse fluorescence microscopy videos were generated, which are presented here. DATA DESCRIPTION: The time lapse fluorescence microscopy data revealed new insights in the interaction of corynebacteria with human macrophage-like THP-1 cells. In contrast to uninfected cells and infections with non-pathogenic C. glutamicum used as a control, pathogenic C. diphtheriae and C. ulcerans showed highly detrimental effects towards human cells and induction of cell death of macrophages.

Treatment with DNases rescues hidden neutrophil elastase from aggregated NETs.

The release of neutrophil extracellular traps (NETs) is one of the weapons neutrophils have in their armory. NETs consist of extracellular chromatin fibers decorated with a plethora of cytoplasmic and granular proteins, such as the antimicrobial serine protease neutrophil elastase (NE). Because the first description of NETs as beneficial to the host, reports on their double-faced role in health and disease have considerably increased recently. On one hand, NETs reportedly trap and kill bacteria and also participate in the resolution of the acute inflammation associated with infection and with tissue damage. On the other hand, numerous negative aspects of NETs contribute to the etiopathogenesis of autoimmune disorders. Employing soluble and solid fluorescent substrates, we demonstrate the interaction of NE with aggregated NETs (aggNETs), the limitation of its enzymatic activity and the containment of the enzyme from surrounding tissues. These events prevent the spread of inflammation and tissue damage. The detection of DNase 1-dependent elevation of NE activity attests the continuous presence of patrolling neutrophils forming NETs and aggNETs even under conditions physiologic conditions.

Neutrophil Extracellular Traps Initiate Gallstone Formation.

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.

Induction of Necrosis in Human Macrophage Cell Lines by Corynebacterium diphtheriae and Corynebacterium ulcerans Strains Isolated from Fatal Cases of Systemic Infections.

When infecting a human host, Corynebacterium diphtheriae and Corynebacterium ulcerans are able to impair macrophage maturation and induce cell death. However, the underlying molecular mechanisms are not well understood. As a framework for this project, a combination of fluorescence microscopy, cytotoxicity assays, live cell imaging, and fluorescence-activated cell sorting was applied to understand the pathogenicity of two Corynebacterium strains isolated from fatal cases of systemic infections. The results showed a clear cytotoxic effect of the bacteria. The observed survival of the pathogens in macrophages and, subsequent, necrotic lysis of cells may be mechanisms explaining dissemination of C. diphtheriae and C. ulcerans to distant organs in the body.

NOX2 mediates quiescent handling of dead cell remnants in phagocytes.

The phagocyte NADPH oxidase (the NOX2 complex) generates superoxide, the precursor to reactive oxygen species (ROS). ROS possess both antimicrobial and immunoregulatory function. Inactivating mutations in alleles of the NOX2 complex cause chronic granulomatous disease (CGD), characterized by an enhanced susceptibility to infections and autoimmune diseases such as Systemic lupus erythematosus (SLE). The latter is characterized by insufficient removal of dead cells, resulting in an autoimmune response against components of the cell's nucleus when non-cleared apoptotic cells lose their membrane integrity and present autoantigenic molecules in an inflammatory context. Here we aimed to shed light on the role of the NOX2 complex in handling of secondary necrotic cells (SNECs) and associated consequences for inflammation and autoimmunity during lupus. We show that individuals with SLE and CGD display accumulation of SNECs in blood monocytes and neutrophils. In a CGD phenotypic mouse strain (Ncf1** mice) build-up of SNECs in Ly6CHI blood monocytes was connected with a delayed degradation of the phagosomal cargo and accompanied by production of inflammatory mediators. Treatment with H2O2 or activators of ROS-formation reconstituted phagosomal abundance of SNECs to normal levels. Induction of experimental lupus further induced increased antibody-dependent uptake of SNECs into neutrophils. Lupus-primed Ncf1** neutrophils took up more SNECs than wild type neutrophils, whereas SNEC-accumulation in regulatory Ly6C-/LO monocytes was lower in Ncf1**mice. We deduce that the inflammatory rerouting of immune-stimulatory necrotic material into inflammatory phagocyte subsets contributes to the connection between low ROS production by the NOX2 complex and SLE.

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.

Papillon-Lefèvre syndrome (PLS) is characterized by nonfunctional neutrophil serine proteases (NSPs) and fulminant periodontal inflammation of unknown cause. Here we investigated neutrophil extracellular trap (NET)-associated aggregation and cytokine/chemokine-release/degradation by normal and NSP-deficient human and mouse granulocytes. Stimulated with solid or soluble NET inducers, normal neutrophils formed aggregates and both released and degraded cytokines/chemokines. With increasing cell density, proteolytic degradation outweighed release. Maximum output of cytokines/chemokines occurred mostly at densities between 2 × 107 and 4 × 107 neutrophils/cm3. Assessment of neutrophil density in vivo showed that these concentrations are surpassed during inflammation. Association with aggregated NETs conferred protection of neutrophil elastase against α1-antitrypsin. In contrast, eosinophils did not influence cytokine/chemokine concentrations. The proteolytic degradation of inflammatory mediators seen in NETs was abrogated in Papillon-Lefèvre syndrome (PLS) neutrophils. In summary, neutrophil-driven proteolysis of inflammatory mediators works as a built-in safeguard for inflammation. The absence of this negative feedback mechanism might be responsible for the nonresolving periodontitis seen in PLS.-Hahn, J., Schauer, C., Czegley, C., Kling, L., Petru, L., Schmid, B., Weidner, D., Reinwald, C., Biermann, M. H. C., Blunder, S., Ernst, J., Lesner, A., Bäuerle, T., Palmisano, R., Christiansen, S., Herrmann, M., Bozec, A., Gruber, R., Schett, G., Hoffmann, M. H. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.

Autoimmune, rheumatic, chronic inflammatory diseases: Neutrophil extracellular traps on parade.

Rheumatic diseases are a group of inflammatory conditions that affect joints and connective tissues and are often accompanied by pain and restriction of motility. In many of these diseases, autoantibodies develop that react with molecules/structures commonly found hidden in neutrophils. Neutrophil extracellular trap (NET) formation and release is considered a defense mechanism against pathogens or endogenous danger signals and it has been associated with initial inflammatory responses. NETs are also endowed with an important resolution potential based on its intrinsic enzymatic activity, but in the case they are not timely removed from the crime scene they might modulate subsequent immune responses and contribute to the pathogenesis of chronic inflammatory diseases. In this review, we will summarize the actual knowledge about the multifaceted roles of NETs in the etiology and pathogenesis of rheumatic autoimmune diseases.

Low-Dose Radiotherapy Ameliorates Advanced Arthritis in hTNF-α tg Mice by Particularly Positively Impacting on Bone Metabolism.

Inflammation and bone erosion are central in rheumatoid arthritis (RA). Even though effective medications for control and treatment of RA are available, remission is only seen in a subset of patients. Treatment with low-dose radiotherapy (LD-RT) which has been already successfully used for amelioration of symptoms in benign diseases should be a promising approach to reduce pain, inflammation, and particularly bone erosion in patients with RA. Even though anti-inflammatory effects of LD-RT are already described with non-linear dose response relationships, and pain-reducing effects have been clinically observed, the underlying mechanisms are widely unknown. Besides immune cells many other cell types, such as fibroblast-like synoviocytes (FLS), osteoclasts, and osteoblast are present in the affected joint and might be modulated by LD-RT. For this study, these cell types were obtained from human tumor necrosis factor-α transgenic (hTNF-α tg) mice and were consecutively exposed to different doses of ionizing radiation (0.1, 0.5, 1.0, and 2.0 Gy, respectively) in vitro. In order to study the in vivo effects of LD-RT within the arthritic joint, hind paws of arthritic hTNF-α tg mice were locally irradiated with 0.5 Gy, a single dose per fraction that is known for good clinical responses. Starting at a dose of 0.5 Gy, proliferation of FLS was reduced and apoptosis significantly enhanced with no changes in necrosis. Further, expression of RANK-L was slightly reduced following irradiation with particularly 0.5 Gy. Starting from 0.5 Gy, the numbers of differentiated osteoclasts were significantly reduced, and a lower bone resorbing activity of treated osteoclasts was also observed, as monitored via pit formation and Cross Laps presence. LD-RT had further a positive effect on osteoblast-induced mineralization in a discontinuous dose response relationship with 0.5 Gy being most efficient. An increase of the gene expression ratio of OPG/RANK-L at 0.1 and 0.5 Gy and of production of OPG at 0.5 and 1.0 Gy was observed. In vivo, LD-RT resulted in less severe arthritis in arthritic hTNF-α tg mice and in significant reduction of inflammatory and erosive area with reduced osteoclasts and neutrophils. Locally applied LD-RT can, therefore, induce a beneficial micro-environment within arthritic joints by predominantly positively impacting on bone metabolism.

Chemical Tools for Targeted Amplification of Reactive Oxygen Species in Neutrophils.

A number of chemical compounds are known, which amplify the availability of reactive oxygen species (ROS) in neutrophils both in vitro and in vivo. They can be roughly classified into NADPH oxidase 2 (NOX2)-dependent and NOX2-independent reagents. NOX2 activation is triggered by protein kinase C agonists (e.g., phorbol esters, transition metal ions), redox mediators (e.g., paraquat) or formyl peptide receptor (FPR) agonists (e.g., aromatic hydrazine derivatives). NOX2-independent mechanisms are realized by reagents affecting glutathione homeostasis (e.g., l-buthionine sulfoximine), modulators of the mitochondrial respiratory chain (e.g., ionophores, inositol mimics, and agonists of peroxisome proliferator-activated receptor γ) and chemical ROS amplifiers [e.g., aminoferrocene-based prodrugs (ABPs)]. Since a number of inflammatory and autoimmune diseases, as well as cancer and bacterial infections, are triggered or enhanced by aberrant ROS production in neutrophils, it is tempting to use ROS amplifiers as drugs for the treatment of these diseases. However, since the known reagents are not cell specific, their application for treatment likely causes systemic enhancement of oxidative stress, leading to severe side effects. Cell-targeted ROS enhancement can be achieved either by using conjugates of ROS amplifiers with ligands binding to receptors expressed on neutrophils (e.g., the GPI-anchored myeloid differentiation marker Ly6G or FPR) or by designing reagents activated by neutrophil function [e.g., phagocytic activity or enzymatic activity of neutrophil elastase (NE)]. Since binding of an artificial ligand to a receptor may trigger or inhibit priming of neutrophils the latter approach has a smaller potential for severe side effects and is probably better suitable for therapy. Here, we review current approaches for the use of ROS amplifiers and discuss their applicability for treatment. As an example, we suggest a possible design of neutrophil-specific ROS amplifiers, which are based on NE-activated ABPs.

Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps.

Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death-inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4-deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.

Capability of Neutrophils to Form NETs Is Not Directly Influenced by a CMA-Targeting Peptide.

During inflammatory reaction, neutrophils exhibit numerous cellular and immunological functions, notably the formation of neutrophil extracellular traps (NETs) and autophagy. NETs are composed of decondensed chromatin fibers coated with various antimicrobial molecules derived from neutrophil granules. NETs participate in antimicrobial defense and can also display detrimental roles and notably trigger some of the immune features of systemic lupus erythematosus (SLE) and other autoimmune diseases. Autophagy is a complex and finely regulated mechanism involved in the cell survival/death balance that may be connected to NET formation. To shed some light on the connection between autophagy and NET formation, we designed a number of experiments in human neutrophils and both in normal and lupus-prone MRL/lpr mice to determine whether the synthetic peptide P140, which is capable of selectively modulating chaperone-mediated autophagy (CMA) in lymphocytes, could alter NET formation. P140/Lupuzor™ is currently being evaluated in phase III clinical trials involving SLE patients. Overall our in vitro and in vivo studies established that P140 does not influence NET formation, cytokine/chemokine production, or CMA in neutrophils. Thus, the beneficial effect of P140/Lupuzor™ in SLE is apparently not directly related to modulation of neutrophil function.

Neurodegeneration Enhances the Development of Arthritis.

The prevalence of neurodegenerative disease and arthritis increases with age. Despite both processes being associated with immune activation and inflammation, little is known about the mechanistic interactions between neurodegenerative disease and arthritis. In this article, we show that tau-transgenic (tau-tg) mice that develop neurodegenerative disease characterized by deposition of tau tangles in the brain are highly susceptible to developing arthritis. Already at steady-state conditions, tau-tg mice exhibit peripheral immune activation that is manifested by higher numbers of granulocytes, plasmablasts, and inflammatory Ly6Chi CCR2+ monocytes, as well as increased levels of proinflammatory cytokines, such as TNF-α and IL-17. Upon induction of collagen-induced arthritis (CIA), tau-tg mice displayed an increased incidence and an earlier onset of CIA that was associated with a more pronounced inflammatory cytokine response. Furthermore, induction of CIA led to significantly elevated numbers of Iba-1-expressing cells in the brain, indicative of microglia activation, and the formation of anti-tau Abs in tau-tg mice. These changes were accompanied by the resolution of tau tangles and significantly decreased neurodegenerative pathology. In summary, these data show that neurodegenerative disease enhances the development of arthritis. In addition, arthritis, once induced, triggers innate immune responses in the brain, leading to resolution of neurodegenerative changes.