Aggregated NETs Sequester and Detoxify Extracellular Histones

In response to various infectious and sterile stimuli neutrophils release chromatin decorated with bactericidal proteins, referred to as NETs. Their scaffolds are formed from chromatin fibers which display an apparent diameter of 15–17 nm and mainly consist from DNA (2 nm) and DNA-associated histones (11 nm). The NET-forming strands are thus not naked DNA but higher ordered chromatin structures. The histones may be released from the NET, especially if their tail arginines have been citrullinated. Several studies indicate that extracellular histones are toxic for mammalian epithelia and endothelia and contribute to the microvascular dysfunction observed e.g., in patients suffering from autoimmune diseases or sepsis. NETs formed at sites of very high neutrophil densities tend to clump and form fairly stable enzymatically active aggregates, referred to as aggNETs. The latter are endowed with a bunch of enzymes that cleave, bind, and/or modify autologous as well as foreign macromolecules. The tight binding of the serine proteases to the matrix precludes the spread of these toxic enzymes into the tissue but still allows the access of soluble inflammatory mediators to the enzymatic active internal surfaces of the NETs where they are degraded. Here, we describe that externally added histones are removed from culture supernatants of aggNETs. We will address the fate of the histones and discuss the feature on the background of neutrophil-driven diseases and the resolution of inflammation

Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c+ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3IEC-KO mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22–dependent mucosal wound healing

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19

Editorial: Immune-Epithelial Crosstalk in Inflammatory Bowel Diseases and Mucosal Wound Healing

80% of the bodies’ immune cells are harbored within the intestine. They are only separated from 1014 microorganisms by a single layer of intestinal epithelial cells and a secreted superficial mucus layer. Therefore, the intestinal epithelial surface represents a main frontier in host defense. Providing an intact mucosal barrier is vital for the host to limit bacterial entry and spread to the circulation. This specialized localization requires dynamic responses of intestinal epithelial cells to both pathogen- and immune-derived signals. Moreover, emergency barriers are needed in the setting of epithelial damage, which allow provisional microbial control and a timely restitution of mucosal integrity. Epithelial cells constantly interact with subjacent immune cells and fibroblasts, actively directing the immune response and also shaping the luminal microbiota. Epithelial dysfunction has been appreciated in recent years as a driving element in the pathogenesis of Inflammatory Bowel Diseases (IBD). Additionally, primary immune deficiencies may manifest in the form of chronic intestinal inflammation mimicking features of IBD. Recent advances in the techniques of epithelial cell culture and the discovery of new immune cell types and cellular properties have tremendously advanced the understanding in this interesting field of research. In this research topic, we want to focus on the complex interaction of intestinal epithelial cells, luminal flora and adjacent immune cells and invite manuscripts which highlight the dynamic responses of both epithelium and immune cells under steady-state or inflammatory conditions, and envision how this may be translated to the benefit of patient-care

Detection by flow cytometry of anti-neutrophil cytoplasmic antibodies in a novel approach based on neutrophil extracellular traps

Background: Anti-neutrophil-cytoplasmic antibodies (ANCA) are auto-antibodies directed against components of neutrophil granulocytes and may be found in various inflammatory conditions, like small-vessel vasculitis or ulcerative colitis (UC). Routine ANCA screening is performed on ethanol-fixed neutrophils using indirect immunofluorescence technique. Yet, how neutrophil granule proteins become available to immunologic presentation is a matter of debate. In recent years, various studies have shown that neutrophils are able to extrude their chromatin decorated with granular proteins as neutrophil extracelullar traps (NETs). Aim: We hypothesized that (I) ANCA immunoreactivity may be found on NETs and (II) NETs may serve as a useful tool in a novel approach for ANCA detection. Methods: Sera from patients suffering from either ANCA-associated vasculitis (n = 10), UC (n = 30) or sera from patients without diagnosed ANCA-associated diseases (n = 20), respectively, were subjected to indirect immunofluorescence and a newly developed method to detect ANCA by flow cytometry employing microbead technology. Results: ANCA-related immunofluorescence was readily detectable on ethanol-fixed NETs, establishing NETs as a structure carrying ANCA target antigens. Moreover, we observed that neutrophils form NETs in response to microbeads and stick to the surface of these beads. Using these NET-coated microbeads in flow cytometry, we were capable of reliably detecting p-ANCA, c-ANCA, and a-ANCA in tested patient sera. UC-related complex DNase-1-sensitive ANCA (NET-ANCA) antigens were also detected on NET-coated microbeads. Conclusion: NET-coated microbeads may be commercially developed as a novel tool for automated ANCA screening assays using flow cytometry

Pleiotropic functions of TNF-α in the regulation of the intestinal epithelial response to inflammation

An important function of intestinal epithelial cells (IECs) is to maintain the integrity of the mucosal barrier. Inflammation challenges the integrity of the mucosal barrier and the intestinal epithelium needs to adapt to a multitude of signals in order to perform the complex process of maintenance and restitution of its barrier function. Dysfunctions in epithelial barrier integrity and restoration contribute to the pathogenesis of inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis. Mucosal healing has developed to a significant treatment goal in IBD. In this review, we would like to highlight physiologic and pathologic adaptations of the intestinal epithelium to inflammation, exemplified by its responses to TNF-α. A large body of literature exists that highlights the diverse effects of this cytokine on IECs. TNF-α modulates intestinal mucus secretion and constitution. TNF-α stimulation modulates paracellular flow via tight junctional control. TNF-α induces intracellular signaling cascades that determine significant cell fate decisions such as survival, cell death or proliferation. TNF-α impacts epithelial wound healing in ErbB- and Wnt-dependent pathways while also importantly guiding immune cell attraction and function. We selected important studies from recent years with a focus on functional in vivo data providing crucial insights into the complex process of intestinal homeostasis

Missing in action - the meaning of cell death in tissue damage and inflammation

Billions of cells die every day in higher organisms as part of the normal process of tissue homeostasis. During special conditions like in development, acute infections, mechanical injuries, and immunity, cell death is a common denominator and it exerts profound effects in the outcome of these scenarios. In order to prevent the accumulation of aged, superfluous, infected, damaged and dead cells, professional phagocytes act in a rapid and efficient manner to clear the battle field and avoid spread of the destruction. Neutrophils are the most abundant effector immune cells that extravasate into tissues and can turn injured tissues into gory battle fields. In peace times neutrophils tend to patrol tissues without provoking inflammatory reactions. We discuss in this review actual and forgotten knowledge about the meaning of cell death during homeostatic processes and drive the attention to the importance of the action of neutrophils during patrolling and for the maintenance or recovery of the homeostatic state once the organism gets attacked or injured, respectively. In this fashion we disclose several disease conditions that arise as collateral damage of physiological responses to death