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.

Diagnostic delay in Hidradenitis suppurativa investigated using data from social media and from statutory health insurances.

BACKGROUND: The chronic inflammatory skin disease hidradenitis suppurativa (HS) leads to severe pain and reduced quality of life. Nonetheless, it often takes years until a correct diagnosis is made. In this analysis, disease-related experiences and pathways of patients with HS were investigated and compared with the physicians' perspective. METHODS: Public posts on forums and social media as well as results of a survey conducted among dermatologists and their patients on the actual medical care reality of HS in Germany were analysed. Furthermore, claims data from German health insurance companies were evaluated. RESULTS: Patients with HS suffer from a 43.3% reduction in working ability. Dermatology (26.5%) was the most frequently consulted specialty, with HS diagnosed predominantly in the inpatient setting (43.8%). Abscesses were described as the most frequent alternative diagnosis in HS patients (53.2%). Patient-reported changes of physicians in dermatology (34.1%) and surgery (42.4%) occurred predominantly within the specialty. Dermatology received most referrals from general practitioners (67.1%), but only 12.1% from surgeons. CONCLUSION: There is an urgent need to reduce the delay in diagnosis and the prolonged burden of disease in patients with HS. Therefore, awareness of the disease, its detection and treatment which goes beyond dermatology should be promoted, if possible as part of medical studies.

Lichen planus in Deutschland - Epidemiologie, Behandlung und Komorbidität. Eine retrospektive Krankenkassendatenanalyse.

HINTERGRUND UND ZIELE: Lichen planus (LP) ist eine chronisch entzündliche Hauterkrankung, die eine große Belastung für die betroffenen Patienten darstellt. Es liegen jedoch nur wenige Daten zu dieser Erkrankung vor. Ziel dieser Studie ist es, das Wissen über die Epidemiologie und die Behandlungsmuster des LP anhand von Abrechnungsdaten deutscher Krankenkassen zu erweitern. PATIENTEN UND METHODEN: Diese retrospektive Beobachtungsstudie nutzte die InGef-Forschungsdatenbank. Es wurden prävalente und inzidente LP-Patienten aus den Jahren 2015 und 2018 identifiziert. Für demografische Charakteristika, Behandlungsmuster und Komorbidität wurden deskriptive Statistiken berechnet. ERGEBNISSE: Die Prävalenz des LP lag bei 95,9 und die Inzidenz bei 20,1 pro 100 000 Personen im Jahr 2018, was 79 605 prävalenten LP-Fällen in Deutschland entspricht. Die erste LP-Diagnose wurde in der Regel von einem Dermatologen oder Hausarzt gestellt. Drei Viertel der inzidenten und die Hälfte der prävalenten Patienten erhielten eine topische Therapie, meist ohne zusätzliche systemische Therapie. Die Komorbidität des LP stand im Einklang mit bereits bekannten Assoziationen. SCHLUSSFOLGERUNGEN: Die verfügbaren Therapieoptionen sind nach wie vor begrenzt, was den ungedeckten Bedarf an sicheren und wirksamen systemischen Behandlungsmodalitäten unterstreicht. Der LP ist häufig mit klinisch relevanter systemischer Komorbidität verbunden. Zusammengenommen könnten diese Beobachtungen zu einem verbesserten Verständnis der Krankheitslast führen und das diagnostische Bewusstsein für diese Erkrankung unter Klinikern schärfen.

Lichen planus in Germany - epidemiology, treatment, and comorbidity. A retrospective claims data analysis.

BACKGROUND AND OBJECTIVES: Lichen planus (LP) is a chronic inflammatory skin disease and is a major burden for affected patients. However, data on this condition are scarce. This study aims to expand the knowledge on the epidemiology and treatment patterns of LP using German health claims data. PATIENTS AND METHODS: This retrospective observational study was based on the InGef research database. Prevalent and incident LP patients were identified in the years 2015 and 2018. Descriptive statistics were calculated for demographic characteristics, treatment patterns, and comorbidity. RESULTS: The prevalence of LP was 95.9 and the incidence was 20.1 per 100,000 individuals in 2018, corresponding to 79,605 prevalent LP cases in Germany. The first LP diagnosis was generally documented by a dermatologist or a primary care physician. Three-quarters of the incident and half of the prevalent patients received topical therapy, mostly without further systemic therapy. Comorbidity in LP patients was consistent with previously known associations. CONCLUSIONS: Available treatment options remain limited, underscoring the unmet need for safe and efficacious systemic treatment modalities. Lichen planus is frequently accompanied by clinically relevant systemic comorbidity. Taken together, these observations may improve our understanding of the burden of this disease and increase diagnostic awareness among clinicians.

Epidemiology, Comorbidity and Risk Factors for Psoriatic Arthritis: A Health Insurance Claims Database Analysis.

Psoriatic arthritis is a frequent manifestation of psoriasis, and has a high level of impact on physical func-tioning, work ability and quality of life. However, there have been few studies of the epidemiology, development of and risk factors for concomitant psoriatic arthritis in patients with psoriasis. This study analysed data from a German public health insurance database of > 2 million individuals. Factors influencing the development of psoriatic arthritis were determined by descriptively analysing comorbidities and Cox regression modelling. The prevalences of psoriasis and psoriatic arthritis were 2.63% and 0.29% in adults (18+ years) and, respectively, 0.30% and 0.01% in children (0-17 years). The proportion of adult patients with incident psoriasis who developed concomitant psoriatic arthritis within five years after diagnosis of psoriasis (mean 2.3 years) was 2.6%. Cardiovascular diseases are the most frequent comorbidity in patients with psoriasis with or without concomitant psoriatic arthritis. Depression and neurosis/stress disorder were identified as indicators for the development of psoriatic arthritis.

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.

Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation.

The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100-1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes.

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major 'eat-me' signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Surface code--biophysical signals for apoptotic cell clearance.

Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called 'find-me', 'eat me' and 'tolerate me' signals. Mononuclear phagocytes are attracted by various 'find-me' signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via 'stay away' signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main 'eat me' signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as 'tolerate me' signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

Epidemiology, comorbidity, and use of systemic therapies in patients with paediatric psoriasis in Germany.

INTRODUCTION: Psoriasis is a chronic, inflammatory disorder with a physical and psychosocial burden. Recent epidemiological data on paediatric psoriasis in Germany is lacking. The aim of this study was to examine the prevalence, the incidence and associated epidemiological features of paediatric psoriasis in Germany using health claims data from a national health claims database. METHODS: Continuously insured patients with paediatric psoriasis (2014-2017) from the Institute of Applied Health Research Berlin (InGef) database were included in this analysis. Between 2014 and 2017, we analysed the prevalence and incidence of paediatric psoriasis (aged ≤17 years), relevant comorbidities, the most frequently attended and diagnosing medical specialties, and the number of systemic treatment prescriptions. RESULTS: Overall, psoriasis prevalence (0.15-0.16%) and incidence (0.06-0.07%) remained stable between 2014 and 2017; both prevalence and incidence were higher in female patients. Psoriasis prevalence gradually increased with age between the age groups <6 years and 16-17 years. Obesity (11.5%) and somatoform disorders (7.0%) were the most common comorbidities identified. Overall, 90.4% of the psoriasis cases were diagnosed by either a dermatologist (50.9%), general practitioner (27.8%), or paediatrician (11.7%). Patients most frequently attended general practitioners (74.5%), dermatologists (57.9%) and paediatricians (56.5%). The use of systemic treatment(s) increased from 4.7% to 5.4% between 2014 and 2017. DISCUSSION: The prevalence and incidence of paediatric psoriasis in Germany remained stable between 2014 and 2017, with a higher prevalence and incidence observed in females and older adolescents. Obesity and somatoform disorders were the most common comorbidities. General practitioners, dermatologists, and paediatricians were most often involved in the treatment of patients, highlighting the need for interdisciplinary management of paediatric psoriasis.

Absolute and relative psoriasis area and severity index (PASI) treatment goals and their association with health-related quality of life.

Background: Relative Psoriasis Area and Severity Index (PASI) improvement, also called 'PASI response', is recommended in major guidelines for assessment of treatment response in psoriasis patients. However, under certain circumstances it has some limitations, e.g. when baseline values are missing or during long-term treatment. Improvement of health-related quality of life (HrQoL) can be measured by the Dermatology Life Quality Index (DLQI).Objective: To evaluate the association of HrQoL and relative and absolute PASI outcomes during therapy.Material and methods: Data of plaque psoriasis patients of two clinical trials (CLEAR and SCULPTURE) were pooled. The rates of patients achieving DLQI 0/1 at week 16 were compared for different categories of absolute PASI and PASI response values. The correlation of DLQI and absolute or relative PASI goals was assessed over 52 weeks.Results: One thousand and fifty-four patients with available assessments of PASI and DLQI were included. 76% of the patients with an absolute PASI ≤ 2 (N = 548) and patients with a 90% improvement in PASI (N = 559) achieved DLQI 0/1 at week 16. Achievement of DLQI 0/1 was equally reflected by absolute PASI and PASI response.Conclusion: Absolute PASI appeared to be a feasible alternative to PASI response for determining treatment success, reflecting HrQoL improvements in an equal manner.

Psoriatic arthritis epidemiology, comorbid disease profiles and risk factors: results from a claims database analysis.

OBJECTIVE: Psoriasis is a systemic inflammatory disease often accompanied by comorbidities, including metabolic syndrome, cardiovascular diseases and depression. Up to 41% of psoriasis patients develop psoriatic arthritis (PsA), making it one of the most relevant manifestations. A large health claims data set was analysed to determine the rate of PsA development in psoriasis patients. Furthermore, comorbid disease profiles of psoriasis patients with or without PsA were compared, and potential risk factors for the development of PsA were identified. METHODS: This was a non-interventional, retrospective analysis of anonymized insurance health claims data using a subset of the Institute of Applied Health Research Berlin (InGef) database. The primary outcome was the prevalence and incidence of diagnosed PsA among psoriasis patients in Germany. Risk factors for the development of PsA in psoriasis patients were determined by conditional logistic regression analysis. RESULTS: The cumulative percentage of patients with existing psoriasis developing concomitant PsA over 4 years was 3.44%, with a mean time to diagnosis of PsA of 1.5 years. Psoriasis patients diagnosed with acute rheumatism (odds ratio: 2.93, 95% CI = 1.76, 4.86; P < 0.001) or pain in unspecific joints (odds ratio: 1.74, 95% CI = 1.01, 2.99; P = 0.047) showed an increased risk for development of PsA later on. Interestingly, fewer than half of the patients with concomitant PsA consulted a rheumatologist. CONCLUSIONS: Unspecific arthritic symptoms are likely to precede PsA diagnoses and can develop soon after onset of psoriasis, with accumulating risk over time. There is a high unmet need for early rheumatological assessment of psoriasis patients.

Hidradenitis Suppurativa and Concurrent Psoriasis: Comparison of Epidemiology, Comorbidity Profiles, and Risk Factors.

INTRODUCTION: Hidradenitis suppurativa (HS) is a chronic, debilitating, and inflammatory skin disease. The epidemiology of HS varies greatly, with an estimated prevalence ranging from 0.03% to 4% worldwide. Similar to psoriasis (PsO), HS also exhibits a systemic inflammatory nature with a spectrum of systemic comorbidities. A large health insurance claims (HICs) database is analyzed to determine the demography and epidemiology of HS, PsO, and HS with concurrent PsO (HS-PsO) patients. Furthermore, the comorbidity profiles, including the comorbidity risk of these patient populations, are analyzed. METHODS: This is a noninterventional retrospective analysis of anonymized HICs data using a subset of the Institute of Applied Health Research Berlin (InGef) database. The primary outcome is the prevalence and incidence of HS, PsO, and HS-PsO. Secondary outcomes include comorbidity profiles and a comorbidity risk analysis. RESULTS: The prevalence and incidence of HS were 0.0681% and 0.0101%, respectively. The prevalence of HS-PsO was 0.004% (6% of the total HS population). HS patients frequently suffered from arterial hypertension (45%), nicotine dependence (46%), obesity (41%), and depression (36%), which were more common in HS-PsO patients compared with HS alone. HS patients had an increased prevalence of metabolic, psychiatric, immune-mediated, and cardiovascular diseases, e.g., overweight/obesity [odds ratio (OR): 2.65, 95% confidence interval (CI) 2.37-2.96], depression (OR: 1.55, 95% CI 1.42-1.76), or seronegative rheumatoid arthritis (OR: 2.82, 95% CI 1.61-4.94) compared with the overall population. The increased risk of myocardial infarction in HS patients (OR: 4.1, 95% CI 3.5-4.8, adjusting for age/sex) was largely attributed to patient's current smoking status (OR: 1.1, 95% CI 0.8-1.5, adjusting for smoking/age/sex). CONCLUSIONS: HS patients show a broad spectrum of inflammatory and metabolic syndrome-related comorbidities, with an increased risk by concurrent PsO. Important for clinical practice, the elevated cardiovascular risk of HS patients can be largely attributed to smoking.

Low amounts of bisecting glycans characterize cerebrospinal fluid-borne IgG.

Immunoglobulin G (IgG) harbors a conserved N-glycosylation site which is important for its effector functions. Changes in glycosylation of IgG occur in many autoimmune diseases but also in physiological conditions. Therefore, the glycosylation pattern of serum IgG is well characterized. However, limited data is available on the glycosylation pattern of IgG in cerebrospinal fluid (CSF) compared to serum. Here, we report significantly reduced levels of bisected glycans in CSF IgG. Galactosylation and sialylation of IgG4 also differed significantly. Therefore, we propose a common mechanism mediating glycosylation changes of IgG at the transition from serum to CSF in steady state conditions.

Autoantibodies Recognizing Secondary NEcrotic Cells Promote Neutrophilic Phagocytosis and Identify Patients With Systemic Lupus Erythematosus.

Deficient clearance of apoptotic cells reportedly contributes to the etiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Based on this knowledge, we developed a highly specific and sensitive test for the detection of SLE autoantibodies (AAb) utilizing secondary NEcrotic cell (SNEC)-derived material as a substrate. The goal of the present study was to validate the use of SNEC as an appropriate antigen for the diagnosis of SLE in large cohort of patients. We confirmed the presence of apoptotically modified autoantigens on SNEC (dsDNA, high mobility group box 1 protein, apoptosis-associated chromatin modifications, e.g., histones H3-K27-me3; H2A/H4 AcK8,12,16; and H2B-AcK12). Anti-SNEC AAb were measured in the serum of 155 patients with SLE, 89 normal healthy donors (NHD), and 169 patients with other autoimmune connective tissue diseases employing SNEC-based indirect enzyme-linked immunosorbent assay (SNEC ELISA). We compared the test performance of SNEC ELISA with the routine diagnostic tests dsDNA Farr radioimmunoassay (RIA) and nucleosome-based ELISA (anti-dsDNA-NcX-ELISA). SNEC ELISA distinguished patients with SLE with a specificity of 98.9% and a sensitivity of 70.6% from NHD clearly surpassing RIA and anti-dsDNA-NcX-ELISA. In contrast to the other tests, SNEC ELISA significantly discriminated patients with SLE from patients with rheumatoid arthritis, primary anti-phospholipid syndrome, spondyloarthropathy, psoriatic arthritis, and systemic sclerosis. A positive test result in SNEC ELISA significantly correlated with serological variables and reflected the uptake of opsonized SNEC by neutrophils. This stresses the relevance of SNECs in the pathogenesis of SLE. We conclude that SNEC ELISA allows for the sensitive detection of pathologically relevant AAb, enabling its diagnostic usage. A positive SNEC test reflects the opsonization of cell remnants by AAb, the neutrophil recruitment to tissues, and the enhancement of local and systemic inflammatory responses.

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.

New Insights into Neutrophil Extracellular Traps: Mechanisms of Formation and Role in Inflammation.

Recent data suggest that NETosis plays a crucial role in the innate immune response and disturbs the homeostasis of the immune system. NETosis is a form of neutrophil-specific cell death characterized by the release of large web-like structures referred to as neutrophil extracellular traps (NETs). NETs are composed of DNA strands associated with histones and decorated with about 20 different proteins, including neutrophil elastase, myeloperoxidase, cathepsin G, proteinase 3, high mobility group protein B1, and LL37. Reportedly, NETosis can be induced by several microbes, and particulate matter including sterile stimuli, via distinct cellular mechanisms. Meanwhile, suicidal NETosis and vital NETosis are controversial. As we enter the second decade of research on NETosis, we have partly understood NETs as double-edged swords of innate immunity. In this review, we will discuss the mechanisms of NETosis, its antimicrobial action, and role in autoimmune diseases, as well as the relatively new field of NET-associated mitochondrial DNA.

Ménage-à-Trois: The Ratio of Bicarbonate to CO2 and the pH Regulate the Capacity of Neutrophils to Form NETs.

In this study, we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET) formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (phorbol-2-myristate-13-acetate), ionomycin, monosodium urate, and LPS. In contrast, an acidic environment impaired NET formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to NET formation. Inflammatory foci may be characterized by an acidic environment. Our data indicate that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover, our findings highlight the necessity for strict pH control during assays of NET formation.

Oxidative Burst-Dependent NETosis Is Implicated in the Resolution of Necrosis-Associated Sterile Inflammation.

Necrosis is associated with a profound inflammatory response. The regulation of necrosis-associated inflammation, particularly the mechanisms responsible for resolution of inflammation is incompletely characterized. Nanoparticles are known to induce plasma membrane damage and necrosis followed by sterile inflammation. We observed that injection of metabolically inert nanodiamonds resulted in paw edema in WT and Ncf1** mice. However, while inflammation quickly resolved in WT mice, it persisted over several weeks in Ncf1** mice indicating failure of resolution of inflammation. Mechanistically, NOX2-dependent reactive oxygen species (ROS) production and formation of neutrophil extracellular traps were essential for the resolution of necrosis-induced inflammation: hence, by evaluating the fate of the particles at the site of inflammation, we observed that Ncf1** mice deficient in NADPH-dependent ROS failed to generate granulation tissue therefore being unable to trap the nanodiamonds. These data suggest that NOX2-dependent NETosis is crucial for preventing the chronification of the inflammatory response to tissue necrosis by forming NETosis-dependent barriers between the necrotic and healthy surrounding tissue.