NETosis occurs independently of neutrophil serine proteases.

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

Neutrophil Extracellular Traps (NETs) in Severe SARS-CoV-2 Lung Disease.

Neutrophil extracellular traps (NETs), built from mitochondrial or nuclear DNA, proteinases, and histones, entrap and eliminate pathogens in the course of bacterial or viral infections. Neutrophils' activation and the formation of NETs have been described as major risk factors for acute lung injury, multi-organ damage, and mortality in COVID-19 disease. NETs-related lung injury involves both epithelial and endothelial cells, as well as the alveolar-capillary barrier. The markers for NETs formation, such as circulating DNA, neutrophil elastase (NE) activity, or myeloperoxidase-DNA complexes, were found in lung specimens of COVID-19 victims, as well as in sera and tracheal aspirates obtained from COVID-19 patients. DNA threads form large conglomerates causing local obstruction of the small bronchi and together with NE are responsible for overproduction of mucin by epithelial cells. Various components of NETs are involved in the pathogenesis of cytokine storm in SARS-CoV-2 pulmonary disease. NETs are responsible for the interplay between inflammation and thrombosis in the affected lungs. The immunothrombosis, stimulated by NETs, has a poor prognostic significance. Better understanding of the role of NETs in the course of COVID-19 can help to develop novel approaches to the therapeutic interventions in this condition.

Neutrophil elastase triggers the development of autoimmune diabetes by exacerbating innate immune responses in pancreatic islets of non-obese diabetic mice.

Type 1 diabetes is an autoimmune disease resulted from self-destruction of insulin-producing pancreatic ß cells. However, the pathological pathways that trigger the autoimmune destruction remain poorly understood. Clinical studies have demonstrated close associations of neutrophils and neutrophil elastase (NE) with ß-cell autoimmunity in patients with Type 1 diabetes. The present study aims to investigate the impact of NE inhibition on development of autoimmune diabetes in NOD mice. NE pharmacological inhibitor (sivelestat) or biological inhibitor (elafin) was supplemented into NOD mice to evaluate their effects on islet inflammation and diabetogenesis. The impact of NE inhibition on innate and adaptive immune cells was measured with flow cytometry and immunohistochemistry. A significant but transient increase in neutrophil infiltration accompanied with elevated NE activity was observed in the neonatal period of NOD mice. Treatment of NOD mice with sivelestat or elafin at the early age led to a marked reduction in spontaneous development of insulitis and autoimmune diabetes. Mechanistically, inhibition of NE significantly attenuated infiltration of macrophages and islet inflammation, thus ameliorating cytotoxic T cell-mediated autoimmune attack of pancreatic ß cells. In vitro studies showed that NE directly induced inflammatory responses in both min6 ß cells and RAW264.7 macrophages, and promoted macrophage migration. These findings support an important role of NE in triggering the onset and progression of ß-cell autoimmunity, and suggest that pharmacological inhibition of NE may represent a promising therapeutic strategy for treatment of autoimmune diabetes.

Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation.

Thymic stromal lymphopoetin (TSLP) influences numerous immune functions, including those in the colonic mucosa. Here we report that TSLP-deficient (Tslp(-/-)) mice did not exhibit increased inflammation during dextran sodium sulfate (DSS)-induced colitis but failed to recover from disease, resulting in death. Increased localized neutrophil elastase (NE) activity during overt inflammation was observed in Tslp(-/-) mice and was paralleled by reduced expression of an endogenous inhibitor, secretory leukocyte peptidase inhibitor (SLPI). Pharmacological inhibition of NE or treatment with rSLPI reduced DSS-induced mortality in Tslp(-/-) mice. Signaling through TSLPR on nonhematopoietic cells was sufficient for recovery from DSS-induced colitis. Expression of the receptor occurred on intestinal epithelial cells (IEC), with stimulation inducing SLPI expression. Therefore, TSLP is critical in mediating mucosal healing after insult and functions in a nonredundant capacity that is independent of restraining T helper 1 (Th1) and Th17 cell cytokine production.

Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Mosaicism of an ELANE Mutation in an Asymptomatic Mother.

PURPOSE: We report normal neutrophil count in a mother, who carries the same ELANE mutation as her daughter with severe congenital neutropenia. We hypothesized that the mother possessed wild- and mutant-type clones and the wild-type clones could generate neutrophils, whereas the mutant clones could not. METHODS: We confirmed mutant variant ratio by sequence signals and measured the frequency of the mutant allele by subcloning in various cell types. We established the ELANE-mutated and non-mutated induced pluripotent stem cells (iPSCs) from the mother's T cells and compared granulopoiesis between these iPSCs. RESULTS: In the sequence analysis of isolated peripheral blood (PB), nail and hair, the mutant variant was detected in approximately 40-60% of lymphocytes, monocytes, hematopoietic progenitor cells, and hair as well as in a small percentage of nail, but in none of the neutrophils. In the subcloning analysis of extracted DNA from CD3+ and CD34+ cells, the mutant allele was identified in 37.5% and 38.1%, respectively. We reprogrammed the mother's PB cells and established the ELANE-mutated and non-mutated iPSCs. Granulopoiesis from mutated iPSCs revealed little sensitivity to granulocyte colony-stimulating factor in comparison with non-mutated iPSCs. CONCLUSIONS: These observations strongly suggest that mutant-carrying neutrophils did not appear in the mother's PB because mutated clones could not differentiate into neutrophils. The mother's normal hematological phenotype could be explained by the perseverance of normal, non-mutated granulopoiesis.

IL-1ß activation in response to Staphylococcus aureus lung infection requires inflammasome-dependent and independent mechanisms.

Maintaining balanced levels of IL-1ß is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1-/- mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1-/- mice with S. aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL-1ß. Ifnlr1-/- mice treated with recombinant IL-1ß displayed increased bacterial burdens in the airway and lung. IL-1ß levels in neutrophils from Ifnlr1-/- infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase-1 had reduced IL-1ß levels 4 h after infection, due to reductions or absence of active caspase-1 respectively, but levels at 24 h were comparable to WT infected mice. Ifnlr1-/- infected mice had decreases in both active caspase-1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL-1ß processing. By inhibiting neutrophil elastase, we were able to decrease IL-1ß levels by 39% in Nlrp3-/- infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL-1ß processing, via inflammasome-dependent and -independent mechanisms.

Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus-derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.-Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Neutrophil Elastase Regulates Emergency Myelopoiesis Preceding Systemic Inflammation in Diet-induced Obesity.

Inflammation plays a significant role in the development of obesity-related complications, but the molecular events that initiate and propagate such inflammation remain unclear. Here, we report that mice fed a high-fat diet (HFD) for as little as 1-3 days show increased differentiation of myeloid progenitors into neutrophils and monocytes but reduced B lymphocyte production in the bone marrow. Levels of neutrophil elastase (NE) and the nuclear factors CCAAT/enhancer-binding protein α (C/EBPα) and growth factor-independent 1 (GFI-1) are elevated in hematopoietic stem and progenitor cells from HFD-fed mice, but mice lacking either NE or C/EBPα are resistant to HFD-induced myelopoiesis. NE deletion increases expression of the inhibitory isoform of p30 C/EBPα, impairs the transcriptional activity of p42 C/EBPα, and reduces expression of the C/EBPα target gene GFI-1 in hematopoietic stem and progenitor cells, suggesting a mechanism by which NE regulates myelopoiesis. Furthermore, NE deletion prevents HFD-induced vascular leakage. Thus, HFD feeding rapidly activates bone marrow myelopoiesis through the NE-dependent C/EBPα-GFI-1 pathway preceding vascular damage and systemic inflammation.

Neuropilin-1 mediates neutrophil elastase uptake and cross-presentation in breast cancer cells.

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.

Modulation of γδ T-cell activation by neutrophil elastase.

γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation.

Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.

Neutrophils are a major source of the epithelial barrier disrupting cytokine oncostatin M in patients with mucosal airways disease.

BACKGROUND: We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium. OBJECTIVE: We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease. METHODS: Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF, and hematopoietic cell-specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM-CSF and follistatin-like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase. RESULTS: OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3-5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45+ cells were OSM+, and of the OSM+ cells, 56% ± 7% were CD16+Siglec-8-, indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45+ events from matched blood samples (n = 5) were OSM+, suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM-CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P < .001) in peripheral blood neutrophils in vitro. OSM+ neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P < .05). CONCLUSIONS: Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma.

Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system.

Macrophage-derived IL-1ß enhances monosodium urate crystal-triggered NET formation.

OBJECTIVE AND DESIGN: Arthritic gout is caused by joint inflammation triggered by the damaging effects of monosodium uric acid (MSU) crystal accumulation in the synovial space. Neutrophils play a major role in mediating joint inflammation in gout. Along with neutrophils, other immune cells, such as macrophages, are present in inflamed joints and contribute to gout pathogenesis. Neutrophils form neutrophil extracellular traps (NETs) in response to MSU crystals. In the presence of MSU crystals, macrophages release IL-1ß, a cytokine crucial to initiate gout pathogenesis and neutrophil recruitment. Our research investigated interactions between human macrophages and neutrophils in an in vitro model system and asked how macrophages affect NET formation stimulated by MSU crystals. MATERIALS OR SUBJECTS: Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF. TREATMENT: Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1ß or anakinra prior to stimulation by MSU crystals. METHOD: Interaction of neutrophils with MSU crystals was evaluated by live imaging using confocal microscopy. The presence of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured by ELISA. NET formation was quantitated by Sytox Orange-based extracellular DNA release assay and NE-DNA ELISA. AggNET formation was assessed by macroscopic evaluation. RESULTS: We found that crystal- and cell-free supernatants of macrophages stimulated with MSU crystals promote MSU crystal-stimulated NET formation in human neutrophils. This observation was confirmed by additional assays measuring the release of MPO, NE, and the enzymatic activity of NE. MSU crystal-induced NET formation remained unchanged when neutrophil supernatants were tested. IL-1ß is a crucial cytokine orchestrating the onset of inflammation in gout and is known to be released in large amounts from macrophages following MSU crystal stimulation. We found that recombinant IL-1ß strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1ß alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1ß is mainly responsible for this effect. CONCLUSIONS: Macrophage-derived IL-1ß enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1ß affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra.

The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases.

A comparative study of expression of Fc receptors in relation to the autoantibody-mediated immune response and neutrophil elastase expression in autoimmune blistering dermatoses.

Here we investigated the cutaneous CD32A and CD89 expression in relation to the neutrophil elastase (NE) expression and serum level of anti-desmoglein 1 and 3 (DSG1/DSG3) IgG in pemphigus, anti-BP180/BP230 IgG in bullous pemphigoid (BP), anti-gliadin nonapeptides (npG), tissue (tTG), and epidermal transglutaminases (eTG) IgA in dermatitis herpetiformis (DH). The examined material consisted of skin/mucosal tissues and sera. In total, 87 patients were studied. Immunohistochemistry on paraffin-embedded sections with quantitative digital morphometry was used to measure the intensity of CD32A/CD89/NE expressions. Levels of anti-DSG1/DSG3 IgG, anti-BP180/BP230 IgG, and anti-npG/tTG/eTG IgA were evaluated with ELISAs. CD32A was abundantly expressed in cutaneous lesions in pemphigus and BP. We found no statistically significant correlation between the CD32A/CD89 and NE expression intensities in pemphigus, BP, and DH. There was a significant correlation between CD89 expression and anti-npG IgA in DH. Our results revealed a lack of correlation between CD32A expressions and anti-DSG1/DSG3 IgG levels in pemphigus, anti-BP180/BP230 IgG in BP as well as CD89 expression and anti-tTG/eTG IgA in DH. CD89 seems to be linked with gluten intolerance in DH rather than with proteolytic destruction of dermal-epidermal junction. CD32A appears to play an important role in mediating skin injury in pemphigus and BP, but probably independently from specific autoantibodies.

Neutrophils from F508del cystic fibrosis patients produce IL-17A and express IL-23 - dependent IL-17RC.

Cystic fibrosis (CF) is a chronic pulmonary disease that is associated with persistent microbial infection and chronic neutrophil infiltration, and also with elevated production of the pro-inflammatory cytokine IL-17A (IL-17). In the current study, we examined IL-17 and the inducible IL-17RC receptor subunit in neutrophils from Pseudomonas aeruginosa infected F508del CF patients at the time of pulmonary exacerbation, and again following intravenous antibiotic treatment. Neutrophils expressed Il17a and Il17rc transcripts and protein at the time of pulmonary exacerbation, which were absent following antibiotic treatment. Further, CF sputum induced IL-23 - dependent Il17rc expression in neutrophils from healthy individuals. Similarly, IL-17 producing neutrophils were detected in F508del and Cftr(-/-) mice infected intranasally with P. aeruginosa. In the sputum of CF subjects, the percentage IL-17 producing neutrophils correlated with elastase and MMP9 activity; therefore, this population of neutrophils may be an important contributor to the severity of pulmonary disease in CF patients.

NETosis-associated serum biomarkers are reduced in type 1 diabetes in association with neutrophil count.

As the immune pathways involved in the pathogenesis of type 1 diabetes (T1D) are not fully understood, biomarkers implicating novel mechanisms of disease are of great interest and call for independent evaluation. Recently, it was reported that individuals with T1D display dramatic elevations in circulating components of neutrophil extracellular traps (NETs), indicating a potential role for NETosis in T1D. Our aim was to evaluate further the potential of NET-associated proteins as novel circulating biomarkers in T1D. We tested serum from subjects with T1D (n = 44) with a median age of 26·5 years and a median duration of 2·2 years, along with 38 age-matched controls. T1D subjects did not show elevations in either neutrophil elastase (NE) or proteinase 3 (PR3), as reported previously. In fact, both NE and PR3 levels were reduced significantly in T1D subjects, particularly in subjects within 3 years of diagnosis, consistent with the known reduction in neutrophil counts in recent-onset T1D. Indeed, levels of both NE and PR3 correlated with absolute neutrophil counts. Therefore, while not ruling out potential local or transient spikes in NETosis activity, the levels of these serum markers do not support a role for systemically elevated NETosis in the T1D population we studied. Rather, a modest reduction in these markers may reflect other important aspects of disease activity associated with reduced neutrophil numbers.