Neutrophil Extracellular DNA Research Articles

Bidirectional effects of neutrophils on Streptococcus oralis biofilms in vitro

ABSTRACT Background Streptococcus oralis is a commensal bacterium and an early biofilm coloniser found in the human oral cavity. One of the biofilm matrix constituents is bacterial extracellular DNA (eDNA). Neutrophils are innate immune cells that respond to biofilms, employing antimicrobial mechanisms such as neutrophil extracellular trap (NET) and reactive oxygen species (ROS) release. Here, bidirectional effects of neutrophils on S. oralis biofilms were investigated. Materials and methods Isolated neutrophils were introduced to S. oralis biofilms at different stages of biofilm development. Biofilm quantity was assessed by crystal violet technique, confocal microscopy and CFU enumeration. Surface adhesion during shear stress was quantified by spectrophotometry. Bacterial and neutrophil extracellular DNA within biofilms and ROS production were analysed using fluorescence and luminescence assays, and neutrophil-eDNA interactions were investigated by flow cytometry and fluorescence microscopy. Results Neutrophils decreased S. oralis biofilm quantity transiently and reduced eDNA but did not affect biofilm surface adhesion. Unexpectedly, CFUs were increased by neutrophils. Bacterial DNA was found to co-localise with neutrophil membranes. Neutrophils produced elevated total and intracellular ROS, however, no NETs in response to biofilms. Conclusion Neutrophils in vitro are not excessively activated by S. oralis biofilms but are able to reduce biofilm quantity in the short-term, possibly through interfering with eDNA.

Neutrophil extracellular DNA traps activate the TLR9 signaling pathway of pancreatic ductal epithelial cells in patients with type 2 autoimmune pancreatitis

The presence of neutrophil infiltration around the pancreatic ducts has been found to be associated with type 2 autoimmune pancreatitis (AIP). However, the functional role and clinical significance of neutrophil migration in the progression of pancreatitis is not fully understood. Here, we found that neutrophil extracellular traps (NETs) are abundant around the pancreatic duct in patients with type 2 AIP. We also observed an increased expression of toll-like receptor 9 (TLR9) in pancreatic ductal epithelial cells (HPDEC) in type 2 AIP patients compared to other pancreatic diseases. TLR9 acts as the DNA component of NETs (NET-DNA) receptor in HPDEC, which senses extracellular DNA and subsequently activates the NF-κB pathway to promote neutrophil recruitment and induce NET formation. In addition, our results indicated that the hydroxychloroquine (HCQ), acting as a TLR9 antagonist, could effectively inhibit the activation of inflammatory pathways, reduce neutrophil migration and block the positive feedback loop. The intervention positions HCQ acts as a potential target drug for the clinical treatment of type 2 AIP.

Safety and efficacy indicators of guarana and Brazil nut extract carried in nanoparticles of coenzyme Q10: Evidence from human blood cells and red earthworm experimental model

This study characterized a nanosupplement based on coenzyme Q10 containing guarana (Paullinia cupana) and Brazil nuts oil (Bertholetia excelsa) (G-Nut). Determined cytotoxic and oxi-immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs) and its effect on mortality of red Californian earthworms (Eisenia fetida) and on the immune efficiency of its coelomocytes immune by in vitro exposure to yeast dead microorganism. The cytotoxic and immunomodulatory effects of G-Nut and the GN-Free extract (0.25-3mg/mL) were determined in PBMC cultures. Apoptotic, oxidative, and inflammatory markers were determined using biochemical, immunological, and molecular protocols. The effects of G-Nut and GN-Free extracts on mortality and immune efficiency were investigated in earthworms. G-Nut and GN-Free did not induce cytotoxic events in PBMCs, triggering the decrease in apoptotic (caspases 3 and 8) gene expression, lipid and protein oxidation levels, or pro-inflammatory cytokine levels. G-Nut and GN-Free did not trigger earthworm mortality and improved coelomocyte immune efficiency by increasing Eisenia neutrophil extracellular DNA traps and brown body formation when exposed to dead yeasts. The G-Nut nanoformulation is safe and can be used as a new form of food supplement by oral or transdermal delivery.

Neutrophil Extracellular DNA Traps in Response to Infection or Inflammation, and the Roles of Platelet Interactions.

Neutrophils present the host's first line of defense against bacterial infections. These immune effector cells are mobilized rapidly to destroy invading pathogens by (a) reactive oxygen species (ROS)-mediated oxidative bursts and (b) via phagocytosis. In addition, their antimicrobial service is capped via a distinct cell death mechanism, by the release of their own decondensed nuclear DNA, supplemented with a variety of embedded proteins and enzymes. The extracellular DNA meshwork ensnares the pathogenic bacteria and neutralizes them. Such neutrophil extracellular DNA traps (NETs) have the potential to trigger a hemostatic response to pathogenic infections. The web-like chromatin serves as a prothrombotic scaffold for platelet adhesion and activation. What is less obvious is that platelets can also be involved during the initial release of NETs, forming heterotypic interactions with neutrophils and facilitating their responses to pathogens. Together, the platelet and neutrophil responses can effectively localize an infection until it is cleared. However, not all microbial infections are easily cleared. Certain pathogenic organisms may trigger dysregulated platelet-neutrophil interactions, with a potential to subsequently propagate thromboinflammatory processes. These may also include the release of some NETs. Therefore, in order to make rational intervention easier, further elucidation of platelet, neutrophil, and pathogen interactions is still needed.

Aspergillus Enhances Eosinophil and Neutrophil Extracellular DNA Trap Formation in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is characterized by inflammatory cell infiltration in the sinonasal mucosa. Eosinophil and neutrophil extracellular traps (EETs and NETs, respectively) are prominently found in CRS. This study aimed to investigate the effect of airborne fungi, Alternaria alternata and Aspergillus fumigatus, on EET and NET formation. Nasal epithelial cells, eosinophils, and neutrophils were isolated from eosinophilic CRS (ECRS), non-ECRS (NECRS), and healthy control. We determined eosinophil and neutrophil transepithelial migration after fungal treatment. We then determined the release of EETs and NETs by fungi using Sytox Green staining and determined the role of reactive oxygen species (ROS) using ROS inhibitors. We identified more abundant EETs and NETs in ECRS than in NECRS. A. alternata and A. fumigatus enhanced eosinophil and neutrophil transepithelial migration. A. fumigatus strongly induced EET and NET formation in CRS and, simultaneously, suppressed fungal metabolic activity. EET formation in CRS is associated with nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and NET formation with NADPH-oxidase and mitochondrial ROS. A. fumigatus, but not A. alternata, induced EET and NET formation, and peripheral blood eosinophils and neutrophils exhibited different immune responses against A. fumigatus following the inflammatory status of the host. Aspergillus-fumigatus-induced EET and NET formation plays a crucial role in CRS pathogenesis.

Staphylococcus aureus Multiplexes Death-Effector Deoxyribonucleosides to Neutralize Phagocytes.

Adenosine synthase A (AdsA) is a key virulence factor of Staphylococcus aureus, a dangerous microbe that causes fatal diseases in humans. Together with staphylococcal nuclease, AdsA generates deoxyadenosine (dAdo) from neutrophil extracellular DNA traps thereby igniting caspase-3-dependent cell death in host immune cells that aim at penetrating infectious foci. Powered by a multi-technological approach, we here illustrate that the enzymatic activity of AdsA in abscess-mimicking microenvironments is not restricted to the biogenesis of dAdo but rather comprises excessive biosynthesis of deoxyguanosine (dGuo), a cytotoxic deoxyribonucleoside generated by S. aureus to eradicate macrophages of human and animal origin. Based on a genome-wide CRISPR-Cas9 knock-out screen, we further demonstrate that dGuo-induced cytotoxicity in phagocytes involves targeting of the mammalian purine salvage pathway-apoptosis axis, a signaling cascade that is concomitantly stimulated by staphylococcal dAdo. Strikingly, synchronous targeting of this route by AdsA-derived dGuo and dAdo boosts macrophage cell death, indicating that S. aureus multiplexes death-effector deoxyribonucleosides to maximize intra-host survival. Overall, these data provide unique insights into the cunning lifestyle of a deadly pathogen and may help to design therapeutic intervention strategies to combat multidrug-resistant staphylococci.

Neutrophil extracellular DNA traps promote pancreatic cancer cells migration and invasion by activating EGFR/ERK pathway.

Neutrophil extracellular DNA traps (NETs) are newly discovered forms of activated neutrophils. Increasing researches have shown that NETs play important roles in cancer progression. Our previous study has proved that tumour‐infiltrating NETs could predict postsurgical survival in patients with pancreatic ductal adenocarcinoma (PDAC). However, the roles of NETs on the progression of pancreatic cancer are unknown. Here, we investigated the effects of NETs on pancreatic cancer cells. Results showed that both PDAC patients’ and normal individuals’ neutrophils‐derived NETs could promote migration and invasion of pancreatic cancer cells with epithelial‐mesenchymal transition. Further, study confirmed that EGFR/ERK pathway played an important role in this progression. The addition of neutralizing antibodies for IL‐1β could effectively block the activation of EGFR/ERK companied with reduction of EMT, migration and invasion. Taken together, NETs facilitated EMT, migration and invasion via IL‐1β/EGFR/ERK pathway in pancreatic cancer cells. Our study suggests that NETs may provide promising therapeutic targets for pancreatic cancer.

Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration.

Metastasis is the leading cause of cancer-related deaths, and obesity is associated with increased breast cancer (BC) metastasis. Preclinical studies have shown that obese adipose tissue induces lung neutrophilia associated with enhanced BC metastasis to this site. Here we show that obesity leads to neutrophil-dependent impairment of vascular integrity through loss of endothelial adhesions, enabling cancer cell extravasation into the lung. Mechanistically, neutrophil-produced reactive oxygen species in obese mice increase neutrophil extracellular DNA traps (NETs) and weaken endothelial junctions, facilitating the influx of tumor cells from the peripheral circulation. In vivo treatment with catalase, NET inhibitors or genetic deletion of Nos2 reversed this effect in preclinical models of obesity. Imaging mass cytometry of lung metastasis samples from patients with cancer revealed an enrichment in neutrophils with low catalase levels correlating with elevated body mass index. Our data provide insights into potentially targetable mechanisms that underlie the progression of BC in the obese population.

Anaplastic Thyroid Cancer Cells Induce the Release of Mitochondrial Extracellular DNA Traps by Viable Neutrophils.

Neutrophils are key effector cells that orchestrate inflammatory responses in the tumor microenvironment. Although neutrophil extracellular DNA traps (NETs) entrap and kill pathogens, they also contribute to chronic inflammation and cancer progression. Thyroid cancer (TC) is the most frequently occurring cancer of the endocrine system, accounting for 70% of deaths due to endocrine tumors. Although anaplastic TC (ATC) is rare among TCs, it is highly lethal. We demonstrated in a recent study that tumor-infiltrating neutrophil density correlated with TC size. Moreover, TC-derived soluble mediators modulate the human neutrophil phenotype. Our study aimed to investigate the involvement of NETs in human TC. Highly purified neutrophils from healthy donors were primed in vitro with a papillary TC or ATC cell line conditioned medium (CM) or with a normal thyroid CM as control. NET release was quantified using a High-Content Imaging System. Neutrophil viability was assessed by flow cytometry. Fluorescence microscopy, flow cytometry, and PCR were performed to determine the mitochondrial origin of ATC-induced NETs. ATC CM-primed neutrophils were cocultured with ATC cells to determine the effects exerted by NETs on cell proliferation. ATC CM induce NET release, whereas papillary TC or normal thyroid CM did not. ATC CM-induced NET production occurred in a reactive oxygen species-dependent and cell death-independent manner and was associated with mitochondrial reactive oxygen species production; the NETs contained mitochondrial DNA. ATC CM-primed neutrophils promoted ATC cell proliferation in a NET-dependent manner.

Surfactant Protein D (SP-D) Inhibits Neutrophil Extracellular DNA Trap Formation: Effects of S-nitrosylation

Surfactant Protein D (SP-D) Inhibits Neutrophil Extracellular DNA Trap Formation: Effects of S-nitrosylation

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis

NETosis is a unique form of neutrophil death that differs from apoptosis and necrosis. However, whether NETosis and apoptosis can occur simultaneously in the same neutrophil is unknown. In this paper, we show that increasing doses of ultraviolet (UV) irradiation increases NETosis, which is confirmed by myeloperoxidase colocalisation to neutrophil extracellular DNA. Increasing UV irradiation increases caspase 3 activation, mitochondrial reactive oxygen species (ROS) generation and p38, but not ERK, phosphorylation. Inhibition of mitochondrial ROS production and p38 activation, but not NADPH oxidase (NOX) activity, suppresses UV-induced NETosis, indicating that UV induces NOX-independent NETosis. Like classical NOX-dependent and -independent NETosis, UV-induced NETosis requires transcriptional firing for chromatin decondensation. Cell death-specific inhibitor studies indicate that UV-mediated NETosis is not apoptosis, necrosis or necroptosis. Collectively, these studies indicate that increasing doses of UV irradiation induce both apoptosis and NETosis simultaneously, but the ultimate outcome is the induction of a novel form of NOX-independent NETosis, or “ApoNETosis”.

Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin

The human body is exposed to a wide range of particles of industrial, environmental or internal origin such as asbestos, alum, silica or crystals of urate, calcium phosphate, calcium oxalate, cystine or cholesterol. Phagocytic clearance of such particles involves neutrophils and macrophages. Here we report that neutrophils encountering such particles of diverse sizes and shapes undergo necrotic cell death, a process associated with the formation of neutrophil extracellular trap (NET)-like extracellular DNA. In human neutrophils receptor-interacting protein kinase (RIPK)-1 inhibition with necrostatin-1s or mixed lineage kinase domain-like (MLKL) inhibition with necrosulfonamide abrogated cell death and associated-neutrophil extracellular DNA release induced by all of the aforementioned particles. Similar results were obtained with Mlkl-deficient mice neutrophils for all particles in vitro. Furthermore, Mlkl-deficient mice lacked tophus formation upon injection of MSU crystals into subcutaneous air pouches. These findings imply that nano- or microparticle-induced neutrophil extracellular DNA release is the consequence of neutrophil necroptosis, a regulated form of cell necrosis defined by RIPK1-RIPK3-MLKL signaling. Interestingly, this finding was consistent across different particle sizes and shapes. The RIPK1-RIPK3-MLKL signaling pathway may represent a potential therapeutic target in nano- or microparticle-related diseases (crystallopathies).

Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells.

The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK) 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA). However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs), cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs) to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG) but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE) or myeloperoxidase (MPO) attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC), nonsevere asthma (NSA), and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

Neutrophil autophagy and extracellular DNA traps contribute to airway inflammation in severe asthma.

Autophagy and neutrophil extracellular DNA traps (NETs) are implicated in asthma; however, their roles in asthma pathogenesis have not been elucidated. We compared autophagy and NET production levels from peripheral blood neutrophils (PBNs) of patients with severe asthma (SA) and non-severe asthma (NSA). Additionally, we investigated the inflammatory effects of NETs on human airway epithelial cells (AECs) and peripheral blood eosinophils (PBEs). Peripheral blood neutrophils from patients with SA (n=30) and NSA (n=38) were treated with interleukin (IL)-8 (100ng/mL). Autophagy (light chain 3-II expression) and NET production levels were evaluated by Western blot, immunofluorescence microscopy, and PicoGreen assay. The effects of NETs on AECs were assessed by investigating cell death, cell detachment, expression of occludin and claudin-1, and IL-8 production; the effects of NETs on PBEs were examined by investigating the activation and release of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Untreated and IL-8-treated PBNs from the SA group produced higher autophagy and NET levels compared with those from the NSA group (P<0.01). IL-8 increased autophagy and NET levels in PBNs from the SA group, but not from the NSA group. NET levels were correlated with autophagy levels in PBNs (P<0.001). IL-8-induced NET production levels negatively were correlated with FEV1/FVC (r=-0.700, P=0.016). NETs induced cell death, detachment, degradation of occludin and claudin-1, and IL-8 production from AECs. Higher levels of NET-induced ECP and EDN were released from PBEs in SA compared with NSA groups. Neutrophil autophagy and NETs could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and PBEs. Modulating neutrophil autophagy and NET production may be a new target therapy for SA.

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps.

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

Role of proapoptotic BH3‐only proteins in Listeria monocytogenes infection

The ability of pathogens to influence host cell survival is a crucial virulence factor. Listeria monocytogenes (Lm) infection is known to be associated with severe apoptosis of hepatocytes and spleen cells. This impairs host defense mechanisms and thereby facilitates the spread of intracellular pathogens. The general mechanisms of apoptosis elicited by Lm infection are understood, however, the roles of BH3-only proteins during primary Lm infection have not been examined. To explore the roles of BH3-only proteins in Lm-induced apoptosis, we studied Listeria infections in mice deficient in Bim, Bid, Noxa or double deficient in BimBid or BimNoxa. We found that BimNoxa double knockout mice were highly resistant to high-dose challenge with Listeria. Decreased bacterial burden and decreased host cell apoptosis were found in the spleens of these mice. The ability of the BH3-deficient mice to clear bacterial infection more efficiently than WT was correlated with increased concentrations of ROS, neutrophil extracellular DNA trap release and downregulation of TNF-α. Our data show a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic host cell death during Listeria infection.

Neutrophil extracellular traps, scholarly debates, and public–private partnerships: highlights of the eighth annual Platelet Colloquium

The first of the Colloquium’s three main sessions included discussion of the rising importance of neutrophil extracellular DNA traps (NETs), which represent an intersection of inflammation, infection, and thrombosis. NETs, a network of DNA fibers, histones, and nuclear proteins produced by nucleosome release from activated neutrophils, are a longrecognized component of the body’s innate immune response to infection. It has recently become apparent, however, that NETs form within blood vessels in response to injury and disease, providing a lattice for red cell adherence, platelet activation, and thrombus formation. Both DNAse and heparin have been shown to disrupt the NET structure, preventing thrombosis. This finding might have relevance in the development of novel therapeutic targets. In addition, the fact that polymers have been shown to bind to cell-free DNA in vitro might hold promise in the development of methods to assess thrombotic risk and status. Also in the first session, the Colloquium brought back a popular segment from 2012, a position debate between two leading researchers. This year’s topic was ‘‘Is There a Role for Antiplatelet Therapy in the Management of Atrial Fibrillation?’’ Arguing in favor was Steven R. Steinhubl, MD, of Geisinger Medical Center, Danville, Pennsylvania; arguing against was Elaine Hylek, MD, of Boston University Medical Center. Table 1 summarizes the main points from both sides of the debate. In the end, the debaters agreed that well-controlled studies will be needed to address many of the knowledge gaps relevant to this question. Session II contemplated the evidence regarding the use of antiplatelet therapies in several high-risk groups, with additional discussions about avenues for future investigation. Despite the available data, substantial knowledge gaps persist for the use of antiplatelet agents in persons with hemoglobinopathies, diabetes, other metabolic disturbances, previous stroke, and deep vein thrombosis. In addition, providers are uncertain about the optimal use of antiplatelet agents during transcatheter aortic valve implantation (TAVI), during ‘‘hybrid’’ coronary procedures, and as a bridge to noncardiac surgery. In Session III, the focus turned to regulatory sciences and governmental support of platelet-related research. Regulatory agencies recognize that the clinical trial ecosystem in the United States is under stress (Table 2). Representatives from the U.S. Food and Drug Administration (FDA) and the National Institutes of Health were on hand to outline the roles that government agencies can play in easing these stressors and facilitating the conduct of high-level research that both addresses public health needs and meets regulatory requirements for therapeutics in the U.S. Specific examples of the new approaches and philosophies embraced by the FDA include implementing new concepts to appropriately P. A. French (&) Chapel Hill, NC, USA e-mail: pat.french@leftlanecomm.com

Predominant Role of Host Proteases in Myocardial Damage Associated with Infectious Endocarditis Induced by Enterococcus faecalis in a Rat Model

Infective endocarditis (IE) remains a life-threatening infectious disease with high morbidity and mortality. The objectives of the present study are to assess the host proteolytic activities of the vegetations and their cytotoxic potential in a rat model of experimental IE. Rats were infected with a strain of Enterococcus faecalis of particularly low virulence and weak protease expression. We tested the presence of proteases released by infiltrated leukocytes (matrix metalloproteinases and elastase) or produced in situ within the septic vegetation, such as those linked to the fibrinolytic system (plasmin and plasminogen activators). We also assessed the tissue damage induced by the infective thrombus in vitro and ex vivo. The model of IE was characterized by larger and more extensive vegetations in infected than in nonseptic rats and by an intense neutrophil infiltrate interfacing with the injured underlying tissue. Neutrophil extracellular DNA was shown to trap bacteria and to produce increased levels of cell-free DNA in plasma. Matrix metalloproteinase-9, elastase, and plasminogen activators were increased in septic versus nonseptic vegetations (as shown by zymography and immunohistology). Finally, proteolysis of the extracellular matrix and apoptosis were shown to be associated with host proteases. Bacteria exhibited no detectable proteolytic activity or direct cytotoxic effects. Bacterial membranes/dead bacteria were sufficient to induce leukocyte recruitment and activation that could promote vegetation formation and growth. Our results suggest that, despite the lack of bacterial proteases, the continuous attractant signals coming from bacterial colonies may lead to a chronic and deleterious aggression toward myocardial/valvular tissues by host proteases.

Innate immune recognition and response to pathogens

Our laboratory studies mechanisms of innate immunity. Through generation of knockout mice, we have demonstrated that a family of Toll-like receptors (TLR) recognizes a variety of PAMPs such as lipopolysaccharide, lipoprotein and nucleic acids derived from bacteria, viruses and protozoa to elicit innate immune responses. We have also demonstrated that a family of RIG-I-like RNA helicases (RLR) including RIG-I and MDA5 participates in TLR-independent recognition of nucleic acids derived from different types of RNA viruses in the cytoplasm. NOD-like receptors(NLR) also sense microbial components in the cytoplasm of cells. Several C-type lectin receptors(CLR) are shown to recognize the carbohydrates from pathogens. Here I will talk about the anti-viral response by neutrophil extracellular DNA trap(NET) and ZAP.

Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways

Asthma is a heterogeneous inflammatory airway disorder that involves eosinophilic and noneosinophilic phenotypes. Unlike in healthy lungs, eosinophils are often present in atopic asthmatic airways, although a subpopulation of asthmatic subjects predominantly experience neutrophilic inflammation. Recently, it has been demonstrated that eosinophils and neutrophils generate bactericidal extracellular traps consisting of DNA and cytotoxic granule proteins. We sought to explore whether living eosinophils and neutrophils infiltrating human atopic asthmatic airways actively form extracellular DNA traps in vivo. Quantitative analysis of eosinophils releasing DNA was performed in endobronchial biopsy specimens from 20 human subjects with mild atopic asthma at baseline and after local allergen challenge and 10 healthy subjects. DNA was stained with propidium iodine and major basic protein with specific antibody. Differential cell counts and cytokines/chemokines were assessed in bronchoalveolar lavage fluid. Asthmatic airways were infiltrated with a significantly higher number of eosinophils than healthy airways (39.3 ± 4.6 vs 0.4 ± 0.9, P< .0001). All asthmatic subjects but only 1 control subject expressed eosinophils releasing DNA that colocalized with major basic protein (33.65 ± 20.33 vs 0.3 ± 0.9 per high-power field, P< .0001). Four asthmatic subjects mostly expressed neutrophilic inflammation and neutrophil DNA traps. Allergen challenge had no significant quantitative effect on eosinophil or neutrophil DNA traps. Airway eosinophils or DNA traps did not correlate with either bronchoalveolar lavage levels of IL-5, IFN-γ, or eotaxin or the provoking doses of methacholine or allergen in asthmatic subjects. Extracellular DNA traps are generated by eosinophils and neutrophils in human atopic asthmatic airways in vivo. The mechanism and role of this new finding will necessitate further investigation.