David N. Douda

SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

Significance Formation of neutrophil extracellular traps (NETs) is a recently described process by which neutrophils combat microbial pathogens. Recent studies demonstrate causative relationships between NETs and debilitating disorders such as rheumatoid arthritis, vasculitis, thrombosis, cystic fibrosis, and acute respiratory distress syndrome. However, the understanding of signaling pathways governing the process termed “NETosis” remains elusive. Two major types of NETosis have been reported; however, the mechanistic differences between these two types are not clearly established. Here we describe that NETosis induced by calcium ionophores is fast, NADPH-oxidase independent, and is mediated by mitochondrial reactive oxygen species (ROS) and a calcium-activated small conductance potassium channel. Thus, drugs that target mitochondrial ROS production or the potassium channels may provide previously unidentified therapeutic approaches for combating disorders with unregulated NETosis. Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed “NETosis” is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.

Innate Immune Collectin Surfactant Protein D Simultaneously Binds Both Neutrophil Extracellular Traps and Carbohydrate Ligands and Promotes Bacterial Trapping

Neutrophils release DNA-based extracellular traps to capture and kill bacteria. The mechanism(s) and proteins that promote neutrophil extracellular trap (NET)-mediated bacterial trapping are not clearly established. Surfactant protein D (SP-D) is an innate immune collectin present in many mucosal surfaces. We hypothesized that SP-D can bind both the pathogens and NETs to augment NET-mediated bacterial trapping. To test this hypothesis, we used LPS and Pseudomonas aeruginosa pneumonia mouse models and performed in vivo and ex vivo assays. In this study, we show that NETs are produced by the neutrophils recruited to the airways in response to the bacterial ligand. Notably, NETs are detected as short fragments of DNA–protein complexes in the airways as opposed to the long stringlike structures seen in ex vivo cultures. SP-D recognizes both the short NET fragments and the long NET DNA structures. SP-D–NET copurification studies further show that SP-D can simultaneously recognize NETs and carbohydrate ligands in vivo. Similar to the LPS model, soluble DNA–protein complexes and increased amounts of SP-D are detected in the murine model of P. aeruginosa pneumonia. We then tested the effect of SP-D on NET-mediated trapping of P. aeruginosa by means of Western blots, fluorescence microscopy, and scanning electron microscopy. Results of these experiments show that SP-D microagglutinates P. aeruginosa and allows an efficient bacterial trapping by NETs. Collectively, these findings provide a unique biological relevance for SP-D–DNA interactions and places SP-D as an important innate immune protein that promotes bacterial trapping by NETs during neutrophil-mediated host defense.

Akt is essential to induce NADPH-dependent NETosis and to switch the neutrophil death to apoptosis

To the editor: Neutrophil extracellular traps (NETs) have been recently identified as major contributors of several hematological and vascular diseases. These disorders include thrombosis, small vessel vasculitis, systemic lupus erythematosus, autoimmunity, pneumonia, sepsis, and blood transfusion

CXCL1 Contributes to Host Defense in Polymicrobial Sepsis via Modulating T Cell and Neutrophil Functions

Severe bacterial sepsis leads to a proinflammatory condition that can manifest as septic shock, multiple organ failure, and death. Neutrophils are critical for the rapid elimination of bacteria; however, the role of neutrophil chemoattractant CXCL1 in bacterial clearance during sepsis remains elusive. To test the hypothesis that CXCL1 is critical to host defense during sepsis, we used CXCL1-deficient mice and bone marrow chimeras to demonstrate the importance of this molecule in sepsis. We demonstrate that CXCL1 plays a pivotal role in mediating host defense to polymicrobial sepsis after cecal ligation and puncture in gene-deficient mice. CXCL1 appears to be essential for restricting bacterial outgrowth and death in mice. CXCL1 derived from both hematopoietic and resident cells contributed to bacterial clearance. Moreover, CXCL1 is essential for neutrophil migration, expression of proinflammatory mediators, activation of NF-κB and MAPKs, and upregulation of adhesion molecule ICAM-1. rIL-17 rescued impaired host defenses in cxcl1−/− mice. CXCL1 is important for IL-17A production via Th17 differentiation. CXCL1 is essential for NADPH oxidase–mediated reactive oxygen species production and neutrophil extracellular trap formation. This study reveals a novel role for CXCL1 in neutrophil recruitment via modulating T cell function and neutrophil-related bactericidal functions. These studies suggest that modulation of CXCL1 levels in tissues and blood could reduce bacterial burden in sepsis.

NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases.

SP-D counteracts GM-CSF-mediated increase of granuloma formation by alveolar macrophages in lysinuric protein intolerance

BackgroundPulmonary alveolar proteinosis (PAP) is a syndrome with multiple etiologies and is often deadly in lysinuric protein intolerance (LPI). At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain. We hypothesized that GM-CSF and surfactant protein D (SP-D) would enhance the clearance of proteins and dying cells that are typically present in the airways of PAP lungs.MethodsCells and cell-free supernatant of therapeutic bronchoalveolar lavage fluid (BALF) of a two-year-old patient with LPI were isolated on multiple occasions. Diagnostic BALF samples from an age-matched patient with bronchitis or adult PAP patients were used as controls. SP-D and total protein content of the supernatants were determined by BCA assays and Western blots, respectively. Cholesterol content was determined by a calorimetic assay or Oil Red O staining of cytospin preparations. The cells and surfactant lipids were also analyzed by transmission electron microscopy. Uptake of Alexa-647 conjugated BSA and DiI-labelled apoptotic Jurkat T-cells by BAL cells were studied separately in the presence or absence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml), ex vivo. Specimens were analyzed by light and fluorescence microscopy.ResultsHere we show that large amounts of cholesterol, and large numbers of cholesterol crystals, dying cells, and lipid-laden foamy alveolar macrophages were present in the airways of the LPI patient. Although SP-D is present, its bioavailability is low in the airways. SP-D was partially degraded and entrapped in the unusual surfactant lipid tubules with circular lattice, in vivo. We also show that supplementing SP-D and GM-CSF increases the uptake of protein and dying cells by healthy LPI alveolar macrophages, ex vivo. Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.ConclusionsWe propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI. To improve the lung condition of LPI patients with PAP, it would be useful to explore alternative therapies for increasing dead cell clearance while decreasing cholesterol content in the airways.

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.

JNK Activation Turns on LPS- and Gram-Negative Bacteria-Induced NADPH Oxidase-Dependent Suicidal NETosis

Neutrophils cast neutrophil extracellular traps (NETs) to ensnare microbial pathogens. Nevertheless, the molecular rheostats that regulate NETosis in response to bacteria are not clearly established. We hypothesized that stress-activated protein kinase or c-Jun N-terminal Kinase (SAPK/JNK) is a molecular switch that turns on NETosis in response to increasing concentrations of lipopolysaccharide (LPS)- and Gram-negative bacteria. Here we show that Escherichia coli LPS (0111:B4; 10–25 μg/ml), but not phorbol myristate acetate (PMA), activates JNK in human neutrophils in a dose-dependent manner. JNK inhibitors SP600125 and TCSJNK6o, and a TLR4 inhibitor TAK242 suppress reactive oxygen species production and NETosis in LPS-, but not PMA-treated neutrophils. Diphenyleneiodonium suppresses LPS-induced NETosis, confirming that endotoxin induces NADPH oxidase-dependent NETosis. Immunoblots, Sytox Green assays, and confocal microscopy of cleaved caspase-3 and nuclear morphology show that JNK inhibition does not induce apoptosis in LPS-stimulated neutrophils. JNK inhibition also suppresses NETosis induced by two typical Gram-negative bacteria, E. coli and Pseudomonas aeruginosa. Therefore, we propose that neutrophils use a TLR4-dependent, JNK-mediated molecular sensing mechanism to initiate NADPH oxidase-dependent suicidal NETosis in response to increasing concentrations of LPS, and Gram-negative bacteria. The LPS-TLR4-JNK activation axis determines the fate of these cells: to be or not to be NETotic neutrophils.

Impaired resolution of inflammation in the Endoglin heterozygous mouse model of chronic colitis.

Endoglin is a coreceptor of the TGF-β superfamily predominantly expressed on the vascular endothelium and selective subsets of immune cells. We previously demonstrated that Endoglin heterozygous (Eng +/−) mice subjected to dextran sulfate sodium (DSS) developed persistent gut inflammation and pathological angiogenesis. We now report that colitic Eng +/− mice have low colonic levels of active TGF-β1, which was associated with reduced expression of thrombospondin-1, an angiostatic factor known to activate TGF-β1. We also demonstrate dysregulated expression of BMPER and follistatin, which are extracellular regulators of the TGF-β superfamily that modulate angiogenesis and inflammation. Heightened colonic levels of the neutrophil chemoattractant and proangiogenic factor, CXCL1, were also observed in DSS-treated Eng +/− mice. Interestingly, despite increased macrophage and neutrophil infiltration, a gut-specific reduction in expression of the key phagocytic respiratory burst enzymes, NADPH oxidase 2 (Nox-2) and myeloperoxidase, was seen in Eng +/− mice undergoing persistent inflammation. Taken together, these findings suggest that endoglin is required for TGF-β superfamily mediated resolution of inflammation and fully functional myeloid cells.

Effect of Arginase Inhibition on Pulmonary L-Arginine Metabolism in Murine Pseudomonas Pneumonia

Rationale Infection of the lung with Pseudomonas aeruginosa results in upregulation of nitric oxide synthases (NOS) and arginase expression, and both enzymes compete for L-arginine as substrate. Nitric oxide (NO) production may be regulated by arginase as it controls L-arginine availability for NOS. We here studied the effect of systemic arginase inhibition on pulmonary L-arginine metabolism in Pseudomonas pneumonia in the mouse. Methods Mice (C57BL/6, 8–10 weeks old, female) underwent direct tracheal instillation of Pseudomonas (PAO-1)-coated agar beads and were treated by repeated intra-peritoneal injections of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or PBS until lungs were harvested on day 3 of the infection. L-arginine metabolites were quantified using liquid chromatography-tandem mass spectrometry, NO metabolites nitrate and nitrite by Griess reagent and cytokines by ELISA. Results NO metabolite concentrations (48.5±2.9 vs. 10.9±2.3 µM, p<0.0001), as well as L-ornithine (29.6±1.7 vs 2.3±0.4 µM, p<0.0001), the product of arginase activity, were increased in Pseudomonas infected lungs compared to naïve controls. Concentrations of the NOS inhibitor asymmetric dimethylarginine (ADMA) were also increased (0.44±0.02 vs. 0.16±0.01 µM, p<0.0001). Arginase inhibition in the infected animals resulted in a significant decrease in L-ornithine (14.6±1.6 µM, p<0.0001) but increase in L-arginine concentration (p<0.001), L-arginine/ADMA ratio (p<0.001), L-arginine availability for NOS (p<0.001), and NO metabolite concentrations (67.3±5.7 µM, p<0.05). Arginase inhibitor treatment also resulted in an increase in NO metabolite levels in animals following intratracheal injection of LPS (p = 0.015). Arginase inhibition was not associated with an increase in inflammatory markers (IFN-γ, IL-1β, IL-6, MIP-2, KC or TNF-α) in lung. Concentrations of the L-ornithine-dependent polyamines putrescine, spermidine and spermine were increased in Pseudomonas infected lungs (p<0.001, respectively) but were unaffected by ABH treatment. Conclusions Systemic arginase inhibition with ABH during Pseudomonas pneumonia in mice results in an increase in pulmonary NO formation but no pro-inflammatory effect.