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.

Neutrophil extracellular traps (NETs) are beneficial antimicrobial defense structures that can help fight against invading pathogens in the host. However, recent studies reveal that NETs exert adverse effects in a number of diseases including those of the lung. Many inflammatory lung diseases are characterized with a massive influx of neutrophils into the airways. Neutrophils contribute to the pathology of these diseases. To date, NETs have been identified in the lungs of cystic fibrosis (CF), acute lung injury (ALI), allergic asthma, and lungs infected with bacteria, virus, or fungi. These microbes and several host factors can stimulate NET formation, or NETosis. Different forms of NETosis have been identified and are dependent on varying types of stimuli. All of these pathways however appear to result in the formation of NETs that contain DNA, modified extracellular histones, proteases, and cytotoxic enzymes. Some of the NET components are immunogenic and damaging to host tissue. Innate immune collectins, such as pulmonary surfactant protein D (SP-D), bind NETs, and enhance the clearance of dying cells and DNA by alveolar macrophages. In many inflammatory lung diseases, bronchoalveolar SP-D levels are altered and its deficiency results in the accumulation of DNA in the lungs. Some of the other therapeutic molecules under consideration for treating NET-related diseases include DNases, antiproteases, myeloperoxidase (MPO) inhibitors, peptidylarginine deiminase-4 inhibitors, and anti-histone antibodies. NETs could provide important biological advantage for the host to fight against certain microbial infections. However, too much of a good thing can be a bad thing. Maintaining the right balance of NET formation and reducing the amount of NETs that accumulate in tissues are essential for harnessing the power of NETs with minimal damage to the hosts.

Neutrophil extracellular traps (NETs), a unique DNA framework decorated with antimicrobial peptides, have been in the scientific limelight for their role in a variety of pathologies ranging from cystic fibrosis to cancer. The formation of NETs, as well as relevant regulatory mechanisms, physiological factors, and pharmacological agents have not been systematically discussed in the context of their beneficial and pathological aspects. Novel forms of NET formation including vital NET formation continue to be uncovered, however, there remain fundamental questions around established mechanisms such as NADPH-oxidase (Nox)-dependent and Nox-independent NET formation. Whether NET formation takes place in the tissue versus the bloodstream, internal factors (e.g. reactive oxygen species (ROS) production and transcription factor activation), and external factors (e.g. alkaline pH and hypertonic conditions), have all been demonstrated to influence specific NET pathways. Elements of neutrophil biology such as transcription and mitochondria, which were previously of unknown significance, have been identified as critical mediators of NET formation through facilitating chromatin decondensation and generating ROS, respectively. While promising therapeutics inhibiting ROS, transcription, and gasdermin D are being investigated, neutrophil phagocytosis plays a critical role in host defense and any therapies targeting NET formation must avoid impairing the physiological functions of these cells. This review summarizes what is known in the many domains of NET research, highlights the most relevant challenges in the field, and inspires new questions that can bring us closer to a unified model of NET formation.

Collectins play important roles in the innate immune defense against microorganisms. Recently, a new collectin, collectin 11 (CL-11 or CL-K1), was identified via database searches. In present work, we characterize the structural and functional properties of CL-11. Under nonreducing conditions, in gel permeation chromatography recombinant CL-11 forms disulfide-linked oligomers of 100 and 200 kDa. A mAb-based ELISA estimates the concentration of CL-11 in plasma to be 2.1 μg/ml, and the presence of CL-11 in plasma was further verified by Western blotting and mass spectrometry. Mannan-binding lectin-associated serine protease 1 (MASP-1) copurified with CL-11 and the interaction in plasma with MASP-1 and/or MASP-3 was further demonstrated using ELISA. We identified the adrenal glands, the kidneys, and the liver as primary sites of expression. CL-11 lectin activity was demonstrated by ELISA and showed that CL-11 has preference for l-fucose and d-mannose. We finally show that CL-11 binds to intact bacteria, fungi, and viruses and that CL-11 decreases influenza A virus infectivity and forms complexes with DNA. On the basis of the significant concentration of CL-11 in circulation and CL-11’s interaction with various microorganisms and MASP-1 and/or MASP-3, it is conceivable that CL-11 plays a role in activation of the complement system and in the defense against invading microorganisms.

Reactive oxygen species (ROS) are essential for neutrophil extracellular trap (NET) formation or NETosis. Nevertheless, how ROS induces NETosis is unknown. Neutrophil activation induces excess ROS production and a meaningless genome-wide transcription to facilitate chromatin decondensation. Here we show that the induction of NADPH oxidase-dependent NETosis leads to extensive DNA damage, and the subsequent translocation of proliferating cell nuclear antigen (PCNA), a key DNA repair protein, stored in the cytoplasm into the nucleus. During the activation of NETosis (e.g., by phorbol myristate acetate,Escherichia coliLPS,Staphylococcus aureus(RN4220), orPseudomonas aeruginosa), preventing the DNA-repair-complex assembly leading to nick formation that decondenses chromatin causes the suppression of NETosis (e.g., by inhibitors to, or knockdown of, Apurinic endonuclease APE1, poly ADP ribose polymerase PARP, and DNA ligase). The remaining repair steps involving polymerase activity and PCNA interactions with DNA polymerases β/δ do not suppress agonist-induced NETosis. Therefore, excess ROS produced during neutrophil activation induces NETosis by inducing extensive DNA damage (e.g., oxidising guanine to 8-oxoguanine), and the subsequent DNA repair pathway, leading to chromatin decondensation.

Neutrophils are short-lived innate immune cells. These cells respond quickly to stimuli, and die within minutes to hours; the relevance of DNA transcription in dying neutrophils remains an enigma for several decades. Here we show that the transcriptional activity reflects the degree of DNA decondensation occurring in both NADPH oxidase 2 (Nox)-dependent and Nox-independent neutrophil extracellular trap (NET) formation or NETosis. Transcriptomics analyses show that transcription starts at multiple loci in all chromosomes earlier in the rapid Nox-independent NETosis (induced by calcium ionophore A23187) than Nox-dependent NETosis (induced by PMA). NETosis-specific kinase cascades differentially activate transcription of different sets of genes. Inhibitors of transcription, but not translation, suppress both types of NETosis. In particular, promoter melting step is important to drive NETosis (induced by PMA, E. coli LPS, A23187, Streptomyces conglobatus ionomycin). Extensive citrullination of histones in multiple loci occurs only during calcium-mediated NETosis, suggesting that citrullination of histone contributes to the rapid DNA decondensation seen in Nox-independent NETosis. Furthermore, blocking transcription suppresses both types of NETosis, without affecting the reactive oxygen species production that is necessary for antimicrobial functions. Therefore, we assign a new function for transcription in neutrophils: Transcriptional firing, regulated by NETosis-specific kinases, helps to drive NETosis.