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SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

In patients with antithrombotic prophylaxis employing low molecular weight heparins, a possible benefit for periodontitis can be expected, as they reportedly reduce NETs formation and dissociate histones from the chromatin backbone of NETs, as resumed byLiu et al.However, any clinical studies on low molecular weight heparins in periodontitis are lacking.Irwandi et al.summarised that novel and targeted approaches to manipulate neutrophil numbers and functions are warranted within the context of the treatment of periodontitis and also to mitigate its potential impact on other LGI disease. Cxcr2 plays a crucial role in phagocytic ability, reactive oxygen species production, F-actin and α-tubulin levels, and phosphorylation of ERK1/2 and p38 MAPK, impaired PI3K-AKT, NF-κB, TGFβ and IFNγ pathways. [...]Cxcr2 blockers might also be a possible option to attenuate the neutrophil hyper-responsiveness seen in periodontitisDelobel et al. 4 Conclusion Recently, the attention in mucosal inflammatory disease has been shifted from the topical to systemic point of view i.e., from the concept of dental biofilm dysbiosis to that of dysregulated immunity. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 1LeeJYTsolisRMBäumlerAJ.The microbiome and gut homeostasis.Science(2022)377(6601):eabp9960. doi:10.1126/science.abp9960 2SilvaLMBrenchleyLMoutsopoulosNM.Primary immunodeficiencies reveal the essential role of tissue neutrophils in periodontitis.Immunol Rev(2019)287(1):226–35. doi:10.1111/imr.12724 3SilvaLMDoyleADGreenwell-WildTDutzanNTranCLAbuslemeL.Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.Science(2021)374(6575):eabl5450. doi:10.1126/science.abl5450 4SørensenOEClemmensenSNDahlSLØstergaardOHeegaardNHGlenthøjA.Papillon-lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses.J Clin Invest(2014)124(10):4539–48. doi:10.1172/JCI76009 5VitkovLHartlDMinnichBHannigM.Janus-faced neutrophil extracellular traps in periodontitis.Front Immunol(2017)8:1404. doi:10.3389/fimmu.2017.01404 6WilliamsDWGreenwell-WildTBrenchleyLDutzanNOvermillerASawayaAP.Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.Cell(2021)184(15):4090–104 e15. doi:10.1016/j.cell.2021.05.013 7MatthewsJBWrightHJRobertsALing-MountfordNCooperPRChappleILC.Neutrophil hyper-responsiveness in periodontitis.J Dental Res(2007)86(8):718–22. doi:10.1177/154405910708600806 8LiXWangHYuXSahaGKalafatiLIoannidisC.Maladaptive innate immune training of myelopoiesis links inflammatory comorbidities.Cell(2022)185(10):1709–27 e18. doi:10.1016/j.cell.2022.03.043 9GoodridgeHSAhmedSSCurtisNKollmannTRLevyONeteaMG.Harnessing the beneficial heterologous effects of vaccination.Nat Rev Immunol(2016)16(6):392–400. doi:10.1038/nri.2016.43 10NeteaMGGiamarellos-BourboulisEJDomínguez-AndrésJCurtisNvan CrevelRvan de VeerdonkFL.Trained immunity: a tool for reducing susceptibility to and the severity of SARS-CoV-2 infection.Cell(2020)181(5):969–77. doi:10.1016/j.cell.2020.04.042 11HajishengallisGLiXDivarisKChavakisT.Maladaptive trained immunity and clonal hematopoiesis as potential mechanistic links between periodontitis and inflammatory comorbidities.Periodontol 2000(2022)89(1):215–30. doi:10.1111/prd.12421 12VitkovLMuñozLEKnopfJSchauerCOberthalerHMinnichB.Connection between periodontitis-induced low-grade endotoxemia and systemic diseases: neutrophils as protagonists and targets.Int J Mol Sci(2021)22(9):4647. doi:10.3390/ijms22094647 13TrindadeDCarvalhoRMachadoVChambroneLMendesJJBotelhoJ.Prevalence of periodontitis in dentate people between 2011 and 2020: a systematic review and meta-analysis of epidemiological studies.J Clin Periodontol(2023)50(5):604–26. doi:10.1111/jcpe.13769 14HajishengallisGChavakisT.Local and systemic mechanisms linking periodontal disease and inflammatory comorbidities.Nat Rev Immunol(2021)21(7):426–40. doi:10.1038/s41577-020-00488-6 15AckermannMAndersHJBilyyRBowlinGLDanielCDe LorenzoR.Patients with COVID-19: in the dark-NETs of neutrophils.Cell Death Differ(2021)28(11):3125–39. doi:10.1038/s41418-021-00805-z 16MaroufNCaiWSaidKNDaasHDiabHChintaVR.Association between periodontitis and severity of COVID-19 infection: a case-control study.J Clin Periodontol(2021)48(4):483–91. doi:10.1111/jcpe.13435 17VitkovLKnopfJKrunićJSchauerCSchoenJMinnichB.Periodontitis-derived dark-NETs in severe covid-19.Front Immunol(2022)13:872695. doi:10.3389/fimmu.2022.872695 18AnJYKernsKAOuelletteARobinsonLMorrisHDKaczorowskiC.Rapamycin rejuvenates oral health in aging mice.Elife(2020)9:e54318. doi:10.7554/eLife.54318

The subgingival biofilm attached to tooth surfaces triggers and maintains periodontitis. Previously, late-onset periodontitis has been considered a consequence of dysbiosis and a resultant polymicrobial disruption of host homeostasis. However, a multitude of studies did not show “healthy” oral microbiota pattern, but a high diversity depending on culture, diets, regional differences, age, social state etc. These findings relativise the aetiological role of the dysbiosis in periodontitis. Furthermore, many late-onset periodontitis traits cannot be explained by dysbiosis; e.g. age-relatedness, attenuation by anti-ageing therapy, neutrophil hyper-responsiveness, and microbiota shifting by dysregulated immunity, yet point to the crucial role of dysregulated immunity and neutrophils in particular. Furthermore, patients with neutropenia and neutrophil defects inevitably develop early-onset periodontitis. Intra-gingivally injecting lipopolysaccharide (LPS) alone causes an exaggerated neutrophil response sufficient to precipitate experimental periodontitis. Vice versa to the surplus of LPS, the increased neutrophil responsiveness characteristic for late-onset periodontitis can effectuate gingiva damage likewise. The exaggerated neutrophil extracellular trap (NET) response in late-onset periodontitis is blameable for damage of gingival barrier, its penetration by bacteria and pathogen-associated molecular patterns (PAMPs) as well as stimulation of Th17 cells, resulting in further neutrophil activation. This identifies the dysregulated immunity as the main contributor to periodontal disease.

Periodontitis is characterized by PMN infiltration and formation of neutrophil extracellular traps (NETs). However, their functional role for periodontal health remains complex and partially understood. The main function of NETs appears to be evacuation of dental plaque pathogen-associated molecular patterns. The inability to produce NETs is concomitant with aggressive periodontitis. But in cases with exaggerated NET production, NETs are unable to maintain periodontal health and bystander damages occur. This pathology can be also demonstrated in animal models using lipopolysaccharide as PMN activator. The progress of periodontitis appears to be a consequence of the formation of gingival pockets obstructing the evacuation of both pathogen-associated and damage-associated molecular patterns, which are responsible for the self-perpetuation of inflammation. Thus, besides the pathogenic effects of the periodontal bacteria, the dysregulation of PMN activation appears to play a main role in the periodontal pathology. Consequently, modulation of PMN activation might be a useful approach to periodontal therapy.

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in 'beads-on-a-string' conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of 'beads-on-a-string' DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.

The frequent severe COVID-19 course in patients with periodontitis suggests a link of the aetiopathogenesis of both diseases. The formation of intravascular neutrophil extracellular traps (NETs) is crucial to the pathogenesis of severe COVID-19. Periodontitis is characterised by an increased level of circulating NETs, a propensity for increased NET formation, delayed NET clearance and low-grade endotoxemia (LGE). The latter has an enormous impact on innate immunity and susceptibility to infection with SARS-CoV-2. LPS binds the SARS-CoV-2 spike protein and this complex, which is more active than unbound LPS, precipitates massive NET formation. Thus, circulating NET formation is the common denominator in both COVID-19 and periodontitis and other diseases with low-grade endotoxemia like diabetes, obesity and cardiovascular diseases (CVD) also increase the risk to develop severe COVID-19. Here we discuss the role of propensity for increased NET formation, DNase I deficiency and low-grade endotoxaemia in periodontitis as aggravating factors for the severe course of COVID-19 and possible strategies for the diminution of increased levels of circulating periodontitis-derived NETs in COVID-19 with periodontitis comorbidity.

Periodontitis is a general term for diseases characterised by inflammatory destruction of tooth-supporting tissues, gradual destruction of the marginal periodontal ligament and resorption of alveolar bone. Early-onset periodontitis is due to disturbed neutrophil extracellular trap (NET) formation and clearance. Indeed, mutations that inactivate the cysteine proteases cathepsin C result in the massive periodontal damage seen in patients with deficient NET formation. In contrast, exaggerated NET formation due to polymorphonuclear neutrophil (PMN) hyper-responsiveness drives the pathology of late-onset periodontitis by damaging and ulcerating the gingival epithelium and retarding epithelial healing. Despite the gingival regeneration, periodontitis progression ends with almost complete loss of the periodontal ligament and subsequent tooth loss. Thus, NETs help to maintain periodontal health, and their dysregulation, either insufficiency or surplus, causes heavy periodontal pathology and edentulism.

Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.

Implants trigger an inflammatory response, which is important for osseointegration. Here we studied neutrophil extracellular trap (NET) release of human neutrophils in response to sandblasted large-grit acid etched (SLA) implants using fluorescent, confocal laser scanning and scanning electron microscopy. Our studies demonstrate that human neutrophils rapidly adhered to SLA surfaces, which triggered histone citrullination and NET release. Further studies showed that albumin or acetylsalicylic acid had no significant effects on the inflammatory response to SLA surfaces. In contrast to bioinert materials, which do not osseointegrate, the bioactivity of SLA surfaces is coupled with the ability to release NETs. Further investigations are necessary for clarifying the role of NETosis for osseointegration.