Neutrophil extracellular traps (NETs) : formation and implications

Neutrophils are cells of the immune system which freely circulate in blood vessels and are recruited to the inflammation sites when the human organism responds to microbial infections. One of the mechanisms of neutrophil action is the formation of neutrophil extracellular traps (NETs) The process of NET generation, called netosis, is a specific type of cell death, different from necrosis and apoptosis. NETs are formed by neutrophils upon contact with various bacteria or fungi as well as with activated platelets or under the influence of numerous inflammatory stimuli, and this process is associated with dramatic changes in the morphology of the cells. The main components of NETs, DNA and granular antimicrobial proteins, determine their antimicrobial properties. The pathogens trapped in NETs are killed by oxidative and non-oxidative mechanisms. On the other hand, it was also discovered that chromatin and proteases released into the circulatory system during NET formation can regulate procoagulant and prothrombotic factors and take part in clot formation in blood vessels. NETs have also been detected in lungs where they are involved in chronic inflammation processes in ALI/ARDS patients. Moreover, DNA-proteins complexes have been found in the airway fluids of cystic fibrosis patients where they can increase the viscosity of the sputum and have a negative impact on the lung functions. The DNA-complexed granular proteins and other proteins released by neutrophils during netosis lead to autoimmunity syndromes such as systemic lupus erythematosus (SLE), small-vessel vasculitis (SVV) or autoimmune diseases associated with the formation of autoantibodies against chromatin and neutrophil components. A possible involvement of NETs in metastasis is also considered

Neutrophil Extracellular Traps in Infectious Human Diseases

Neutrophils, as the main cells of the first line of host defense against microbial pathogens, are responsible for pathogen recognition, inhibition of pathogen spreading into the host tissue, and finally, killing the invader cells. Neutrophils carry out these functions via numerous mechanisms, including a relatively recently described activity based on a release of neutrophil extracellular traps (NETs), a process called netosis. NETs are structures composed of DNA backbone, decorated with antimicrobial factors, derived from neutrophil granules. The structure of NETs and their enzymatic and microbicidal inclusions enable efficient trapping and killing of microorganisms within the neutrophil extracellular space. However, the efficiency of NETs depends on neutrophil ability to recognize pathogen signals and to trigger rapid responses. In this chapter, we focus on possible pathways involved in the release of NETs and summarize the current knowledge on triggers of this process during bacterial, fungal, protozoan, and viral infections. We also consider the mechanisms used by microorganisms to evade NET-killing activity and analyze the harmful potential of NETs against the host cells and the contribution of NETs to noninfectious human diseases

Selected mucolytic, anti-inflammatory and cardiovascular drugs change the ability of neutrophils to form extracellular traps (NETs)

Neutrophils form the first line of host defense against infections that combat pathogens using two major mechanisms, the phagocytosis or the release of neutrophil extracellular traps (NETs). The netosis (NET formation) exerts additional, unfavorable effects on the fitness of host cells and is also involved at the sites of lung infection, increasing the mucus viscosity and in the circulatory system where it can influence the intravascular clot formation. Although molecular mechanisms underlying the netosis are still incompletely understood, a role of NADPH oxidase that activates the production of reactive oxygen species (ROS) during the initiation of NETs has been well documented. Since several commonly used drugs can affects the netosis, our current study was aimed to determine the effects of selected mucolytic, anti-inflammatory and cardiovascular drugs on NET formation, with a special emphasis on ROS production and NADPH oxidase activity. The treatment of neutrophils with N-acetylcysteine, ketoprofen and ethamsylate reduced the production of ROS by these cells in a dose-dependent manner. NET formation was also modulated by selected drugs. N-acetylcysteine inhibited the netosis but in the presence of H2O2 this neutrophil ability was restored, indicating that N-acetylcysteine may influence the NET formation by modulating ROS productivity. The administration of ethamsylate led to a significant reduction in NET formation and this effect was not restored by H2O2 or S. aureus, suggesting the unexpected additional side effects of this drug. Ketoprofen seemed to promote ROS-independent NET release, simultaneously inhibiting ROS production. The results, obtained in this study strongly suggest that the therapeutic strategies applied in many neutrophil-mediated diseases should take into account the NET-associated effects

Neutrophil extracellular traps (NETs) in upper respiratory tract secretions : insights into infectious and allergic rhinitis

Neutrophil extracellular traps (NETs) are structures released by neutrophils in response to various infections. NETs have a biocidal role and have been demonstrated to be effective against bacteria, fungi, viruses, and parasites. Depending on the situation, NETs can protect the host from pathogen invasion or contribute to the development of autoimmune diseases such as cystic fibrosis and rheumatoid arthritis. In this study, we aimed to investigate the occurrence of NET as one of the components in upper respiratory tract secretions in infectious and allergic diseases. Nasal mucus was collected from donors diagnosed with infectious rhinitis or allergic rhinitis. The extracellular DNA content was determined using SytoxGreen staining, and the total protein pool was determined using the microBCA method. Micrococcal nuclease was used to digest the samples and ELISA was employed to identify the NET proteins. The enzymatic activity of elastase was determined. Our findings showed that nasal mucus collected from patients with infectious rhinosinusitis contained extracellular DNA that could come from a variety of sources, responsible for increasing the density and viscosity of secretions, as well as NETs proteins. The identified enzymatic activity of NET elastase indicates the possible irritation of nasal tissues. However, the DNA content was not identified in the samples from allergic patients. In addition, we have shown in preliminary studies that therapy using N-acetylcysteine can liquefy nasal secretions.The study suggests that the composition of nasal mucus varies according to the cause of mucosal irritation. The presence of DNA and NET proteins can have severe consequences for the therapeutic process prolonging treatment. The low viscosity of nasal mucus in allergic patients facilitates mucosal flushing and the removal of allergens. Understanding the occurrence and role of NETs in various respiratory diseases is critical for developing effective treatment strategies that consider the complex interaction between the immune system and pathogens. The results of this study suggest that NETs may be present in upper respiratory tract secretions with an infectious background, supporting basic defense mechanisms using eosinophils and EETs. Further research is needed to explore the potential of NETs as a therapeutic target in respiratory diseases

Interactions of Candida albicans Cells with Aerobic and Anaerobic Bacteria during Formation of Mixed Biofilms in the Oral Cavity

Biofilm is a compact coating formed on various artificial and physiologic surfaces by a population of microorganisms which in this habitat establish a close cooperation, exploiting both the physical interactions that stabilize the community and chemical cooperation, engaging numerous agents to modify the environment, i.e., to influence the acidity, nutrient acquisition, or oxygen availability. Microorganisms can also communicate using quorum-sensing molecules carrying specific messages. Some microbes temporarily dominate, while others are constantly replaced by different community members. But these co-operations or competitions have a deep sense—they serve to protect the whole community against the defense system of the host to assure survival. The oral cavity is inhabited by diverse microorganisms, including bacteria, but also yeast-like fungi from the genus Candida that stay under a tight control of the host as long as its immune system is not weakened; then these relatively mild commensals convert to dangerous pathogens that start the invasion often in collaboration with other microbes. Elongated hyphal forms of fungal cells favor the biofilm type of growth and communication with other microbes supporting immune resistance of the biofilm. In this chapter, we discuss the mechanisms of interactions between bacteria and C. albicans in the oral cavity, their communication, host responses, and possible strategies of anti-biofilm treatment

Aspartic proteases and major cell wall components in Candida albicans trigger the release of neutrophil extracellular traps

Neutrophils use different mechanisms to cope with pathogens that invade the host organism. The most intriguing of these responses is a release of neutrophil extracellular traps (NETs) composed of decondensed chromatin and granular proteins with antimicrobial activity. An important potential target of NETs is Candida albicans—an opportunistic fungal pathogen that employs morphological and phenotype switches and biofilm formation during contact with neutrophils, accompanied by changes in epitope exposition that mask the pathogen from host recognition. These processes differ depending on infection conditions and are thus influenced by the surrounding environment. In the current study, we compared the NET release by neutrophils upon contact with purified main candidal cell surface components. We show here for the first time that in addition to the main cell wall-building polysaccharides (mannans and β-glucans), secreted aspartic proteases (Saps) trigger NETs with variable intensities. The most efficient NET-releasing response is with Sap4 and Sap6, which are known to be secreted by fungal hyphae. This involves mixed, ROS-dependent and ROS-independent signaling pathways, mainly through interactions with the CD11b receptor. In comparison, upon contact with the cell wall-bound Sap9 and Sap10, neutrophils responded via a ROS-dependent mechanism using CD16 and CD18 receptors for protease recognition. In addition to the Saps tested, the actuation of selected mediating kinases (Src, Syk, PI3K, and ERK) was also investigated. β-Glucans were found to trigger a ROS-dependent process of NET production with engagement of Dectin-1 as well as CD11b and CD18 receptors. Mannans were observed to be recognized by TLRs, CD14, and Dectin-1 receptors and triggered NET release mainly via a ROS-independent pathway. Our results thus strongly suggest that neutrophils activate NET production in response to different candidal components that are presented locally at low concentrations at the initial stages of infection. However, NET release seemed to be blocked by increasing numbers of fungal cells

Candida albicans shields the periodontal killer Porphyromonas gingivalis from recognition by the host immune system and supports the bacterial infection of gingival tissue

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen—Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host–biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection

Adhesive protein-mediated crosstalk between <i>Candida albicans</i> and <i>Porphyromonas gingivalis</i> in dual species biofilm protects the anaerobic bacterium in unfavorable oxic environment

Abstract The oral cavity contains different types of microbial species that colonize human host via extensive cell-to-cell interactions and biofilm formation. Candida albicans —a yeast-like fungus that inhabits mucosal surfaces—is also a significant colonizer of subgingival sites in patients with chronic periodontitis. It is notable however that one of the main infectious agents that causes periodontal disease is an anaerobic bacterium— Porphyromonas gingivalis. In our study, we evaluated the different strategies of both pathogens in the mutual colonization of an artificial surface and confirmed that a protective environment existed for P. gingivalis within developed fungal biofilm formed under oxic conditions where fungal cells grow mainly in their filamentous form i.e. hyphae. A direct physical contact between fungi and P. gingivalis was initiated via a modulation of gene expression for the major fungal cell surface adhesin Als3 and the aspartic proteases Sap6 and Sap9. Proteomic identification of the fungal surfaceome suggested also an involvement of the Mp65 adhesin and a “moonlighting” protein, enolase, as partners for the interaction with P. gingivalis. Using mutant strains of these bacteria that are defective in the production of the gingipains—the proteolytic enzymes that also harbor hemagglutinin domains—significant roles of these proteins in the formation of bacteria-protecting biofilm were clearly demonstrated

Farnesol, a Quorum-Sensing Molecule of Candida albicans Triggers the Release of Neutrophil Extracellular Traps

The efficient growth of pathogenic bacteria and fungi in the host organism is possible due to the formation of microbial biofilms that cover the host tissues. Biofilms provide optimal local environmental conditions for fungal cell growth and increased their protection against the immune system. A common biofilm-forming fungus&mdash;Candida albicans&mdash;uses the quorum sensing (QS) mechanism in the cell-to-cell communication, which determines the biofilm development and, in consequence, host colonization. In the presented work, we focused on the ability of neutrophils&mdash;the main cells of the host&rsquo;s immune system to recognize quorum sensing molecules (QSMs) produced by C. albicans, especially farnesol (FOH), farnesoic acid (FA), and tyrosol (TR), with emphasis on the neutrophil extracellular traps (NETs) formation in a process called netosis. Our results showed for the first time that only farnesol but not farnesolic acid or tyrosol is capable of activating the NET production. By using selective inhibitors of the NET signaling pathway and analyzing the activity of selected enzymes such as Protein Kinase C (PKC), ERK1/2, and NADPH oxidase, we showed that the Mac&minus;1 and TLR2 receptors are responsible for FOH recognizing and activating the reactive oxygen species (ROS)-dependent netosis pathway