Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects mediated by these proteases in inflammatory/auto-immune disorders. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome (PLS). The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials.

BackgroundThe majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms.Objective/designIn this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed.Results/conclusionBiofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown.

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Dental care professionals (DCPs) are thought to be at enhanced risk of occupational exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, robust data to support this from large-scale seroepidemiological studies are lacking. We report a longitudinal seroprevalence analysis of antibodies to SARS-CoV-2 spike glycoprotein, with baseline sampling prior to large-scale practice reopening in July 2020 and follow-up postimplementation of new public health guidance on infection prevention control (IPC) and enhanced personal protective equipment (PPE). In total, 1,507 West Midlands DCPs were recruited into this study in June 2020. Baseline seroprevalence was determined using a combined IgGAM enzyme-linked immunosorbent assay and the cohort followed longitudinally for 6 mo until January/February 2021 through the second wave of the coronavirus disease 2019 pandemic in the United Kingdom and vaccination commencement. Baseline seroprevalence was 16.3%, compared to estimates in the regional population of 6% to 7%. Seropositivity was retained in over 70% of participants at 3- and 6-mo follow-up and conferred a 75% reduced risk of infection. Nonwhite ethnicity and living in areas of greater deprivation were associated with increased baseline seroprevalence. During follow-up, no polymerase chain reaction–proven infections occurred in individuals with a baseline anti–SARS-CoV-2 IgG level greater than 147.6 IU/ml with respect to the World Health Organization international standard 20-136. After vaccination, antibody responses were more rapid and of higher magnitude in those individuals who were seropositive at baseline. Natural infection with SARS-CoV-2 prior to enhanced PPE was significantly higher in DCPs than the regional population. Natural infection leads to a serological response that remains detectable in over 70% of individuals 6 mo after initial sampling and 9 mo from the peak of the first wave of the pandemic. This response is associated with protection from future infection. Even if serological responses wane, a single dose of the Pfizer-BioNTech 162b vaccine is associated with an antibody response indicative of immunological memory.

Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii, stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated.KEYWORDS neutrophils, NETs, reactive oxygen species, periodontitis, oral bacteria P eriodontitis is initiated by the accumulation of microbial biofilms at and below the gingival margin. Indeed, it has been estimated that ϳ700 oral bacterial species and ϳ1,200 predominant phylotypes exist (1-3). Of these bacterial species, 5 major bacterial complexes (red, orange, yellow, green, and purple) have been identified by Socransky et al. using DNA probes (4) and are referred to here as Socransky complexes. The clustering and ordination analysis allowed them to assign microbial species to a color complex on the basis of the strength of the association with each other and the clinical staging of periodontitis. The biofilms which develop during disease are orchestrated to maximize their adherence, communication, and survival. The accumulation of bacterial species within the biofilm enables its development and perseverance, and certain bacteria, such as Fusobacterium nucleatum, are key orchestrators of biofilm formation and maturation (5).In susceptible individuals, dysbiosis and an aberrant host-microbe equilibrium can result in the onset of disease (6), where the microbial biofilm thrives by exploiting the

BackgroundAggressive periodontitis is associated with the presence of Aggregatibacter actinomycetemcomitans, a leukotoxin (Ltx)-producing periodontal pathogen. Ltx has the ability to lyse white blood cells including neutrophils.ObjectivesThis study was aimed at investigating the interactions between neutrophils and Ltx with regard to the chemotactic properties of Ltx and the release of neutrophil extracellular traps (NETs).MethodsNeutrophils from healthy blood donors were isolated and incubated for 30 min and 3 h with increasing concentrations of Ltx (1, 10, and 100 ng/mL) as well as with A. actinomycetemcomitans strains (NCTC 9710 and HK 1651) producing different levels of Ltx. Formation of NETs and cell lysis were assessed by microscopy, fluorescence-based assays, and measurement of released lactate dehydrogenase. Neutrophil migration in response to different Ltx gradients was monitored by real-time video microscopy, and image analysis was performed using ImageJ software.ResultsAlthough Ltx (10 and 100 ng/mL) and the leukotoxic A. actinomycetemcomitans strain HK 1651 lysed some neutrophils, other cells were still capable of performing NETosis in a concentration-dependent manner. Low doses of Ltx and the weakly leukotoxic strain NCTC 9710 did not lead to neutrophil lysis, but did induce some NETosis. Furthermore, all three concentrations of Ltx enhanced random neutrophil movement; however, low directional accuracy was observed compared with the positive control (fMLP).ConclusionsThe results indicate that Ltx acts both as a neutrophil activator and also causes cell death. In addition, Ltx directly induces NETosis in neutrophils prior to cell lysis. In future studies, the underlying pathways involved in Ltx-meditated neutrophil activation and NETosis need to be investigated further.

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Maintenance of oral health is in part managed by the immune-surveillance and antimicrobial functions of polymorphonuclear leukocytes (PMNs), which migrate from the circulatory system through the oral mucosal tissues as oral PMNs (oPMNs). In any microorganism-rich ecosystem, such as the oral cavity, PMNs migrate toward various exogenous chemoattractants, phagocytose bacteria, and produce neutrophil extracellular traps (NETs) to immobilize and eliminate pathogens. PMNs obtained from the circulation through venipuncture (hereafter called cPMNs) have been widely studied using various functional assays. We aimed to study the potential of oPMNs in maintaining oral health and therefore compared their chemotactic and antimicrobial functions with cPMNs. To establish chemotactic, phagocytic, and NET forming capacities, oPMNs and cPMNs were isolated from healthy subjects without obvious oral inflammation. Directional chemotaxis toward the chemoattractant fMLP was analyzed using an Insall chamber and video microscopy. fMLP expression was assessed by flow cytometry. Phagocytosis was analyzed by flow cytometry, following PMN incubation with heat-inactivated FITC-labeled micro-organisms. Furthermore, agar plate-based killing assays were performed with Escherichia coli ( Ec ). NET formation by oPMNs and cPMNs was quantified fluorimetrically using SYTOX™ Green, following stimulation with either PMA or RPMI medium (unstimulated control). In contrast to cPMNs, the chemotactic responses of oPMNs to fMLP did not differ from controls (mean velocity ± SEM of cPMNs: 0.79 ± 0.24; of oPMNs; 0.10 ± 0.07 micrometer/min). The impaired directional movement toward fMLP by oPMNs was explained by significantly lower fMLP receptor expression. Increased adhesion and internalization of various micro-organisms by oPMNs was observed. oPMNs formed 13 times more NETs than stimulated cPMNs, in both unstimulated and stimulated conditions. Compared to cPMNs, oPMNs showed a limited ability for intracellular killing of Ec . In conclusion, oPMNs showed exhausted capacity for efficient chemotaxis toward fMLP which may be the result of migration through the oral tissues into the oral cavity, being a highly “hostile” ecosystem. Overall, oPMNs' behavior is consistent with hyperactivity and frustrated killing. Nevertheless, oPMNs most likely contribute to maintaining a balanced oral ecosystem, as their ability to internalize microbes in conjunction with their abundant NET production remains after entering the oral cavity.