Fusobacterium nucleatum is recognized as a master species in the development of periodontitis, with many strain-specific functions. In order to enhance the multiplication of late colonizers, it is crucial to create favorable conditions with priority to decrease neutrophil efficiency. This is carried out by decreasing superoxide generation and apoptosis induction, limiting the number of counter-attacking immune cells (Kurgan et al., 2017). The production and release of reactive oxygen species (ROS) is part of the pathogen elimination strategies and can be induced by the phagocytosis of the invaders (El-Benna et al., 2016; Zeng et al., 2019). In contrast to well-characterized pathogens, the amount of F. nucleatum differs within the timeline of disease progression in accordance with its bridging-specie nature (Tomšič et al., 2021). Moreover, it has been proved that F. nucleatum exaggerates NET formation in comparison to other bacterial species, such as P. gingivalis, in strong connection with NOD-like receptors (Alyami et al., 2019). Triggering NETosis, instead of phagocytosis, as a neutrophil defense mechanism is probably associated with the huge size of the pathogen (Urban et al., 2006). Another antimicrobial mechanism strongly exploited by F. nucleatum is the release of Human Neutrophil Peptide-1 (HNP-1) (Musrati et al., 2016). Increased concentrations of HNP-1 peptide trigger epithelial cell death and bacterial attachment to keratinocytes (Gursoy et al., 2013). Exposure of F. nucleatum to defensins, a class of antimicrobial peptides released for instance by neutrophils, can result in decreased bacterial membrane permeability and elevated dental plaque biofilm formation. These strategies are destined to aid the resistance against the membrane disruption and lysis caused by the above-mentioned proteins. As a consequence, the proliferation level is secured (Keskin et al., 2014; Xu and Lu, 2020). Importantly, secreted serine protease fusolisin degrades extracellular matrix proteins as well as cleaves the most abundant immunoglobulin at the mucosal surface (IgA), contributing to the inactivation of the host defense and disease progression (Bachrach et al., 2004; Doron et al., 2014). To add insult to injury, observed tissue environment modulation can give rise to other pathological conditions, such as colorectal cancer (Luo et al., 2019).

Pathogens can also have an indirect effect on the function of neutrophils. In the case of Prevotella intermedia and Tannerella forsythia, it has been proved that they can adhere to and internalize into human dental follicle stem cells (hDFSCs). This infection modulates the environment and diminishes the expression of cytokines, however, it does not change hDFSC differentiation capacity. Consequently, a reduced release of IL-8 can contribute to limited chemotaxis of polymorphonuclear leukocytes (PMNs). Moreover, in the presence of infected hDFSCs neutrophil phagocytic activity and NET formation are also decreased, which gives pathogens enough time for gingival colonization (Hieke et al., 2016). Importantly, a challenge with T. forsythia induces a strong immune response as indicated by the number of immune cells accumulated at the lesion of administration in a murine model. These in vivo experiments showed, that unlike in the presence of P. gingivalis, the neutrophil migration is not inhibited (Gosling et al., 2005). However, some comorbidity, such as glycogen storage diseases (GSDs), can in few cases further enhance gingival tissue destruction. A case report of a GSD patient manifesting with the subversion of the neutrophil chemotaxis and neutropenia described an evident T. forsythia-mediated intraoral bone loss (Ma et al., 2018).

Interestingly, Ksiazek et al. discovered that T. forsythia expresses a serpin (protease inhibitor) called miropin that can contribute to its survival and ability to avoid protease activity of neutrophils (Ksiazek et al., 2015). Strikingly, another newly discovered metalloproteinase called mirolysin together with the previously characterized, secreted metalloproteinase called karilysin, represent important virulence factors of T. forsythia. Both proteinases show a synergistic inhibitory effect on many pathways in the host immune system. Significantly, T. forsythia with a mutation in the expression of these enzymes presented with a strongly diminished survival rate (Jusko et al., 2012, 2015). Apart from proteinases, an outer surface layer (S-layer) is a T. forsythia-associated virulence factor that can contribute to serum resistance and has a significant role in coaggregation with other oral pathogens, such as P. gingivalis. The S-layer significantly reduces the deposition of C3b on the bacterial surface, which would act as a tag for phagocytosis (Shimotahira et al., 2013). Strikingly, a bacterial glycan found linked to the S-layer can modulate dendritic cells and suppress T helper 17 response (Settem et al., 2013). As an anaerobic bacteria, T. forsythia lacks a complex enzymatic system against oxidative stress. However, the genome of this pathogen encodes an oxidative stress response sensor protein (OxyR) homolog, that acts as a positive regulator for antioxidant gene expression. This can contribute to the resistance of the bacterial community to oxidative stress in the aerobic oral cavity and protect against oxidative burst in leukocytes, which is essential in the dental plaque biofilm formation (Honma et al., 2009; Moriguchi et al., 2017).

The main component of NETs is DNA (Brinkmann et al., 2004). Among other species (P. gingivalis, F. nucleatum and Aggregatibacter actinomycetemcomitans), P. intermedia has the highest nuclease activity, enabling it to block the capture and subsequent phagocytosis by neutrophils. Two genes have been identified as responsible for this feature, nucA and nucD, encoding enzymes that require cations for their activity (Doke et al., 2017). Still, this strategy itself cannot be considered unique, as numerous pathogens associated with periodontal disease are able to express such enzymes, including members of the red and the orange complexes (Palmer et al., 2012). Bacteria species are grouped based on chronological coexistence during diseases progression. Members of the red complex (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) are late colonizers and the multiplication of them relies on, and is tightly connected to the conquest of the members of the orange complex (e.g., Fusobacterium nucleatum, Prevotella intermedia) (Mohanty et al., 2019).

Secreted extracellular adenosine triphosphate (eATP) is a distinctive virulence factor characterized in A. actinomycetemcomitans. eATP is predominantly an intracellular signaling molecule involved in the recruitment of immune cells (Ding et al., 2016). However, in periodontitis, eATP secreted by A. actinomycetemcomitans induces an upregulation of cytokine expression, resulting in the massive recruitment of inflammatory cells via mainly the p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) pathways (Herbert et al., 2017). Therefore, would be of great importance to investigate the effects of eATP on p38 or MK2 kinases not only in macrophages, but also in neutrophils Interestingly, macrophages challenge with A. actinomycetemcomitans induced autophagic influx, restricting the expression of the proinflammatory cytokine IL-1β and ROS production, which ensure protection for this pathogen (Lee et al., 2020). Of importance, bacterial metabolites, such as short-chain fatty acids (SCFAs), are widely spread among different species and connected to a great number of immunological disorders (Ferreira et al., 2014). During bacterial infection, A. actinomycetemcomitans-associated SCFAs can also alter neutrophil effector mechanisms by downregulating cytokine production and phagocytic activity (Corrêa et al., 2017).

Leukotoxins are virulence factors expressed e.g., by some Staphylococcus spps. or by Mannheimia haemolytica, altering both the innate and the adaptive immune system (Futagawa-Saito et al., 2004; Aulik et al., 2010). Strikingly, leukotoxin A (LtxA) secreted by A. actinomycetemcomitans triggers a dysregulation in neutrophils, resulting in the release of citrullinated proteins (Konig et al., 2016). The hypercitrullination of host proteins, on one hand leads to diminished functions, such as in the case of histone proteins embedded in NETs can be responsible for decreased anti-microbial activity (Li et al., 2010). On the other hand, the development of rheumatoid arthritis (RA) is the consequence of an elevated level of citrullinated proteins, that leads to the hyperactivity of the immune system resulting in the destruction of the host tissue (Kuhn et al., 2006). The link between RA and periodontitis has long been under the scope of scientific research due to the numerous shared immune-pathological similarities, including overall disease progression, cytokine profile and risk factors (Koziel et al., 2014; de Molon et al., 2019). As these two diseases can present with similar symptoms, some approaches can be applied in both cases to control disease manifestations, i.e., the melanocortin agonism can be a potential way to overcome excessive oral inflammation (Madeira et al., 2016). Melanocortin proteins upon receptor biding elevate the resolution of inflammation by reducing the amount of released pro-inflammatory cytokines and induce efferocytosis, the clearance of neutrophils by macrophages (Montero-Melendez et al., 2011). Besides the induction of citrullinating enzymes in neutrophils, LtxA has a strong toxic effect on leukocytes and induces NET formation in a dose-dependent manner. Additionally, the activity of neutrophil elastase (NE), a principal proteinase in bacterial defense, is exploited. Normally, NE is localized in the cytoplasm, however, upon LtxA exposure, neutrophil lysis is triggered, followed by the release of high amounts of elastase. As a consequence, human gingival epithelial cells and fibroblasts detach and die (Madeira et al., 2016; Hiyoshi et al., 2019).

Of note, A. actinomycetemcomitans activates many more neutrophil defense mechanisms, such as ROS production, the release of proteases and the already mentioned NET formation that can be considered a successful defense strategy in the absence of bacterial nuclease activity (Mikolai et al., 2021). Although, the coin has two sides, the overactivation of the above-mentioned mechanisms results in the destruction of the host tissues. Among other virulence factors, this pathogen produces a toxin called the Cytolethal Distending Toxin (CDT), which causes cell cycle arrest in vitro and in vivo as well as blocks proliferation of the target cells. These disease-promoting effects of CTD are noted towards the periodontal epithelial cells in the rat model (Ohara et al., 2011). Unfortunately, amoxicillin, azithromycin, and metronidazole show an attenuated efficiency against A. actinomycetemcomitans, while phagocytosis of the pathogen is only effective at a lower MOI (Multiplicity of Infection) (Ardila and Bedoya-García, 2020). Fortunately, when neutrophils are highly outnumbered by bacterial cells, internalized azithromycin significantly increases the phagocytic elimination efficiency of PMNs (Lai et al., 2015).

Peptoanaerobacter stomatis is a newly characterized member of the destructive oral microbiome. In contrast to well-characterized periodontal pathogens, this one is Gram-positive (Sizova et al., 2015). Infection with this pathogen promotes migration of not just neutrophils, but also monocytes, which additionally strongly fuel inflammation, along with the vigorously induced granule content exocytosis (Vashishta et al., 2019). Furthermore, a significant induction of NET formation is observed upon neutrophil challenge with this pathogen (Armstrong et al., 2018). Moreover, P. stomatis is relatively resistant to phagocytosis, while ROS production is significantly increased. These types of defense mechanisms are a double-edged sword, because they simultaneously induce the degradation of the host gingival tissue and periodontitis progression (Flores et al., 2017).

Another Gram-positive member of the community is Filifactor alocis that shows an extraordinary resilience to oxidative stress as mentioned in Table 1. This provides a substantial colonization advantage over the host defense system and competing pathogens. Similar to other victorious pathogens, F. alocis manipulates the neutrophil immune responses. The analysis of global changes in the transcriptome of neutrophils challenged with F. alocis reveals strong effects on the PMNs. A delayed apoptosis is accompanied by a prolonged inflammatory response and activated migration through the MAPK cascade and the TNF-α signaling pathways (Miralda et al., 2020). Strikingly, F. alocis fails to induce NET formation, but doesn’t influence the P. stomatis-mediated NETosis. In contrast, an earlier challenge of neutrophils with F. alocis decreases NET formation triggered by PMA (Armstrong et al., 2018).