key: cord-0017475-3zsjne0f
authors: Vitkov, Ljubomir; Muñoz, Luis E.; Knopf, Jasmin; Schauer, Christine; Oberthaler, Hannah; Minnich, Bernd; Hannig, Matthias; Herrmann, Martin
title: Connection between Periodontitis-Induced Low-Grade Endotoxemia and Systemic Diseases: Neutrophils as Protagonists and Targets
date: 2021-04-28
journal: Int J Mol Sci
DOI: 10.3390/ijms22094647
sha: ff172a6e1a8c683d4ace3ae1e031a25eec7ed598
doc_id: 17475
cord_uid: 3zsjne0f

Periodontitis is considered a promoter of many systemic diseases, but the signaling pathways of this interconnection remain elusive. Recently, it became evident that certain microbial challenges promote a heightened response of myeloid cell populations to subsequent infections either with the same or other pathogens. This phenomenon involves changes in the cell epigenetic and transcription, and is referred to as ‘‘trained immunity’’. It acts via modulation of hematopoietic stem and progenitor cells (HSPCs). A main modulation driver is the sustained, persistent low-level transmission of lipopolysaccharide from the periodontal pocket into the peripheral blood. Subsequently, the neutrophil phenotype changes and neutrophils become hyper-responsive and prone to boosted formation of neutrophil extracellular traps (NET). Cytotoxic neutrophil proteases and histones are responsible for ulcer formations on the pocket epithelium, which foster bacteremia and endoxemia. The latter promote systemic low-grade inflammation (SLGI), a precondition for many systemic diseases and some of them, e.g., atherosclerosis, diabetes etc., can be triggered by SLGI alone. Either reverting the polarized neutrophils back to the homeostatic state or attenuation of neutrophil hyper-responsiveness in periodontitis might be an approach to diminish or even to prevent systemic diseases.

A multitude of clinical findings demonstrated an unambiguous correlation between periodontitis and systemic diseases, like atherosclerosis, diabetes, and cardiovascular diseases [1] . The main link between systemic diseases and periodontitis is considered to be the systemic low-grade inflammation (SLGI); apparently a consequence of low-grade endoxemia (LGE) [2] [3] [4] [5] [6] [7] . Endotoxins like lipopolysaccharide (LPS) are involved in the pathogenesis of many diseases such as atherosclerosis, obesity, chronic fatigue, metabolic syndrome, and many other inflammation-driven conditions. The detection of elevated plasma endotoxin levels provides evidence for each of these diseases [8] . Indeed, periodontitis [9, 10] and even gingivitis [11] is characterized by LGE, which is considered a possible precondition for SLGI [12] . The exact signaling pathway responsible for triggering the systemic diseases caused by periodontitis-related LGE is not yet clarified. Understanding trained immunity progressed greatly in the last few years. Earlier, the idea of immune memory was reserved for the adaptive immunity. Growing in vivo and clinical evidences now indicate the ability of innate immunity to memorize bacterial challenges and to adjust its response to recurrent challenges, metabolically, epigenetically, and transcriptionally [13] [14] [15] [16] . Certain microbial infections or vaccines promote a heightened response of myeloid cell populations to a subsequent infection with the same or even different pathogens. This process involves changes in cell transcription and is referred to as "trained immunity" [17, 18] . The innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite the short lifespan of some of these cells in circulation; trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs) [14] .

This review discusses the accomplishment of LGE as the main link between periodontitis and systemic diseases. Long-term LGE in periodontitis is a consequence of disturbance of gingival barrier function, development of trained immunity, and emergence of hyper-responsive neutrophils. The trained immunity triggered by periodontitis-reliant LGE appears to be a crucial deteriorating factor in some systemic diseases, such as diabetes, atherosclerosis, and cardiovascular diseases [2] [3] [4] [5] [6] [7] . This highlights the importance of periodontitis-dependent LGE and suggests the necessity of neutrophil calming as a new prophylactic approach to systemic diseases. Due to the huge domain of trained innate immunity (for references see [19] ), we focused on the role of neutrophils in periodontitis, as they are responsible for the persistent periodontitis-reliant LGE, which leads to disbalanced trained immunity and systemic disease pathology.

The concept of trained immunity describes the long-term functional reprogramming of the progenitors of innate immune cells evoked by exogenous or endogenous insults. Subsequently, this leads to an altered response of differentiated cells towards a second challenge, after returning to a non-activated state [19] . The secondary response to the subsequent non-specific stimulus can be altered in such a way that the cells respond more or less strongly, when compared to the primary response, conferring context-and timeadjusted responses [19] . Different stimuli, such as β-glucan [20] , bacillus Calmette-Guérin (BCG vaccine) [16] , and LPS [15] induce different programs of trained immunity. However, as periodontitis preferentially supplies LPS, we focus on the effects of LPS on innate immunity, in this review. HSPCs directly respond to LPS, e.g., via the toll-like receptor 4 (TLR4) [21, 22] or indirectly through LPS-induced cytokines [23] . The pioneering transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) is induced by LPS in HSPCs and is required for the epigenetic chromatin marks and altered sensitivity of the associated genes to secondary challenges [15] . C/EBPβ can act as a pioneer factor triggering chromatin opening of myeloid genes, without inducing their transcription [24] . It can pre-program chromatin accessibility for other transcription factors, leading to a facilitated response of DNA regulatory elements to stimulation [25] . LPS-exposed HSPCs become epigenetically primed for a myeloid lineage bias with the enhancers remaining more accessible than naive HSPCs. Furthermore, LPS-exposed HSPCs keep increased accessibility of numerous genes that predispose one to a more rapid activation of myeloid lineage commitment, in response to secondary stimulation [15] . As the innate memory responses depend solely on epigenetic remodeling, the trained immunity appears to lack specificity ( Figure 1 ).

The molecular basis of the epigenetic modifications includes changes in chromatin organization at the level of the topologically associated domains, transcription of long non-coding RNAs, methylation and acetylation of genes involved in the innate immune responses, and reprogramming of cellular metabolism [19] . Most proinflammatory gene loci in the quiescent myeloid cells are in a repressed configuration [26] , hindering access of the transcriptional machinery to the regulatory regions that drive the expression of inflammatory factors [27] . Two key epigenetic marks characterize trained immunity-the acetylation of histone 3 lysine 27 (H3K27ac) at distal enhancers (marked by histone 3 lysine 4 methylation (H3K4me1)) and the consolidation of histone 3 lysine 4 trimethylation (H3K4me3) at the promoters of stimulated genes [19] (Figure 2 ). The transmission of these marks through DNA replication and the cell cycle is robustly required to maintain trained immunity [28] .

(a) (b) Figure 1 . Concept of trained immunity. (a) The LPS signaling pathway via Toll-like receptor 4 (TLR4) and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) constitutes a hallmark of both innate and adaptive immune activation. NF-κB is a protein complex controlling the transcription of DNA, cytokine production, cell survival. It orchestrates the immune response to infection. The activation of NF-κB is initiated by the signal-induced degradation of Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (I-κB) proteins; and (b) comparison between innate and adaptive immunity. Innate and adaptive immunity provide broad and antigen-specific protection, respectively. (Top) innate immunity can provide protection against heterologous stimuli. Adaptive memory (bottom) is elicited with primary exposure to a specific antigen, and then, a secondary exposure and subsequent protection requires exposure to the same antigenic epitopes.

The molecular basis of the epigenetic modifications includes changes in chromatin organization at the level of the topologically associated domains, transcription of long non-coding RNAs, methylation and acetylation of genes involved in the innate immune responses, and reprogramming of cellular metabolism [19] . Most proinflammatory gene loci in the quiescent myeloid cells are in a repressed configuration [26] , hindering access of the transcriptional machinery to the regulatory regions that drive the expression of inflammatory factors [27] . Two key epigenetic marks characterize trained immunity-the acetylation of histone 3 lysine 27 (H3K27ac) at distal enhancers (marked by histone 3 lysine 4 methylation (H3K4me1)) and the consolidation of histone 3 lysine 4 trimethylation (H3K4me3) at the promoters of stimulated genes [19] (Figure 2 ). The transmission of these marks through DNA replication and the cell cycle is robustly required to maintain trained immunity [28] . NF-κB is a protein complex controlling the transcription of DNA, cytokine production, cell survival. It orchestrates the immune response to infection. The activation of NF-κB is initiated by the signal-induced degradation of Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (I-κB) proteins; and (b) comparison between innate and adaptive immunity. Innate and adaptive immunity provide broad and antigen-specific protection, respectively. (Top) innate immunity can provide protection against heterologous stimuli. Adaptive memory (bottom) is elicited with primary exposure to a specific antigen, and then, a secondary exposure and subsequent protection requires exposure to the same antigenic epitopes. 

LGE is a consequence of either metabolic disorders like metabolic endotoxemia or chronic infections like periodontitis. Prolonged LGE results in trained immunity, a longlasting proinflammatory phenotype of leukocytes. LGE represents a strong risk factor for the occurrence of chronic inflammatory diseases such as diabetes, atherosclerosis, cardio- 

LGE is a consequence of either metabolic disorders like metabolic endotoxemia or chronic infections like periodontitis. Prolonged LGE results in trained immunity, a longlasting proinflammatory phenotype of leukocytes. LGE represents a strong risk factor for the occurrence of chronic inflammatory diseases such as diabetes, atherosclerosis, cardiovascular diseases, and others [29, 30] . Here, we provide a concise overview of mechanisms responsible for initiations of systemic diseases by LGE.

Diabetes: Triggering of TLR4 activates proinflammatory pathways via activation of NF-κB and activator protein 1, which, in turn, promote production and release of multitudes of proinflammatory cytokines, including TNF-α and IL-6. This causes serine phosphorylation of the IRS-1, resulting in insulin resistance [31] . Thus, low doses of LPSs induce a biphasic change in glucose uptake in normal-weight volunteers. Insulin sensitivity enhances in the first few hours after injection [32] and significantly reduces later [33] . Doses of LPSs as low as 3 ng/kg are sufficient to induce a significant reduction of insulin sensitivity and increase circulation of insulin and glucose, 24 h after the LPS injection [33] .

Cardiovascular diseases:

LGE is associated with a significantly increased risk of cardiovascular diseases; however, their mutual relationship is still elusive. In cardiovascular disease patients, LGE triggers TLR4-mediated inflammatory responses, leading to SLGI [34] . A main representative of cardiovascular disease is the coronary artery disease resulting from a reduced blood flow to the heart muscle caused by luminal obstruction by atherosclerotic plaques in the arteries of the heart.

Atherosclerosis: Trained immunity is mechanistically linked to atherosclerosis [35] .

LGE induces atherosclerotic plaque formation and progression of atherosclerotic lesions, and release of proinflammatory molecules from endothelial cells [36] . LPS appears to increase endothelial lipase, which was suggested to cause a reduction in HDL [37] . The latter supports the growth of atherosclerotic plaques [38] . A short-term treatment with LPS in a super-low-dose that mimics chronic infection elicits the polarization of monocytes into a sustained pro-inflammatory state. This is characterized by upregulation of lymphocyte antigen 6C, C-C chemokine receptor type 5, monocyte chemoattractant protein 1, and decreased expression levels of scavenger receptor class B type 1. It ultimately aggravates atherosclerosis [39] . LGE skews neutrophils into a non-resolving inflammatory state with elevated and reduced levels of inflammatory and homeostatic mediators, respectively [40] . These neutrophils are prone to NET formation [41, 42] , which further fosters atherosclerosis [43] and cardiovascular complications [44] . This implies that LGE drives many systemic diseases, predominantly acting through the over-activation of innate immunity.

While a (non-lethal) high-dose of LPS induces innate immune tolerance with an attenuated immune response to a second challenge, a super-low-dose of LPS induces trained immunity [39, 45, 46] . The latter is achieved via the reprogramming of HSPCs. Although trained immunity usually improves the host's defense against subsequent pathogenic threats, in chronic inflammatory disease it might be maladaptive [19] . Thus, neutrophils and other leukocytes derived from HSPCs with trained immunity might develop in a distinct state of hyper-responsiveness; this might be responsible for the development of non-resolving inflammation. The nature of non-resolving inflammatory neutrophils is reflected in elevated reactive oxygen species, due to disrupted peroxisome homeostasis. This results in the skewed activation of oxidized calmodulin-dependent protein kinase II, downstream expression of LTB4, matrix-metalloproteinase (MMP) 9, miR24, and reduced ATF4/KLF2-mediated expression of resolving mediators, such as LRRC32 and miR-126 [40] . Indeed, neutrophil hyper-responsiveness is a hallmark of periodontitis [47] [48] [49] [50] . Epigenetic reprogramming of HSPCs due to LGE makes neutrophils prone to exaggerated neutrophil extracellular trap (NET) formation [41, 42] . NETs are evolutionarily conserved innate immunity structures produced by activated neutrophils, often in response to bacterial challenge. NETs have a backbone of DNA and contain neutrophil proteases, as well as other bactericidal agents [51] [52] [53] [54] . Exaggerated NET formation causes crevicular ulcers (see Section 6.2), subsequent bacteremia and LGE. Diabetes can induce late-onset periodontitis [55, 56] , as it promotes neutrophils to form NETs [57] . The boosted NET formation is a part of trained immunity and is epigenetically reprogrammed through the acetylation of histone 4 in neutrophils [41, 42] . Subsequently, the transcriptomes of hyper-responsive neutrophils in periodontitis are altered [58] . Due to trained immunity, the neutrophil hyper-responsiveness remains in edentulous patients with a history of periodontitis [47] [48] [49] [50] , despite the disappearance of bacteremia after teeth exfoliation [59] . The fact that the hyper-responsiveness of neutrophils from patients with periodontitis is evident in vitro, where the influence of adaptive immunity is excluded, cannot be explained in other way than by trained immunity. However, neutrophils are comprised of certain subsets [60] , so they might have different effects on periodontal and systemic disease (SD) pathology. In vivo, many components modulate neutrophil response, e.g., innate immunity, adaptive immunity, environment, etc. Besides the innate immunity mechanisms responsible for deactivating LPS (see Section 6), many adaptive immunity mechanisms are able to counteract the neutrophil hyper-reactivity in vivo. Therefore, patients with periodontitis are de facto immunized with the LPS of periodontal pathogens [61] . Normal human serum contains polyreactive "natural" antibodies that bind LPS [62] . LPS antibodies to periodontal pathogens [63] are elevated in periodontal patients, as compared to subjects with a healthy periodontium. Regulatory T cells (Treg)/neutrophil interactions in inflammatory and autoimmune diseases play a crucial role for the neutrophil regulation and suppression [64] . The pro-inflammatory function of neutrophils in the promotion and pathogenesis of several autoimmune diseases like vasculitis [65] , rheumatoid arthritis [66] etc., was reported. Defective Treg function were demonstrated in all these diseases, and Treg therapy ameliorated them [67] . Reduced Treg numbers in mice leads to exaggerated neutrophil activity resulting in mortality in endotoxic shock [68] . Tregs regulate survival and activity of human and murine neutrophils, and co-cultures of Tregs and neutrophils increases neutrophil apoptosis [68] . In addition, LPS-activated Tregs inhibit neutrophil functions [69] [70] [71] and even promote their apoptosis [69] . Thus, despite the periodontitis-induced hyper-responsibility, neutrophils cannot autonomously determine the entire immune response since it is orchestrated by the adaptive immunity. Therefore, it is not surprising that periodontitis is not ever associated to LGE-related SDs. Due to the huge complexity of the interactions between innate immunity and adaptive immunity, it is impossible to currently draw conclusions regarding the extent to which the periodontitisrelated trained immunity define LGE-related SDs. Future investigations might show to which extent this connection is clinically relevant.

Increasing the periodontitis severity carries a proportionally higher risk of coronary artery disease [72] . The relationship between periodontitis and coronary artery calcification indicates that periodontitis correlates positively and linearly with the presence of coronary artery calcification [73] . A relationship between periodontitis and aortic vascular inflammation exists, which in turn is a surrogate of coronary disease [74] . Live Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were isolated from atheromatous lesions [75, 76] . Furthermore, periodontal pathogens have the ability to induce atherosclerosis in animal models [77] [78] [79] .

Many reports clearly demonstrated the association between diabetes and periodontal disease in both animals and humans. LGE might induce diabetes predominantly due to the bacterial burden of periodontitis (see Section 3). The resulting proinflammatory cytokines cause insulin resistance [31, 33, 80] . In addition, P. gingivalis contributes to the development of insulin resistance via an impaired adaptive immune response [81] . Interestingly, treating the periodontal disease reduced glycated hemoglobin in diabetic patients [82] [83] [84] .

The high co-incidence between periodontitis and LGE-related SDs, e.g., cardiovascular diseases, diabetes etc., as well as the ability to induce some SDs via periodontitis in animal model, leads to the question as to how periodontitis might yield an initial ignition of LGE-related SDs? Three periodontitis aspects might be considered:

(i) Transient supply of LPS as bacterial fragments and whole bacteria in periodontitis [85] [86] [87] [88] . As an enduring LPS supplier, this aspect of periodontitis appears to be a crucial factor in triggering the LGE-relied SDs. (ii) Trained immunity marked by periodontitis-induced neutrophil hyper-responsiveness [47] [48] [49] [50] . This aspect might be responsible for the gingiva ulceration, and hence might act as a promoter of both LGE and bacteremia (see Section 6.2.); (iii) An independent settlement of periodontal pathogens in the atherosclerotic plaques [79, [89] [90] [91] . The periodontal pathogens appear able to induce and promote atherosclerosis in humans [75] .

LGE can be induced in many different ways. However, the mechanisms of LPS transition into the blood of patients with periodontitis remain elusive. The enteral LPS content is the main reservoir of LPS within the human body [92] , but the intestinal barrier function prevents LPS transmission into the blood of healthy individuals. (I) The mucin layer limits the access of LPS to the epithelium. (II) A multitude of cationic antimicrobial host proteins binds the negatively charged LPS [93] . (III) Intestinal alkaline phosphatase detoxifies LPS by the cleavage of the phosphate groups in position 1 and 4 of lipid A. The dephosphorylated LPS does not trigger TLR4 signaling [94] . (IV) The LPS-binding protein (LBP) forms a large LPS-LBP complex, with a reduced ability to trigger TLR4 signaling [95] . (V) LPS that enters the bloodstream, i.e., into the portal vein, is detoxified in the liver and excreted into the bile. Taken together, intestinal LPS does not abundantly transit into the peripheral blood of individuals with healthy intestines [92] . Alterations of the intestinal microbiota (dysbiosis) lead to increased intestinal permeability and translocation of LPS to the blood circulation. This disorder is referred to as metabolic endotoxemia ME. Dysbiosis also leads to the production of trimethylamine-N-oxide, a gut bacterial metabolite discussed as a new risk factor for the development of cardiovascular diseases [96] . Hence the question arises as to how LPS of periodontitis origin transmits into the blood of intestinally heathy individuals? LPS is able to even penetrate healthy gingival epithelium in very small quantities [97] , as it is detoxified by blood proteins, blood enzymes, and neutrophil-derived enzymes [98] . This, consequently, does not contribute to the elevation of the blood LPS level. However, increased blood plasma LPS levels were reported both in late [9] and early-onset periodontitis [10] and even transient bacteremia during daily hygienic procedures [86] [87] [88] . These clinical findings imply three key points:

• LPS penetration occurs via the inflamed periodontal pocket. • LPS penetration might be to, a large part, a consequence of transient bacteremia.

Intermittent pressure on the pocket epithelium appears to be crucial for bacteremia and subsequent endotoxemia.

Increased blood plasma levels of LPS were reported in gingivitis [11] and characterize periodontitis [9, 10] . The source of the LPS is the subgingival plaque (biofilm). A part of the LPS unleashed into the crevice is bound by blood proteins, neutrophil cationic antimicrobial proteins [99, 100] , neutrophil enzymes, and by LPS antibodies. Thus, a diverse repertoire of inactivation mechanisms modulates the ability of LPS to activate neutrophils via TLR4 [101] . The concentrations of soluble CD14 (sCD14) [102] , LPS-binding protein [103] , and antibodies to LPS of periodontal pathogens [63] are elevated in periodontal patients, as compared to subjects harboring a healthy periodontium.

In vitro P. gingivalis induces an increase of the gingival permeability through the secreted gingipains, enabling macro-molecules to diffuse through the tight junctions [104] . LPS within polar, aqueous fluid forms aggregates several tens of nanometers in size [105] . Moreover, LPS is discharged by bacteria in the form of outer membrane vesicles in a similar size range [106] , or as fragments of the outer bacterial membrane in cases of bacterial death. The periodontal crevice, where the dental plaque grows, is confined between the dental root surface and the crevicular gingival epithelium. The latter continuously produces blood transudate denoted gingival crevicular fluid (GCF) [107, 108] . The incessant GCF flow that drains off the LPS. P. gingivalis is not a prerequisite for periodontitis. The host inactivation mechanisms operate on mucosal surfaces and in tissues, lymph, and blood, and might profoundly influence LPS bioactivity in vivo [8, 109] .

LPS is first encountered by plasma proteins and the enzymes of the GCF, which is just blood plasma transudate. Alkaline phosphatase is abundant in GCF [110, 111] and inactivates LPS by dephosphorylation [112] . Acyloxyacyl hydrolase, produced by crevicular neutrophils [113] deactivates LPS through the removal of the secondary acyl chains from lipid A. Even though LPS penetrates the epithelium in gingivitis [11] , it is partially bound by the blood plasma components like HDL and LDL [114] [115] [116] ; sCD14 binds and transfers LPS from leukocytes to HDL [116, 117] . The bactericidal-permeability protein binds the LPS produced by many Gram-negative bacteria and blocks lipid A bioactivity [118, 119] ; Gelsolin, a highly conserved plasma protein, binds and neutralizes LPS [120] ; human serum from healthy individuals contains polyreactive "natural" antibodies that bind and neutralize LPS antigens [62] .

Gingivitis is a transitory inflammation and is not yet connected to trained immunity. Sustained untreated chronic gingivitis leads to pocket formation and consequently to transition into periodontitis. Due to the features of the periodontal pocket, which is a pathological formation, periodontitis must be considered a non-resolving chronic inflammation [121] . It is characterized by the formation of crevicular NETs, [122, 123] , multiple gingival micro-ulcerations [124] and bacteremia [85] [86] [87] [88] . The periodontitis-related bacteremia is responsible for the direct contact of LPS with HSPCs.

The exaggerated NET formation in the crevice drastically changes the situation, as NETs damage the epithelium, such that both bacteria and their pathogen-associated molecular pattern (PAMPs) overcome the epithelial barrier. On one hand, the topically-liberated LPS of the gingiva binds host proteins (LPS, antibodies as well as enzymes), and on the other hand, the periodontitis-related endotoxemia appears to be concomitant with bacteremia. Thus, the question arises, whether periodontitis-related endoxemia is just a consequence of the transitory bacteremia? Bacteremia requires discontinuations of crevicular epithelium in order to transmit the whole bacteria into connective tissue. Indeed, the crevicular epithelium is characterized by multiple micro-ulcerations [124] . Epithelial ulcerations enable the invasion of microorganisms and the unrestricted penetration of their PAMPs into the connective tissue, and thus aggravate the course of periodontitis. The influx of neutrophil and NET-derived proteases into the connective tissue facilitates bacterial spread. Therefore, both in late-onset [125, 126] and experimental periodontitis [59] , crevicular NETs are unable to completely prevent periodontal pathogen dissemination into connective tissues and peripheral blood. The latter becomes evident as bacteremia (number of species and positive cultures), which increases with the severity of the gingival inflammation [127] . The periodontitis-reliant bacteremia disappears after teeth exfoliation [59] . Epithelial ulcerations are of interest insofar as they represent the entry points for oral pathogens and their PAMPs, especially LPS. This results in bacteremia and endotoxemia. Epithelial disruptions such as insect bites or minor skin scratches [128] and gingival micro-ulcerations [86] [87] [88] are also accompanied by transient bacteremia and endotoxemia. In experimental periodontitis, disseminated oral pathogens are regularly observed within both liver and spleen, but not in mice after complete teeth loss [59] . Ulcers of oral epithelium other than the crevicular epithelium are very common, but they are mostly due to hereditary and environmental factors. Only in cases of acute necrotizing gingivitis, syphilis and tuberculosis are caused by bacteria [129, 130] . Indeed, oral pathogens penetrate crevicular epithelium either by bacterial internalization [131] or mechanically (yeasts) [132] without the formation of ulcers.

Thus, bacterial pathogens appear to not directly cause gingival ulcerations. Otherwise, hyper-responsive neutrophils and in particular exaggerated NET formation are able to damage mucosal epithelium. The main task of NETs is to limit the bacterial spread [51] , but exaggerated NET formation is destructive [133] . Exaggerated NET formation in the periodontal crevice [99] results in oversupply of neutrophil proteases [134] [135] [136] . These proteases damage the epithelial basal lamina if the NETs are not aggregated [137] and laminin-332 breakdown products foster neutrophil recruitment, which damage epithelia and subsequently weaken the epithelial barrier [138] . NET-derived components such as histones [139] [140] [141] [142] and myeloperoxidase (MPO) [140] are cytotoxic to epithelial cells; neutrophil proteases damage and even kill the epithelial cells. Some periodontal pathogens might even promote epithelial ulceration by stimulating the release of neutrophil elastase (NE) [143] . Indeed, increased GCF levels of laminin-332 [144] [145] [146] and neutrophil proteases [134] [135] [136] correlate with the epithelial ulceration in periodontitis [121, 124] . NETs also cause epitheliopathy via Oncostatin M [147] . Gingival epithelium becomes ulcerated in late-onset [121] and in experimental periodontitis [148, 149] . Obviously, epithelial ulcerations enable the contact between connective tissue matrices and NET-bound NE and MMPs, which are much more aggressive than the soluble enzymes [138] . High NET concentrations reportedly suppress keratinocyte proliferation, delay wound closure [57, 150] , and hence prolong the persistence of ulcers. However, aggNETs proteolytically inactivate several soluble pro-inflammatory mediators. Except for limited histological examinations, the ulcers of crevicular epithelium are not extensively studied and their role for systemic diseases remains elusive.

The continuously secreted GCF [107, 108] produces GCF flow, which drains off the disseminated bacteria and PAMPs. However, the retention of GCF in the pocket rises with deepening the periodontal pocket and increasing the GCF viscosity, particularly due to suppuration [151] . Intermittent pressure on the gingiva during mastication and dental hygiene is an underestimated problem and publications on this topic are scarce. However, clinicians should never forget to instruct dental patients after intra-pocket application of medications [152] and guided bone regeneration [153] to restrain from chewing. During mastication and dental hygiene procedures, the food bolus or the tooth brush, exerts intermittent pressure onto the oral gingiva, whereby the crevicular content (GCF and dispersed bacteria) is also pressed towards the pocket epithelium. In the ulcers, where matrices of the connective tissue are partly dissolved by exaggerated NETs, bacteria might be deeply inserted and even reach the venules. The high tendency of the venules to bleed in periodontitis and the transitory bacteremia/endotoxemia during mastication [85] , tooth brushing and flossing [86] [87] [88] , suggests such a portal of entry for pocket bacteria. Thus, the periodontal pocket might be considered to be a pump, which presses bacteria and their metabolic products into gingival connective tissues. From there they are pushed by intermittent mastication pressure into the gingival venules and reach the blood circulation via vena cava superior, conditioning both periodontitis-related bacteremia and endotoxemia. Thus, endotoxemia might be largely a consequence of transient bacteremia and lysis of the blood-borne bacteria, as the circulating LPS is continuously detoxified by the liver [92] .

Intermittent pressure on the pocket epithelium and consequently within the crevice appears to be crucial for bacteremia and endotoxemia. Despite the fact that the phenomenon of intermittent pressure is familiar, up to now it was considered clinically irrelevant and is not yet examined. Thus, its examination and the exact mechanism of bacterial penetration through the gingiva into blood are indispensable to enable further progress in the periodontal pathology.

It is empirically known that the treatment of periodontitis attenuates systemic inflammatory diseases [154] . A main reason for this appears to be the reduction of the periodontitis-related LGE. The latter skews neutrophils into a non-resolving inflammatory state with elevated the levels of the inflammatory mediators dectin-1, MMP9, and leukotriene B4 (LTB4), and reduced the levels of the homeostatic/anti-inflammatory mediator leucine-rich repeat containing 32 (LRRC32), transforming growth factor-β, and ferroportin (FPN) [40] . The reduction of LGE by the treatment of periodontitis attenuates the LPS effects on neutrophils and might be a favorable approach for the mitigation of systemic diseases. Treatment options of periodontitis and their limitations are well-known in clinical periodontology. Therefore, here we focus on mitigating the inflammatory effects of LPS on neutrophils. In general, two strategies are conceivable-(i) reverting the polarized neutrophils back to the homeostatic state or (ii) diminishing the exaggerated crevicular NET formation.

A potential candidate is 4-phenylbutyrate (4-PBA), known to restore peroxisome homeostasis in other cells [155] [156] [157] [158] . Recently, the application of 4-PBA was demonstrated to be a novel and effective strategy to revert polarized neutrophils back to their homeostatic state [40] . Thus, 4-PBA restores peroxisome-lysosome fusion in neutrophils and reduces the LPS-mediated elevation of neutrophil-derived ROS. In in vitro cultures of neutrophils, 4-PBA effectively reduced the induction of oxidized calmodulin-dependent protein kinase II, LTB4, MPO, dectin-1, CD11b, and miR-24 through a super-low-dose LPS and also restored the expression of ATF4, FPN, LRRC32, and miR-126 that was suppressed by LPS [40] . This functional rejuvenation of homeostatic neutrophils by 4-PBA treatment might reduce the pathogenesis of experimental atherosclerosis [40] and of related cardiovascular complications.

Despite the ability of aggNETs to prevent and even resolve inflammation on mucosal surfaces [109] and other tissues [159, 160] , they also cause an increase in the viscosity of GCF [122] . This hinders the GCF evacuation from deep periodontal pockets and potentially drives the formation of periodontal abscesses [151] . Diminishing the exaggerated NET formation, which appears to be a part of some cases of trained immunity [41, 42] by inhibitors of peptidylarginine deiminases 4 (PADI4), can reduce the periodontitis-related LGE and subsequently attenuate systemic diseases. As NETs directly promote atherosclerosis, the use of specific PADI4-inhibitors, in order to suppress the PADI4-dependent NET formation, might have therapeutic benefits in atherosclerosis [43] and cardiovascular complications [44] . In humans, metformin treatment reduces the concentrations of NET components independent of glucose control. In vitro this effect was related to the inhibitory effect exerted on the protein kinase C-nicotinamide adenine dinucleotide phosphate oxidase pathway [161] . Using topical treatment in periodontitis might prevent the systemic side effects of neutrophil inhibitors.

Taken together, both strategies aim at reducing the periodontitis-related LGE. Future treatment strategies are needed to diminish neutrophil hyper-responsiveness and, in particular, their propensity for NET formation, as neutrophil hyper-responsiveness fosters both non-resolving inflammation and LPS penetration through the gingival epithelium.

LGE is blamed for many systemic diseases, like diabetes, atherosclerosis, cardiovascular diseases, and others. Periodontitis, similar to other chronic infections, causes LGE and hence might foster systemic diseases. LGE might turn innate immunity into a state of trained immunity with the hyper-responsive neutrophils prone to exaggerated NET formation. The latter is responsible on one hand for gingival ulcerations and subsequent bacteremia/endotoxemia, and on the other hand for damages of blood vessels and other host tissues observed in systemic diseases. Attenuation of periodontitis reduces the periodontitis-related LGE and is prone to ameliorate the LGE-related systemic diseases.

Modulation of innate immunity might be a promising approach to diminish systemic inflammatory diseases and in the treatment of periodontitis. Acknowledgments: We acknowledge support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) and Saarland University, within the funding program Open Access Publishing.

Authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 

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BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils

Harnessing the Beneficial Heterologous Effects of Vaccination

Trained Immunity: A Program of Innate Immune Memory in Health and Disease

Defining Trained Immunity and Its Role in Health and Disease

Trained Immunity: A Tool for Reducing Susceptibility to and the Severity of SARS-CoV-2 Infection

Toll-like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

Pathogen-Induced TLR4-TRIF Innate Immune Signaling in Hematopoietic Stem Cells Promotes Proliferation but Reduces Competitive Fitness

Hematopoietic Progenitor Cells as Integrative Hubs for Adaptation to and Fine-Tuning of Inflammation

EBPβ Induces Chromatin Opening at a Cell-Type-Specific Enhancer

C/EBP Maintains Chromatin Accessibility in Liver and Facilitates Glucocorticoid Receptor Recruitment to Steroid Response Elements

Chromatin Contributions to the Regulation of Innate Immunity

Identification and Characterization of Enhancers Controlling the Inflammatory Gene Expression Program in Macrophages

Adaptation and Memory in Immune Responses

Potential Role of Endotoxin as a Proinflammatory Mediator of Atherosclerosis

Induction of Cardiovascular Pathology in a Novel Model of Low-Grade Chronic Inflammation

Gut Microbiota, Lipopolysaccharides, and Innate Immunity in the Pathogenesis of Obesity and Cardiovascular Risk

Early Endotoxemia Increases Peripheral and Hepatic Insulin Sensitivity in Healthy Humans

Innate Immunity Modulates Adipokines in Humans

Circulating Endotoxemia: A Novel Factor in Systemic Inflammation and Cardiovascular Disease in Chronic Kidney Disease

Trained Immunity: An Underlying Driver of Inflammatory Atherosclerosis

The Neutrophil-Activating Protein (NAP-1) Is Also Chemotactic for T Lymphocytes

Triglyceride-Rich Lipoproteins as Agents of Innate Immunity

The Persistence of Low-Grade Inflammatory Monocytes Contributes to Aggravated Atherosclerosis

Novel Reprogramming of Neutrophils Modulates Inflammation Resolution during Atherosclerosis

Histone Acetylation Promotes Neutrophil Extracellular Trap Formation

Histone Deacetylase Inhibitors Dose-Dependently Switch Neutrophil Death from NETosis to

Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

Neutrophil Extracellular Traps and Their Implications in Cardiovascular and Inflammatory Disease

Molecular Mechanisms Responsible for the Selective and Low-Grade Induction of Proinflammatory Mediators in Murine Macrophages by Lipopolysaccharide

Molecular Mechanism Responsible for the Priming of Macrophage Activation

Constitutionally Hyperreactive Neutrophils in Periodontitis

Hyper-Reactive Mononuclear Cells and Neutrophils in Chronic Periodontitis

Hyperactivity and Reactivity of Peripheral Blood Neutrophils in Chronic Periodontitis: Neutrophil Hyperactivity and Reactivity in Chronic Periodontitis

A Hyperactive Neutrophil Phenotype in Patients With Refractory Periodontitis

Novel Cell Death Program Leads to Neutrophil Extracellular Traps

Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida Albicans

To NET or Not to NET:Current Opinions and State of the Science Regarding the Formation of Neutrophil Extracellular Traps

Diabetes as a Risk Factor for Periodontal Disease-Plausible Mechanisms

Diabetes as a Potential Risk for Periodontitis: Association Studies

Diabetes Primes Neutrophils to Undergo NETosis, Which Impairs Wound Healing

Oral Neutrophil Transcriptome Changes Result in a Pro-Survival Phenotype in Periodontal Diseases

Host Defense against Oral Microbiota by Bone-Damaging T Cells

Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States

The Histopathological Comparison on the Destruction of the Periodontal Tissue between Normal Junctional Epithelium and Long Junctional Epithelium

Polyreactive Antibodies: Function and Quantification

Characteristics and Utilization of Antibody Measurements in Clinical Studies of Periodontal Disease

The Pivotal Role of Regulatory T Cells in the Regulation of Innate Immune Cells

IL-1beta Production by Human Polymorphonuclear Leucocytes Stimulated by Anti-Neutrophil Cytoplasmic Autoantibodies: Relevance to Systemic Vasculitis

Seeing the Wood for the Trees: The Forgotten Role of Neutrophils in Rheumatoid Arthritis

Human Regulatory T Cells: Role in Autoimmune Disease and Therapeutic Opportunities

Deficiency of Phosphatidylinositol 3-Kinase δ Signaling Leads to Diminished Numbers of Regulatory T Cells and Increased Neutrophil Activity Resulting in Mortality Due to Endotoxic Shock

Lipopolysaccharide-Activated CD4 + CD25 + T Regulatory Cells Inhibit Neutrophil Function and Promote Their Apoptosis and Death

Induction of Human IL-10-Producing Neutrophils by LPS-Stimulated Treg Cells and IL-10

Neutrophil-CD4+CD25+ T Regulatory Cell Interactions: A Possible New Mechanism of Infectious Tolerance

Association Between Periodontal Disease and Atherosclerotic Cardiovascular Diseases: Revisited. Front. Cardiovasc

Periodontal Disease Is an Independent Predictor of Intracardiac Calcification

Periodontal Disease Associates With Arterial Inflammation Via Potentiation of a Hematopoietic-Arterial Axis

Progulske-Fox, A. Periodontal Bacterial Invasion and Infection: Contribution to Atherosclerotic Pathology

Periodontal Diseases and Association with Atherosclerotic Disease

Active Invasion of Oral and Aortic Tissues by Porphyromonas Gingivalis in Mice Causally Links Periodontitis and Atherosclerosis

Periodontal Pathogens Invade Gingiva and Aortic Adventitia and Elicit Inflammasome Activation in Avβ6 Integrin-Deficient Mice

Polymicrobial Oral Infection with Four Periodontal Bacteria Orchestrates a Distinct Inflammatory Response and Atherosclerosis in ApoEnull Mice

Periodontitis Aggravated Pancreatic B-cell Dysfunction in Diabetic Mice through Interleukin-12 Regulation on Klotho

Periodontitis Induced by Porphyromonas Gingivalis Drives Periodontal Microbiota Dysbiosis and Insulin Resistance via an Impaired Adaptive Immune Response

Periodontal Therapy and Systemic Inflammation in Type 2 Diabetes Mellitus: A Meta-Analysis

An Overview of Systematic Reviews on the Effectiveness of Periodontal Treatment to Improve Glycaemic Control

The Effect of Periodontal Therapy on Glycemic Control and Fasting Plasma Glucose Level in Type 2 Diabetic Patients: Systematic Review and Meta-Analysis

Systemic Release of Endotoxins Induced by Gentle Mastication: Association With Periodontitis Severity

Associated With Toothbrushing and Dental Extraction

Bacteraemia Due to Dental Flossing

Periodontal Health Status and Bacteraemia from Daily Oral Activities: Systematic Review/Meta-Analysis

Sequential Colonization of Periodontal Pathogens in Induction of Periodontal Disease and Atherosclerosis in LDLR null Mice

Microbial Diversity Similarities in Periodontal Pockets and Atheromatous Plaques of Cardiovascular Disease Patients

Molecular Analysis of Oral Bacteria in Dental Biofilm and Atherosclerotic Plaques of Patients with Vascular Disease

Gastrointestinal and Hepatic Mechanisms Limiting Entry and Dissemination of Lipopolysaccharide into the Systemic Circulation

Antimicrobial Peptides and Maintenance of Intestinal Homeostasis

On the Role and Fate of LPS-Dephosphorylating Activity in the Rat Liver

Lipopolysaccharide-Binding Protein

Metabolic Endotoxemia and Cardiovascular Disease: A Systematic Review about Potential Roles of Prebiotics and Probiotics

The Passage of Tritiated Bacterial Endotoxin across Intact Gingival Crevicular Epithelium

Koenderman, L. Functional Heterogeneity and Differential Priming of Circulating Neutrophils in Human Experimental Endotoxemia

Binding Interactions of Bacterial Lipopolysaccharide and the Cationic Amphiphilic Peptides Polymyxin B and WLBU2

Lipo)Polysaccharide Interactions of Antimicrobial Peptides

Detoxifying Endotoxin: Time, Place and Person

Increased Levels of Soluble CD14 in Sera of Periodontitis Patients

Porphyromonas Gingivalis LPS Stimulates the Expression of LPS-Binding Protein in Human Oral Keratinocytes in Vitro

Porphyromonas Gingivalis Induces Penetration of Lipopolysaccharide and Peptidoglycan through the Gingival Epithelium via Degradation of Junctional Adhesion Molecule 1

The Dispersion of Gram-Negative Lipopolysaccharide by Deoxycholate. Subunit Molecular Weight

Outer-Membrane Vesicles from Gram-Negative Bacteria: Biogenesis and Functions

Gingival Crevice Fluid Flow

Methodological Evaluation of Gingival Crevicular Fluid Volume and Neutrophil Elastase Levels: Sequential Sampling, Length of Sampling Time and Two Different Sampling Methods

Frontline Science: Aggregated Neutrophil Extracellular Traps Prevent Inflammation on the Neutrophil-rich Ocular Surface

Biomarker Levels in Gingival Crevicular Fluid of Subjects with Different Periodontal Conditions: A Cross-Sectional Study

Evaluation of Biochemical Parameters Present in the Saliva of Patients with Chronic Periodontitis: Results from a Meta-Analysis

A Role for Intestinal Alkaline Phosphatase in the Maintenance of Local Gut Immunity

A Host Lipase Detoxifies Bacterial Lipopolysaccharides in the Liver and Spleen

Distribution of Bacterial Endotoxin in Human and Rabbit Blood and Effects of Stroma-Free Hemoglobin

Bacterial lipopolysaccharide binds and stimulates cytokine-producing cells before neutralization by endogenous lipoproteins can occur

Plasma CD14 Decreases Monocyte Responses to LPS by Transferring Cell-Bound LPS to Plasma Lipoproteins

Plasma Lipoproteins Promote the Release of Bacterial Lipopolysaccharide from the Monocyte Cell Surface

The Bactericidal/Permeability-Increasing Protein (BPI), a Potent Element in Host-Defense Against Gram-Negative Bacteria and Lipopolysaccharide

Mechanisms of Disposal of Bacterial Lipopolysaccharides by Animal Hosts

Inactivation of Endotoxin by Human Plasma Gelsolin † . Biochemistry

The Periodontal Pocket: Pathogenesis, Histopathology and Consequences

Extracellular Neutrophil Traps in Periodontitis

Neutrophil Fate in Gingival Crevicular Fluid

Progulske-Fox, A. Human Atherosclerotic Plaque Contains Viable Invasive Actinobacillus Actinomycetemcomitans and Porphyromonas Gingivalis

Oral Bacteria in Placental Tissues: Increased Molecular Detection in Pregnant Periodontitis Patients

Experimental Transient Bacteraemias in Human Subjects with Varying Degrees of Plaque Accumulation and Gingival Inflammation

The Effect of Gentamicin in Irrigating Solutions on Articular Infection Prophylaxis during Arthroscopic ACL Reconstruction

The Basis of Diagnosis and Treatment

Bacterial Internalization in Periodontitis

Candida Attachment to Oral Epithelium: Candida Attachment to Oral Epithelium

Neutrophils and NETs in Modulating Acute and Chronic Inflammation

Gingival Crevicular Fluid Collagenase-2 (MMP-8) Test Stick for Chair-Side Monitoring of Periodontitis: MMP-8 Test in Monitoring Periodontitis

Aberrant Neutrophil Reactions in Periodontitis

Analysis of Matrix Metalloproteinases, Especially MMP-8, in Gingival Crevicular Fluid, Mouthrinse and Saliva for Monitoring Periodontal Diseases

Treatment with DNases Rescues Hidden Neutrophil Elastase from Aggregated NETs

Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4

Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

Aggregatibacter Actinomycetemcomitans Induces Detachment and Death of Human Gingival Epithelial Cells and Fibroblasts via Elastase Release Following Leukotoxin-dependent Neutrophil Lysis

Increased Amounts of Laminin in GCF from Untreated Patients with Periodontitis

Effectiveness of Adjunctive Low-Dose Doxycycline Therapy on Clinical Parameters and Gingival Crevicular Fluid Laminin-5 Γ2 Chain Levels in Chronic Periodontitis

Gingival Crevicular Fluid Laminin-5 Gamma2-Chain Levels in Periodontal Disease

Neutrophil Extracellular Traps (NETs) Contribute to Pathological Changes of Ocular Graft-vs.-Host Disease (OGVHD) Dry Eye: Implications for Novel Biomarkers and Therapeutic Strategies

Age-Related Periodontitis and Alveolar Bone Loss in Rice Rats

Cimetidine Reduces Interleukin-6, Matrix Metalloproteinases-1 and -9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease

Low Concentrations of Neutrophil Extracellular Traps Induce Proliferation in Human Keratinocytes via NF-KB Activation

Clinical Evaluation of Periodontal Diseases

Clinical Effect of Subgingivally Delivered Simvastatin in the Treatment of Patients With Chronic Periodontitis: A Randomized Clinical Trial

Immediately Restored, Single-Tapered Implants in the Anterior Maxilla: Prosthodontic and Aesthetic Outcomes After 1 Year

Treatment of Periodontitis Improves the Atherosclerotic Profile: A Systematic Review and Meta-Analysis

The Peroxisome Proliferator Phenylbutyric Acid (PBA) Protects Astrocytes from Ts 1 MoMuLV-Induced Oxidative Cell Death

PEX11α Is Required for Peroxisome Proliferation in Response to 4-Phenylbutyrate but Is Dispensable for Peroxisome Proliferator-Activated Receptor Alpha-Mediated Peroxisome Proliferation

Phenylbutyrate Up-Regulates the Adrenoleukodystrophy-Related Gene as a Nonclassical Peroxisome Proliferator

Hif-2α Promotes Degradation of Mammalian Peroxisomes by Selective Autophagy

Aggregated Neutrophil Extracellular Traps Limit Inflammation by Degrading Cytokines and Chemokines

Aggregated Neutrophil Extracellular Traps Resolve Inflammation by Proteolysis of Cytokines and Chemokines and Protection from Antiproteases

The Antidiabetic Drug Metformin Blunts NETosis in Vitro and Reduces Circulating NETosis Biomarkers in Vivo