Fimmu 13 915081

REVIEW
published: 07 July 2022

doi: 10.3389/fimmu.2022.915081
The Roles of Neutrophils Linking

Periodontitis and Atherosclerotic
Cardiovascular Diseases
Rizky A. Irwandi 1, Scott T. Chiesa 2, George Hajishengallis 3, Venizelos Papayannopoulos 4,
John E. Deanfield 2 and Francesco D’Aiuto 1*
1 Periodontology Unit, UCL Eastman Dental Institute, University College London, London, United Kingdom, 2 UCL Institute of
Cardiovascular Science, University College London, London, United Kingdom, 3 Department of Basic & Translational
Sciences, Laboratory of Innate Immunity & Inflammation, Penn Dental Medicine, University of Pennsylvania, Philadelphia, PA,
United States, 4 Antimicrobial Defense Laboratory, The Francis Crick Institute, London, United Kingdom
Inflammation plays a crucial role in the onset and development of atherosclerosis.

Periodontitis is a common chronic disease linked to other chronic inflammatory
diseases such as atherosclerotic cardiovascular disease (ASCVD). The mechanistic
pathways underlying this association are yet to be fully understood. This critical review
aims at discuss the role of neutrophils in mediating the relationship between periodontitis
Edited by:
Martin Herrmann, and ASCVD. Systemic inflammation triggered by periodontitis could lead to adaptations in
University Hospital Erlangen, Germany hematopoietic stem and progenitor cells (HSPCs) resulting in trained granulopoiesis in the
Reviewed by: bone marrow, thereby increasing the production of neutrophils and driving the hyper-
Mallary Greenlee-Wacker,
Central Michigan University,
responsiveness of these abundant innate-immune cells. These alterations may contribute
United States to the onset, progression, and complications of atherosclerosis. Despite the emerging
Katherine C. MacNamara,
evidence suggesting that the treatment of periodontitis improves surrogate markers of
Albany Medical College, United States
cardiovascular disease, the resolution of periodontitis may not necessarily reverse
*Correspondence:
Francesco D’Aiuto neutrophil hyper-responsiveness since the hyper-inflammatory re-programming of
f.daiuto@ucl.ac.uk granulopoiesis can persist long after the inflammatory inducers are removed. Novel and
targeted approaches to manipulate neutrophil numbers and functions are warranted
Specialty section:
This article was submitted to within the context of the treatment of periodontitis and also to mitigate its potential impact
Molecular Innate Immunity, on ASCVD.
a section of the journal
Frontiers in Immunology Keywords: neutrophils, systemic inflammation, trained immunity, innate immune memory, periodontitis,
periodontal disease, atherosclerosis, atherosclerotic cardiovascular disease
Received: 07 April 2022
Accepted: 13 June 2022
Published: 07 July 2022
Citation: INTRODUCTION
Irwandi RA, Chiesa ST,
Hajishengallis G, Papayannopoulos V, Atherosclerotic cardiovascular disease (ASCVD) consists of a group of disorders that affect the heart
Deanfield JE and D’Aiuto F (2022) The
and blood vessels (1) and include coronary heart disease, cerebrovascular disease, and peripheral
Roles of Neutrophils Linking
Periodontitis and Atherosclerotic
vascular disease (2). ASCVD is a major cause of global mortality and a leading contributor to
Cardiovascular Diseases. disability as it causes 18.6 million deaths and contributes to 34.4 million people living with disability
Front. Immunol. 13:915081. in 2019 (3). Although its pathogenesis, progression, and complications comprise multiple complex
doi: 10.3389/fimmu.2022.915081 processes, inflammation plays a key role in each stage of the disease (4).
Frontiers in Immunology | www.frontiersin.org 1 July 2022 | Volume 13 | Article 915081

Irwandi et al. Neutrophils Link Periodontitis and ASCVD
Periodontitis is a common chronic inflammatory disease two diseases have been extensively explored (2, 14). However, the
caused by oral microbial dysbiosis. The onset and progression causal mechanistic pathways between these two common non-
of the disease could span over decades and is influenced by communicable diseases are yet to be fully understood. In this
genetic and environmental factors. This prevalent oral disease is review, we aim to critically address the role of neutrophils in
characterized by progressive destruction of hard and soft tissues linking periodontitis to ASCVD. Systemic inflammation
supporting the tooth, including the periodontal ligament and triggered by different causes, including periodontitis, may drive
alveolar bone (5). Untreated periodontitis leads inevitably not inflammatory adaptation of HSPCs and trained granulopoiesis in
only to tooth loss but also to masticatory impairment and the bone marrow, resulting in increased production of
negative influences on the quality of life of a patient (6). Like neutrophils with a hyper-responsive phenotype (12, 30). This
ASCVD, periodontitis is a major public health concern as it systemic inflammation-driven modification of granulopoiesis
affects over half of the population of the world (7) and 5–15% of can contribute to atherosclerosis in a stage-dependent manner.
the global population presents a severe form of the disease (8), However, although the periodontitis treatment successfully
causing increased costs of oral healthcare (9). achieves the resolution of the periodontal tissue site and
The evidence linking periodontitis to systemic diseases has improves surrogate markers of ASCVD, studies reveal that the
previously focused on the findings that periodontal bacteria and hyper-responsive function in neutrophils may persist (23, 31).
their endotoxins disseminate physically through the blood Therefore, novel approaches to target neutrophils by
circulation (10, 11). However, periodontitis also triggers manipulating their numbers and functions are warranted in
systemic inflammation, indicated by an increased level of C- periodontitis treatment and to mitigate its impact on ASCVD.
reactive protein (CRP), TNFa, IL-1b, and IL-6 in the serum of
patients (2). Because of chronic inflammation occurring at the
periodontium, endotoxemia, bacteremia, and systemic THE LINK BETWEEN PERIODONTITIS
inflammation are collectively implicated in numerous systemic AND ASCVD
diseases, including atherosclerotic cardiovascular disease
(ASCVD) (12–14). The impact of the treatment of periodontitis on cardiovascular
Neutrophils are the most abundant inflammatory cells in outcomes and surrogate markers of ASCVD has been extensively
humans and the first-line defense against infection in the innate- explored. Patients with periodontitis exhibit an increased risk of
arm of the immune system. They are derived from the myeloid coronary and cerebrovascular events compared with
differentiation lineage of hematopoietic stem cells (HSCs) in the periodontally healthy individuals. These findings may not
bone marrow. Upon detection of pathogens, neutrophils capture apply to the whole population as influenced by the
and destroy invading pathogens via phagocytosis and demographic characteristics, individuals, studies, and case
intracellular degradation, degranulation, and the formation of definition of periodontitis (2, 32). In the ARIC study on 6736
neutrophil extracellular traps (NETs) (15). Moreover, the dentate participants with 299 incidents of ischemic stroke, it was
emerging evidence over the past decade reveals that revealed that seven periodontal profile classes, that were used to
neutrophils are involved in chronic inflammation and are assess the participants were associated with an increased risk of
implicated in chronic inflammatory disorders, including cardioembolic and thrombotic stroke subtypes compared with
periodontitis and ASCVD (16–20). Periodontitis appears to be periodontally healthy participants. The assessment in this study
associated with hyper-responsive neutrophils (21–23), which was based on seven tooth-clinical parameters, resulting in seven
might, at least in part, be attributed to the notion that oral different periodontal profile classes, and the greater class
disease could influence hematopoietic tissue activity and trained indicates a more severe form of periodontitis (33). Lastly,
immunity (12). Trained immunity represents a non-specific based on the 1999–2010 Taiwanese National Health Insurance
memory in innate immune cells that is induced by earlier Research Database involving 393,745 patients with periodontitis
encounters with infectious or inflammatory stimuli and which and 393,745 non-periodontitis individuals, there was a
promotes increased immune responses to future challenges with significantly increased incidence of arterial fibrillation in
the same or different stimuli (24, 25). Meanwhile, in ASCVD, patients with periodontitis compared with controls (34). The
neutrophils contribute to different stages and clinical findings from all studies had been adjusted for a wide range of
manifestations of atherosclerosis (26) and literature also potential confounders, indicating that periodontitis is an
suggests that inflammation-adapted hematopoietic stem and independent risk factor for an increased risk of ASCVD events.
progenitor cells (HSPCs) may contribute to the disease Treatment of periodontitis may influence the progression of
pathogenesis (27–29). As such, recent consensus between the ASCVD. The study involving 511,630 periodontitis patients and
European Federation of Periodontology and the World Heart 208,713 individuals without periodontitis from The Longitudinal
Federation includes neutrophil hyper-responsiveness as one of Database of Taiwan’s National Health Insurance demonstrated
the mechanisms to explain the epidemiological association that patients receiving dental prophylaxis had a lower hazard
between periodontitis and ASCVD (2). ratio of acute myocardial infarction compared to periodontally
Mechanisms linking periodontitis to ASCVD and the effect of healthy controls, suggesting an almost 10% reduction in the risk
periodontitis treatment in improving the surrogate markers of of a new ASCVD event (35). These improvements were not
ASCVD in an attempt to show causal interactions between the observed across all types of periodontitis and controls, suggesting

a difference in host susceptibility and response even after the enhanced risk of CVD events as well as an elevated level of
treatment of peridontitis when looking at future incidence of CRP and fibrinogen in the serum (70).
ASCVD events (36). Systemic inflammation triggered by periodontitis potentially
Consistent evidence suggests that periodontitis is associated occurs because of bacterial dissemination or periodontal tissue-
with higher blood pressure and endothelial dysfunction (2, 37–39). derived inflammatory mediator leakage into the blood
A recent review and subsequent meta-analysis of intervention circulation. The ulceration of the epithelium owing to local
studies confirmed that the management of periodontitis can be a periodontal inflammation along with the support of its rich
novel non-drug approach for the treatment of hypertension. The vascularization may provide greater access for bacteria and
evidence is still inconclusive as to whether the treatment of their endotoxins such as lipopolysaccharides (LPS) to the
periodontitis influences blood pressure even in the absence of circulation, leading to bacteremia (13). This event has been
hypertension (40). The basis of this improvement in vascular reported in patients with periodontitis during mastication,
function could be linked to the effect of the treatment of toothbrushing, and dental scaling (10, 11). Bacteremia would
periodontitis in improving endothelial function as assessed by induce inflammatory alterations in the endothelium, which
flow-mediated dilation (FMD) (41–44). A possible mechanism by include enhanced expression of adhesion molecules and the
which local periodontal treatment improves endothelial function production of pro-inflammatory cytokines. With regard to the
in periodontitis patients is endothelial nitric oxide synthase/nitric spillover of inflammatory mediators into the bloodstream, this
oxide (eNOS/NO) activation. NO is mainly produced by eNOS in can affect vascular tissues as well as other distant organs,
endothelial cells and induces the relaxation of smooth muscle cells including the liver, which then may initiate an acute-phase
(45, 46). Meanwhile, IL-6, TNFa, IL-1b, and CRP directly reduced response (14).
eNOS at both mRNA and protein levels in human endothelial cells
(47–49). As a result, NO bioavailability in the serum is reduced,
leading to endothelial dysfunction and periodontitis is associated
with this surrogate marker of ASCVD (50, 51). Hepatocytes are
INFLAMMATION AND NEUTROPHILS:
the major producers of CRP triggered by IL-6 and IL-1b IMPLICATIONS FOR ATHEROSCLEROSIS
stimulation, while TNFa also upregulates CRP production in
Atherosclerosis is a cause of myocardial infarction, ischemic
human coronary artery smooth muscle cells (52–55). A recent
cardiomyopathy, and ischemic stroke that contribute to most
meta-analysis reveals a progressive CRP level reduction up to 6
deaths in the population worldwide (4). Arterial wall damage due
months in patients with periodontitis following effective treatment
to blood lipid profile imbalance, oscillating shear stress, and pro-
(37). This was also associated with a reduction of reduced serum
inflammatory mediators initiates this underlying ASCVD
IL-6, TNFa, and IL-1b in patients with periodontitis compared to
pathology. The subsequent processes are endothelial cell
baseline (56–59). Collectively, these findings suggest that reduced
activation in arterial tissue, myeloid cell adhesion to the
levels of CRP, IL-6, TNFa, and IL-1b after treatment of
endothelium, and infiltration into the arterial intima (26). At
periodontitis could restore eNOS activity and NO bioavailability,
the late stage of atherosclerosis, inflammatory cell accumulation,
resulting in improved endothelial dysfunction.
lipoprotein deposition, and cellular debris buildup are
responsible for the arterial plaque formation followed by the
plaque instability leading to atherosclerotic plaque rupture.
PERIODONTITIS AS A TRIGGER OF Atherosclerosis is a decades-long process with many stages of
SYSTEMIC INFLAMMATION evolution, and evidence suggests that neutrophils are involved in
different stages of atherosclerosis (Figure 1) (26).
Periodontitis and ASCVD share similar hallmarks of The onset of atherosclerosis is characterized by endothelial
inflammatory mechanisms (60), genetic (2, 61), and common dysfunction, which induces neutrophil recruitment to the
risk factors (62). However, a significant body of evidence endothelium. Specifically, the dysfunction up-regulates the
supports an independent association between periodontitis and expression of various endothelial cell adhesion molecules,
ASCVD following adjustment for confounders and shared risk including E-selectin, P-selectin, and intracellular adhesion
factors (63, 64). This independent association can be explained molecule-1 (ICAM-1) (71). Platelets then deliver CCL5, the
by the capability of periodontitis to trigger a low-grade but primary ligand of CCR5 on the endothelium and promote
consistent systemic inflammation, which may contribute to the cathepsin G secretion by neutrophils, resulting in the firm
development of ASCVD (65). Patients with periodontitis exhibit adhesion to and accumulation of the cells in the endothelium
an elevated level of systemic pro-inflammatory mediators, which (72, 73). Furthermore, neutrophils aggravate endothelial
include CRP, TNFa, IL-1b, IL-6, as well as increased neutrophil dysfunction by secreting reactive oxygen species (ROS),
numbers in the blood (2, 14, 66–69). A retrospective study azurocidin, proteinase 3, cathelicidin, and cathepsin G in the
involving 60,174 participants revealed that even after the arterial lumen (26, 74). ROS and proteases activate and
adjustment of confounders, participants with periodontitis dysregulate the endothelial cell layer and degrade the
were 1.59 times more likely to have ASCVD (63). Previously, underlying extracellular matrix, enabling leukocyte infiltration
an 8-year prospective cohort study involving 11,869 participants and low-density lipoprotein (LDL) extravasation (26).
also showed that those reporting poor oral hygiene had an Azurocidin also contributes to increasing endothelial

FIGURE 1 | Stage-dependent role of neutrophils in atherosclerosis. During the early stage of atherosclerosis, upregulation of E-selectin, P-selectin, and ICAM-1
induces neutrophil recruitment. Platelet-derived CCL5 activates neutrophils to release cathepsin G, leading to the firm adhesion to and accumulation of neutrophils in
the endothelium. ROS and proteases secreted by neutrophils activate and dysregulate the endothelial cell layer and degrade the underlying extracellular matrix,
resulting in monocyte infiltration and LDL extravasation. Neutrophils secrete MPO that mediates LDL oxidation and promotes foam cell formation. Cathelicidin- and a-
defensin-derived neutrophils activate macrophages towards pro-inflammatory state, while NETs stimulate macrophage to release IL-6 and IL-1b that promote Th17
differentiation followed by the amplification of neutrophil recruitment. At the late stage of atherosclerosis, activated VSMCs induce neutrophil chemotaxis and release
CCL7 to stimulate NETs. Histone H4-derived from NETs induces VSMC lysis and the secretion of proteases by neutrophils degrades collagen and lyses VSMCs,
leading to the plaque instability. Neutrophils contribute to plaque erosion through NET release as well as colocalization with TLR2 of endothelial cells to induce
endothelial cell stress and apoptosis.
permeability (26, 74–76), whereas azurocidin, proteinase 3, endothelial cells, and an in vitro experiments showed that co-culture
cathelicidin, a-defensin, and cathepsin G all promote myeloid of neutrophils with endothelial cells potentiates endothelial stress
cell recruitment and facilitate monocyte entry into the and apoptosis, resulting in endothelial cell detachment followed by
atherosclerotic lesions (74, 77–82). luminal endothelial cell desquamation (plaque erosion) (19). Lastly,
The progression of the lesion continues following the NETs are also involve during endothelial erosion as the disruption
aggravation of endothelial dysfunction by neutrophils, where of NETs by either peptidyl arginine deiminase 4 (PAD4) gene
macrophage activation and foam cell formation occur in the knockout or DNase I treatment in atherosclerotic mice attenuates
arterial intima. Neutrophil-derived granule proteins, cathelicidin, endothelial disintegration and endothelial cell apoptosis (87).
and a-defensin activate macrophages toward a pro-inflammatory Neutrophils play dual roles, which are both adverse and
state (M1 macrophage phenotype). Neutrophils also secrete favorable for cardiac tissue repair following myocardial
myeloperoxidase (MPO) to generate oxygen radicals that oxidize infarction (Figure 2A). Tissue necrosis/ischemia post-acute
apolipoprotein B, a protein structure in LDL (82). Subsequently, myocardial infarction releases alarmins and inflammatory
macrophages take up this oxidized LDL (oxLDL), resulting in foam signals that attract neutrophils to the site of infarction (88).
cell formation (83). Moreover, NETs stimulate macrophages by Activated neutrophils secrete ROS, proteases, NETs, and IL-1b.
turning on transcription factors encoding IL-6 and IL-1b. These Granulopoiesis stimulated by IL-1b leads to neutrophil
cytokines promote the differentiation of Th17 cells, which in turn accumulation in the injured site, which is harmful to the
amplify neutrophil recruitment in the lesion (84). At this stage, remodeling of the ischemic area, resulting in eventual heart
because of sustained myeloid cell recruitment and foam cell failure (89, 90). Following neutrophil accumulation, monocytes
generation, the atheroma becomes pronounced. and monocyte-derived macrophages infiltrate the infarcted site
In the late stage of atherosclerosis, neutrophils destabilize the to phagocytose cell debris and apoptotic neutrophils, activating
atherosclerotic plaque. Mechanistically, activated vascular smooth cardiac repair (88). Intriguingly, neutrophils also contribute to
muscle cells (VSMCs) in advanced atherosclerotic lesions induce this cardiac healing as their damage-associated molecular
neutrophil chemotaxis and secrete CCL7, which stimulates NET patterns (DAMPs), such as neutrophil gelatinase-associated
release. One of the NET cytotoxic components, histone H4, disrupts lipocalin (NGAL) and S100A8/A9, stimulate macrophages to
the integrity of the VSMC plasma membrane, leading to cell lysis shift toward reparative phenotypes (91, 92). These anti-
(85). Moreover, endotoxemia in a mouse model of atherosclerosis inflammatory macrophages aid in the resolution of
revealed that leukotriene B4-induced neutrophil recruitment to inflammation (88). Neutrophils also secrete annexin A1, which
atherosclerotic plaques induces collagen degradation and VSMC stimulates pro-angiogenic macrophage polarization. These
lysis, leading to the feature of plaque instability (86). In eroded macrophage phenotypes release vascular endothelial growth
human plaques, neutrophils colocalized with toll-like receptor 2 of factor A (VEGFA) to support angiogenesis in the ischemic site of

FIGURE 2 | Neurophils in ASCVD complications. (A) After acute myocardial infarction, ischemic/necrotic tissues release alarmins and inflammatory signals to induce
neutrophil recruitment. Activated neutrophils secrete ROS, proteases, and NETs. S100A8/A9-derived from NETs induces IL-1b release following NLRP3
inflammasome priming in naïve neutrophils. IL-1b reaches the bone marrow to stimulate granulopoiesis leading to the amplification of neutrophil production and
accumulation, which in turn, detriments the ischemic heart and eventual heart failure. During cardiac tissue repair, NGAL and S100A8/A9 stimulate macrophages to
shift towards reparative phenotypes, resulting in increased clearance of apoptotic cells/debris. Neutrophils also secrete Annexin A1 to favor the shift of macrophages
towards pro-angiogenic phenotypes that release VEGFA to promote angiogenesis in the ischemic site of cardiac tissue. (B) Following ischemic stroke, dying neurons
attract neutrophils to the ischemic area, and neutrophil ROS and elastase promote endothelial cell dysfunction and vascular permeability. NETs contribute to
thrombus growth, thereby increasing stroke volume. Neutrophils also promote neuronal cell death that is likely mediated by NETs.
the myocardium (93). Besides cardiac tissue repair, neutrophils trained myeloid cells, including neutrophils (trained
aggravate other atherosclerosis complications, namely, ischemic myelopoiesis/granulopoiesis) (95, 96). This training is based
stroke (Figure 2B). Following an episode of ischemic stroke, dying on the ability of HSPCs to sense numerous inflammatory cues
neurons attract neutrophils to the area to release ROS and elastase to in response to hematopoietic stress such as systemic
enhance endothelial cell dysfunction and permeability. NETs inflammation (12, 96). HSPCs can directly sense pathogen-
promote thrombus growth, thereby increasing stroke volume. associated molecular patterns (PAMPs), such as LPS, via their
Finally, neutrophils increase neuronal cell death in a process that pattern recognition receptors (PRRs), such as toll-like receptors
is likely to involve NETs (26, 94). (for example, TLR-4 for sensing LPS). Regarding the direct
mechanism, in the context of periodontitis-induced bacteremia,
it could be envisioned that systemically disseminated
IMPACT OF PERIODONTITIS-INDUCED periodontal pathogens or their products (for example, LPS or
SYSTEMIC INFLAMMATION ON BONE lipopeptides) may also reach the bone marrow, resulting in
innate immune training of HSPCs. However, indirect activation
MARROW ACTIVITY AND SUBSEQUENT relies on specialized cells residing in either the peripheral tissue
CIRCULATING NEUTROPHIL or bone marrow to affect the hematopoietic system through the
ALTERATIONS: PLAUSIBLE MECHANISMS release of cytokines (97, 98).
In addition to direct sensing of pathogens, cytokines and
Modulation of HSPCs in the Bone Marrow: growth factors derived from both the bone marrow niche and
A Key Role for Periodontitis-Induced peripheral tissue can mediate indirect adaptation of HSPCs (97,
Systemic Inflammation 98). IL-1b promotes myeloid differentiation and self-renewal of
Trained immunity can be initiated in the bone marrow via HSPCs as chronic administration of this cytokine elevates the
sustained epigenetic, metabolic, and transcriptional number of myeloid-bias HSPCs. This is also consistent with the
adaptations in HSPCs, leading to enhanced myeloid-biased recent finding that enhanced myelopoiesis of HSPCs in a mouse
differentiation and production of increased numbers of model of b-glucan or experimental periodontitis-induced trained

immunity is mediated by IL-1b (95, 99). TNFa exhibits different LIP-subjected mice to healthy recipient mice resulted in
actions in HSPCs as it promotes both the survival and myeloid increased severity of arthritis in the latter, as compared with
differentiation of HSCs while inducing the apoptosis of myeloid the transplantation of bone marrow from periodontally healthy
progenitors (100). The increased level of IL-6 in the bone mice (99). The implication of this finding (if a similar
marrow niche promotes myelopoiesis, indicated by an elevated phenomenon is confirmed in humans) is that clinicians should
number of multipotent progenitors (MPPs) and common take inflammatory memory in the bone marrow into
myeloid progenitors (CMPs) (101). Type 1 IFN mediates consideration when selecting appropriate donors for
trained granulopoiesis in mice following b-glucan treatment hematopoietic transplantation (99).
resulting in the production of neutrophils with an enhanced Indeed, the notion that periodontitis might trigger an
ROS-dependent anti-tumor phenotype (102). IFNg promotes adaptation of HSPCs is supported by clinical imaging studies
HSC self-renewal and myeloid differentiation in a mouse using 18F-fluorodeoxyglucose positron emission tomography/
model of repeated Mycobacterium avium infection (103). computed tomography ( 18 F-FDG-PET/CT). One study
Granulocyte colony-stimulating factor (G-CSF) is a key growth revealed that periodontal inflammation was associated with
factor that drives granulopoiesis. Specifically, G-CSF produced hematopoietic activity in the bone marrow and arterial
by monocytes in the bone marrow niche is responsible for HSPC inflammation. Furthermore, the authors used mediation path
mobilization from the bone marrow to the circulation (104). analysis to show that the relationship between periodontal and
Moreover, in emergency granulopoiesis, G-CSF promotes the arterial inflammation was significantly mediated by bone
expansion and granulocyte lineage specification of granulocyte- marrow activity (109). More recently, another study using the
monocyte progenitors (GMPs) (105). same cohort of 304 participants as the aforementioned study
Systemic inflammation, indicated by elevated levels of TNFa, showed that periodontal inflammation (as determined by 18F-
IL-1b, IL-6, and IFNg in the serum, is clinically present in FDG-PET/CT) is independently correlated with not only
patients with periodontitis (66, 67). Plasma IFNa, a type 1 increased arterial inflammation but also increased an risk of
IFN, is also higher in periodontitis patients compared to future cardiovascular events (64). Additionally, another study
healthy controls (106). However, the role of type 1 IFN- harnessing the same imaging technique also reported a trend for
mediated trained granulopoiesis in inducing hyper-responsive increased periodontal inflammation and femur bone marrow
neutrophils in periodontitis patients needs to be addressed activity in patients with periodontitis (relative to controls), albeit
experimentally. Recent evidence indicates an increased level of no differences were observed in vascular inflammation between
serum G-CSF in ligature-induced periodontitis mice (107). the two groups (110). The lack of clear differences in this study is
Fibroblasts in the periodontal tissue contribute to the release of likely attributed (a) to the small sample size (14 participants as
G-CSF during periodontal inflammation, resulting in the opposed to >300 participants in the above-discussed studies) and
promotion of granulopoiesis (108). Based on the cumulative (b) to the fact that participants with severe periodontal disease
evidence of the effect of cytokines and growth factors on HSPCs were under supportive periodontal therapy, which could have
and the abovementioned clinical studies, it could be shown that mitigated inflammation and associated parameters, including
periodontitis-associated systemic inflammation may modulate surrogate markers of ASCVD (discussed below) (110).
HSPCs toward trained granulopoiesis (Table 1). This notion was Moreover, the control group also included individuals with
recently confirmed in a preclinical model. Specifically, it was mild periodontitis, which could also impact systemic
shown that ligature-induced periodontitis (LIP)-associated inflammation and hematopoietic tissue activity, thus reducing
systemic inflammation leads to maladaptive innate immune potential differences compared with the experimental group.
training in the bone marrow (i.e., generating inflammatory
memory) and the generation of increased numbers of hyper- Alteration of Circulating Neutrophils in
responsive neutrophils; these populate oral and non-oral tissues Periodontitis and its Putative Effect on
and promote the emergence of inflammatory comorbidities, as Atherosclerosis
exemplified by the periodontitis-arthritis axis (99). This is Patients with periodontitis exhibit elevated numbers of
consistent with available clinical observations as outlined neutrophils and altered phenotypes presenting hyper-reactive
below. Intriguingly, the transplantation of bone marrow from features in cellular functions (discussed below) (21–23, 68, 69,
TABLE 1 | Summary of circulating molecules that are elevated in periodontitis and involved in hematopoietic tissue adaptation.
No. Molecules Action on hematopoietic tissue adaptation References
1. IL-1b Promotes myeloid differentiation and self-renewal of HSPCs (95, 97–99)

2. TNF-a Promotes survival of HSPCs and myeloid differentiation (100)
Induces myeloid progenitor apoptosis
3. IL-6 Enhances myelopoiesis by elevating MPPs and CMPs (101)
4. Type 1 IFN Mediates trained granulopoiesis with hyper-responsive neutrophils (102)
5. IFN-g Promotes HSC self-renewal and myeloid differentiation (103)
6. G-CSF Drives granulopoiesis (104)
Promotes GMP expansion and granulocyte lineage specification (105)

110–117). These include increased levels of ROS production in effect is uncertain. A reduction of several serum inflammatory
response to fMLP, PMA, or periodontal pathogens (21–23, 102, markers that is expected to reduce the risk of ASCVD is not
111–115), elevated TNFa production following stimulation with observed in serum IFNg and IL-10 because both markers remain
the periodontal pathogen, Fusobacterium nucleatum, in vitro unchanged following periodontitis treatment (40). As these
(116), and elevated neutrophil elastase levels linked to markers are predominantly generated and released into blood
periodontal tissue destruction (117, 118). The systemic effect of circulation by inflammatory cells, it is interesting to speculate
this alteration has been recently confirmed in an experimental that while successful treatment indeed achieves the resolution of
study with mice and experimental periodontitis that exhibited periodontal inflammation, circulating inflammatory cells still
hyper-inflammatory neutrophil response following the exposure retain their periodontitis-induced altered phenotypes. The
to secondary peritonitis compared with mice without latter is supported by clinical studies that investigated the
periodontitis (119). functions of peripheral blood-derived neutrophils and
These altered features in neutrophils present a hallmark of monocytes in periodontitis patients after the treatment. The
trained myelopoiesis, which includes increased numbers of hyper-responsiveness of these myeloid cells persisted as the
myeloid cells with enhanced inflammatory responsiveness (24, levels of cytokines produced by the cells in response to
95). As such, these changes may implicate the response of future pathogen or LPS stimulation were comparable between
inflammatory stimuli that drive certain pathological processes neutrophils from patients before and after periodontal
such as atherosclerosis. treatment (31, 116). Similarly, a longitudinal study on the
Neutrophils that are increased in number and hyper- circulating neutrophil profiles of patients with periodontitis
responsiveness due to periodontitis can contribute to any stage of showed that neutrophils (as a proportion of total cells isolated
this ASCVD pathology. Further research should address this from peripheral blood of periodontitis patients) did not change
hypothesis experimentally. However, it is still interesting to between baseline (before treatment) and after 3-, 6-, and 12-
speculate that periodontitis-induced neutrophil alteration month post-periodontitis treatment (124). The retained
contributes to the association between periodontitis and ASCVD neutrophil phenotypes might be because of the trained
because of several clinical studies that support this notion. An myelopoiesis induced by periodontitis-triggered systemic
elevated number of neutrophils in peripheral blood may increase inflammation. In murine experiments, long-lasting changes in
the risk of ASCVD in periodontitis patients because neutrophil myelopoiesis were observed following either microbial- or
counts from peripheral blood are positively correlated with ASCVD sterile-induced inflammation (29, 95). Similarly, a recent report
risk (120, 121). Moreover, periodontitis patients consistently shows that upon the LIP resolution, HSPCs in the bone marrow
present with endothelial dysfunction, which is a key feature of retain a myeloid differentiation bias (99).
ASCVD (2). This disturbed vascular function and elevated
neutrophil ROS production triggered by periodontitis can
aggravate the initiation of atherosclerosis. The latter may also
potentiate the progression of atherosclerosis, particularly in TARGETING NEUTROPHILS AS A NOVEL
oxidizing LDL. Hyper-responsiveness of neutrophils THERAPEUTIC APPROACH: DUAL
characterized by excessive production of neutrophil elastase and BENEFIT ON PERIODONTITIS AND ASCVD
ROS in periodontitis patients could also contribute to the late stage
of atherosclerosis as both hyper-reactive features induce endothelial The targeted therapeutic approach for ASCVD has been
apoptosis, resulting in endothelial desquamation (plaque erosion), established by focusing on the reduction of inflammation
fibrous cap thinning and plaque ruptures. Finally, whereas the (Table 2). The first clinical trial, CANTOS, in an attempt to
production of NETs by circulating neutrophils has been shown to be close the gap between pre-clinical studies and clinical practice,
comparable between patients with periodontitis and healthy provided evidence that reducing inflammation could be relevant
controls, plasma NET degradation was lower in patients with to treating atherosclerosis in humans. In this trial, the anti-IL-1b
periodontitis than in controls, suggesting an impaired NET antibody, Canakinumab, was administered subcutaneously to
degradation process in the plasma of periodontitis patients (122). individuals with a sustained acute myocardial infarction. The
This impairment might favor atherosclerosis complications, trial revealed a significant reduction in the rates of recurrent
especially in post-ischemic stroke where NET accumulation cardiovascular events, hospitalization for heart failure, and heart
promotes thrombus formation and expands stroke volume. In failure-associated death (125, 126). However, the adverse event
this context, a case-control study revealed that periodontitis was in the group receiving Canakinumab was more significant death
an independent predictor of poor outcome in post-ischemic stroke due to infection or sepsis compared to the placebo group (125).
patients (123). Moreover, oral administration of colchicine in patients with a
recent myocardial infarction significantly reduces the risk of
ischemic cardiovascular events (127). The benefits of colchicine
IMPACT OF PERIODONTITIS TREATMENT were also observed in patients with chronic coronary disease
ON NEUTROPHILS (128). Unfortunately, patients in the colchicine group showed
higher incidents of pneumonia and non-cardiovascular-caused
Whereas the treatment of periodontitis improves the surrogate death than those in the placebo group (127, 128). Other clinical
markers of ASCVD, its long-term cardiovascular-protective studies on targeting inflammation in ASVD treatment have been

TABLE 2 | Summary of clinical trials.
No. Name of Mechanism of action Phase Identifier (Trial Outcome

drug registration)
1. Canakinumab Binds to IL-1b resulting in blocking the interaction between IL-1b and Phase NCT01327846 Reduction in cardiovascular events,
IL-1 receptor 3 hospitalization for heart failure, and heart
failure-associated death
Emergence of death due to sepsis
2. Colchicine Prevents microtubule formation resulting in tubulin disruption Phase NCT02551094 Reduction in ischemic cardiovascular events
3 Increase in pneumonia
3. Metoprolol Attenuates neutrophil migration and infiltration by impairing the Phase NCT01311700 Reduction of infarct size
neutrophil-platelet interaction that is crucial during early phases of 4 Improvement of cardiac function
neutrophil recruitment
4. AZD5069 Prevents neutrophil recruitment to the site of inflammation Phase NCT01480739 No adverse events and safety concerns
(a CXCR2 1 ISRCTN48328178 Currently on going
antagonist) Phase
2
5. AMY-101 Inhibits downstream activation of the anaphylatoxin C3a and C5a Phase NCT03694444 Reduction in gingival inflammation
(a receptors 2a Reduction of MMP-8 and MMP-9 levels in
complement gingival crevicular fluids
C3 inhibitor)
reviewed elsewhere (129). These clinical trials indicate that in reduced atherosclerotic plaque size. However, a similar
therapies with alternative strategies are required to achieve treatment did not affect neutrophil adhesion in lung
outcomes in which the benefits outweigh the risks. microcirculation following an LPS-induced lung inflammation
Besides the necessity of different strategies to reduce the risk– model in mice (73). Furthermore, the elucidation of heteromeric
benefit ratio, a refined understanding of the potential role of interactions between neutrophil-derived human neutrophil
periodontitis-induced neutrophil alteration in the pathology of peptide-1 (HNP1) and platelet-borne CCL5 that must
ASCVD and its retained phenotypes following successful stimulate monocyte adhesion via CCR5 ligation allows the
periodontitis treatment highlights the importance of selectively design of a peptide that can disturb these neutrophil–platelet
targeting neutrophils during the treatment of periodontitis. This interactions. This specifically designed short peptide alleviated
therapeutic approach is warranted to mitigate the potential inflammation in a mouse model of myocardial infarction (20).
impact of periodontitis on ASCVD. The main endpoint of this Finally, inducing endogenous inhibitors in leukocytes can
intervention is to either manipulate the number of neutrophils overcome integrin activation by chemokine, thereby
and/or their functional activities. We only discuss the approaches suppressing myeloid cell adhesion. For example, growth
to block neutrophil recruitment and prevent NET-driven differentiation factor-15 (GDF-15) and annexin A1 inhibit
inflammation, while other neutrophil-targeted therapeutics chemokine-induced b2 integrin activation and subsequently
have been extensively reviewed elsewhere (130). reduce neutrophil recruitment in a mouse model of chronic
While the results of neutrophil recruitment blockage are inflammation (72, 132). Moreover, recombinant developmental
promising in preclinical studies, clinical trials exhibit endothelial locus-1 (DEL-1), the first identified endogenous
unsuccessful outcomes due to the redundancy of signals during inhibitor of the leukocyte adhesion cascade (133), inhibited
neutrophil recruitment and off-target effects caused by receptor neutrophil recruitment in mouse and non-human primate
cross-linking. Recent studies have provided strategies for models (17, 134).
overcoming such difficulties. The combined inhibition of Important roles of NETs in both atherosclerosis progression
several endothelial cell molecules interrupts redundant signals (atherogenesis, plaque destabilization and erosion) and
that recruit neutrophils (131). Intravenous injection of complication (atherothrombosis) stand out as a potential
nanoparticles carrying small interfering RNAs (siRNAs) that therapeutic target to manipulate cardiovascular inflammation.
target endothelial adhesion molecules including ICAM-1, E-, P- Peptidyl arginine deiminase 4 (PAD4) citrullinates histone to
selectin, and vascular cellular adhesion molecule-1 (VCAM-1) disrupt electrostatic bonds in nucleosomes, decondensing
decreased leukocyte recruitment to ischemic myocardium in a chromatin that leads to NET release (135). Cl-amidine, a PAD
mouse model of post-myocardial infarction (131). Moreover, inhibitor administered intravenously in an atherosclerotic mouse
specific blockage of neutrophils that traffic to certain vascular model, prevented NET formation, leading to reduced
tissue requires a refined understanding of recruitment patterns at atherosclerotic lesion area and thrombosis (136). However, the
particular sites (16). A neutrophil granule protein, cathepsin G mechanism of NETosis in mice is different from that in humans
promotes myeloid cell adhesion to only arterial but not as ex vivo experiments of human neutrophils showed that PMA-
microvascular tissue (73). Indeed, antibody-assisted cathepsin induced NETosis of the cells was not affected following the PAD4
G neutralization in an atherosclerotic mouse model specifically inhibitor, Cl-amidine (137). Meanwhile, DNase 1 treatment
alleviated neutrophil recruitment to the carotid artery, resulting might be considered to mitigate ASCVD complications like

thrombosis due to NET deposition in the vascular lumen. This A clinical trial targeting neutrophils to lower ASCVD risk is
notion is supported by a study where the treatment protected still lacking. However, two randomized clinical trials reported
mice from deep vein thrombosis following an inferior vena cava that metoprolol provides a cardio-protective effect following
stenosis model (138). A reduction in lesion size was also observed acute myocardial infarction, which is one of the atherosclerosis
in an atherosclerosis mouse model after DNase injections (84). In complications (Table 2). Specifically, intravenous administration
humans, DNAse 1 treatment to eliminate NET deposition might of metoprolol reduced infarct size and improved cardiac function
need a combination with other substances, as DNAse 1 alone was in patients with acute myocardial infarction (146, 147). Studies
not adequate to degrade NETs in vitro (139). Lastly, NET using a myocardial infarct mouse model revealed the mechanism
chromatin can stimulate macrophages by activating AIM2 of this protection in which metoprolol attenuates neutrophil
inflammation, causing the release of IL-8 and IL-1b in migration and infiltration by impairing the neutrophil–platelet
atherosclerotic lesions. The use of an AIM2 inhibitor could interaction that is crucial during early phases of neutrophil
also attenuate ASCVD complications because reduced plaque recruitment (148, 149). Another drug candidate, AZD5069, a
vulnerability through the thickening of the fibrous cap was CXCR2 antagonist, could be a potent drug candidate in treating
observed in an atherosclerotic mouse model after the treatment patients with advanced atherosclerotic lesions. CXCR2 is a
of an AIM2 inhibitor (140). ApoE-deficient mice on a 6-week chemokine receptor 2 in neutrophils that regulates neutrophil
high fat diet and injected with anti-chromatin antibodies also migration, and the inhibition of this receptor successfully
showed a reduced plaque area per lumen, suggesting the prevents neutrophil recruitment to the site of inflammation
potential of chromatin blockage to hinder atherosclerosis (150). No adverse effects on neutrophil function were observed,
(141). The use of substances to block NETs in humans should and no safety concerns were raised in participants receiving oral
be implemented with caution because studies about the pro- administration of AZD5069 (151). The CICADA trial was
inflammatory features of NETs in humans are still conflicting, as proposed to investigate the effect of a CXCR2 antagonist
in vitro NET clearance by human macrophages did not induce (administered orally) on coronary flow, structure, and function
pro-inflammatory cytokines (139), but NET transfection to in patients with coronary heart disease (152).
mouse macrophages did (142). Mechanical intervention in periodontitis management with
Targeting neutrophils as an approach to mitigate the potential adjunctive neutrophil-targeted therapeutic approach could also
impact of periodontitis on ASCVD is appealing considering the provide benefit in reducing periodontal-induced systemic
numerous efforts outlined previously. This targeted strategy can inflammation that can implicate all stages of atherosclerosis. A
not only have direct effects on ASCVD but also reduce phase IIa clinical trial of the complement C3 inhibitor, AMY-
periodontitis, which triggers inflammation that primes 101, summarized in Table 2, shows promising results in reducing
neutrophils for ASCVD pathology. However, safety and local inflammation in periodontal tissue (153). The
specificity issues are challenges that need to be solved as pharmacological blockade of the central complement
neutrophils interact with other myeloid lineages (130). The component, C3, inhibits downstream activation of the
solution to the former concern is that the intervention should anaphylatoxin C3a and C5a receptors (C3aR and C5aR,
consider the therapeutic window, where the attenuation of the respectively), which induce inflammatory bone loss in a
inflammatory process mediated by neutrophils does not interfere preclinical model (154). C5aR and TLR2 coactivation in
with neutrophil capacity during host defense (130). Recent neutrophils contribute to oral microbiota dysbiosis leading to
studies revealed that the use of antibodies to inhibit NET- overt periodontal inflammation and subsequent periodontal
derived histones reduced the amplification of NET-induced tissue destruction (155). Local injection of AMY-101 to the
inflammation rather than completely blocking NETosis or gingiva reduced gingival inflammation without adverse events,
inflammation (141). Few studies have presented strategies to warranting phase III clinical trials for further investigation (153,
obtain the specificity to therapeutically target neutrophils. For 156). Importantly, AMY-101 significantly reduced the gingival
example, the conjugation of siRNA targeting Bruton’s tyrosine crevicular levels of MMP-8 and MMP-9 (152), which are the
kinase (BTK) to the F(ab’)2 fragment of an anti-neutrophil major neutrophil-derived proteases and are considered
monoclonal antibody specifically targeted alveolar neutrophils biomarkers of periodontal tissue destruction (157). Meanwhile,
in an acute lung injury mouse model. This treatment was other local neutrophil-targeted treatments, including resolvin E1,
administered locally using a technique called intranasal developmental endothelial locus-1, and milk fat globule
instillation (143). Meanwhile, others harness nanoparticle epidermal growth factor 8, are still in pre-clinical studies.
technology to enhance specificity. One of them reported that These proteins reduce neutrophil recruitment to the site of
neutrophils adhered to activated endothelium-engulfed albumin inflammation and prevent animal models of ligature-induced
nanoparticles carrying piceatannol, leading to the inactivation of periodontitis (134, 158, 159). Additionally, resolving E1 also
these adherent neutrophils and consequently preventing vascular promotes neutrophil apoptosis and its clearance (efferocytosis),
inflammation (144). Additionally, another report exhibited lipid- resulting in the resolution of periodontal inflammation (157).
based nanoparticles that were successfully incorporated with Focusing on the termination of periodontitis may prevent its
identified peptides that interact with the neutrophil-specific systemic impact, which is heightened systemic inflammation.
surface marker, CD177 (145). These strategies of local intervention potentially further improve

FIGURE 3 | Periodontitis-induced systemic inflammation, inflammatory modulation of bone marrow progenitor cells, and implications for atherosclerosis.
Periodontitis triggers systemic inflammation, which causes inflammatory modulation of hematopoietic stem and progenitor cells, resulting in a trained myelopoiesis. In
turn, as a hallmark of trained myelopoiesis, an increased number of myeloid cells, including neutrophils with enhanced inflammatory responsiveness, may contribute
to all stages of the ASCVD pathology, atherosclerosis.
the current periodontal treatment in providing a sustained reduce periodontitis-triggered systemic inflammation and
protection to cardiovascular health. reverse the periodontitis-induced neutrophil changes. Such
targeted approaches can be harnessed as a direct treatment for
ASCVD and indirect intervention of the disease through the
CONCLUSION reduction of heightened systemic inflammation triggered
by periodontitis.
Periodontitis triggers systemic inflammation which could
modulate hematopoietic tissue activity in the bone marrow,
re sulting in trained my elopoiesis. Clinical s tudies
demonstrating increased numbers as well as enhanced AUTHOR CONTRIBUTIONS
inflammatory responsiveness in neutrophils back this notion.
The evidence of persistent elevated neutrophil numbers and their RI wrote the original draft. RI, SC, GH, VP, JD, and FD provided
altered phenotypes, despite the resolution of periodontitis critical revisions to the article. All authors listed have made a
following local treatment, also supports the speculation that substantial, direct, and intellectual contribution to the work and
periodontitis-induced systemic inflammation can induce long- approved it for publication.
term myelopoiesis bias, a hallmark of innate immune training in
the bone marrow. The quantitative and qualitative alterations in
neutrophils may contribute to all stages of the ASCVD FUNDING
pathology, atherosclerosis (Figure 3). Although clinical
intervention studies suggest that periodontal therapy improves This work was undertaken at the UCL Biomedical Research
surrogate markers of ASCVD, the long-term effects of this oral Centre which receives funding from the UK National Institute
treatment to maintain such improvement and convincing for Health and Care Research. GH receives a grant from the US
evidence that successful periodontitis treatment can reduce the National Institutes of Health (DE031206). RI receives funding
risk or incidence of ASCVD are yet to be investigated. from the Indonesia Endowment Fund for Education (S-1692/
Meanwhile, targeting neutrophils is warranted to improve local LPDP.4/2019 —LPDP—Indonesia Scholarship). The figures
periodontal therapy, eliminate periodontitis effectively, that can were created using Biorender.com
2019: Update From the GBD 2019 Study. J Am Coll Cardiol (2020) 76
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published: 07 July 2022

The Roles of Neutrophils Linking

Inﬂammation plays a crucial role in the onset and development of atherosclerosis.

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