Maladaptive trained immunity and clonal hematopoiesis as potential mechanistic links between periodontitis and inflammatory comorbidities

Abstract

Periodontitis is bidirectionally associated with systemic inflammatory disorders. The prevalence and severity of this oral disease and linked comorbidities increases with aging. Here we review two newly emerged concepts, trained innate immunity (TII) and clonal hematopoiesis of indeterminate potential (CHIP), which together support a potential hypothesis on how periodontitis affects and is affected by comorbidities and why the susceptibility to periodontitis and comorbidities increases with aging. Given that chronic diseases are largely triggered by the action of inflammatory immune cells, modulation of their bone marrow precursors, the hematopoietic stem and progenitor cells (HSPCs), may affect multiple disorders that emerge as comorbidities. Such alterations in HSPCs can be mediated by TII and/or CHIP, two non-mutually exclusive processes sharing a bias for enhanced myelopoiesis and production of innate immune cells with heightened proinflammatory potential. TII is a state of elevated immune responsiveness based on innate immune (epigenetic) memory. Systemic inflammation can initiate TII in the bone marrow via sustained rewiring of HSPCs, which thereby display a skewing towards the myeloid lineage, resulting in generation of hyper-reactive or ‘trained’ myeloid cells. CHIP arises from aging-related somatic mutations in HSPCs which confer a survival and proliferation advantage to the mutant HSPCs and give rise to an outsized fraction of hyper-inflammatory mutant myeloid cells in the circulation and tissues. This review discusses emerging evidence that supports the notion that TII and CHIP may underlie a causal and age-related association between periodontitis and comorbidities. A holistic mechanistic understanding of the periodontitis-systemic disease connection may offer novel diagnostic and therapeutic targets for treating inflammatory comorbidities.

1. Introduction

Periodontal disease is an exemplar of a dysregulated balance in the crosstalk between the local subgingival microbiome and the immune response of the host that is induced to ostensibly control the pathogen challenge. In periodontitis patients, however, the inflammatory response not only fails to mediate protective immunity, but also fuels the selective expansion of pathogenic species leading to dysbiosis ¹. Indeed, the inflammatory destruction of connective and bone tissue is not simply a hallmark of the disease but also generates a nutritionally favorable environment (enriched with degraded proteins such as collagen and heme-containing compounds, which are sources of amino-acids and iron, respectively) for periodontitis-associated ‘inflammophilic’ species ². The flourishing of periodontitis-associated microbial communities under inflammatory conditions exacerbates the inflammatory destruction process, generating a feed-forward loop that promotes the persistence of the dysbiotic microbiome and the chronicity of periodontitis. This oral disease has a significant socioeconomic impact ^3,4 and the global prevalence of its severe form is approximately 10% of the adult population ⁵.

Multiple clinical studies have associated periodontitis with certain inflammatory disorders (comorbidities), such as, cardiovascular disease (CVD), rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease (IBD), type 2 diabetes (T2D), osteoporosis, Alzheimer’s disease, and non-alcoholic fatty liver disease (NAFLD) ^6–13. In this regard, periodontal disease shares inflammatory effector mechanisms with these comorbidities; for instance, interleukin (IL)-23– and IL-17–driven destructive inflammation participates in the pathogenesis of not only periodontitis but also of RA, psoriasis and IBD ^14–19. Periodontitis also has common genetic or environmental risk factors (e.g., age, socioeconomic status, smoking, diabetes, obesity, hypertension) with other chronic inflammatory disorders ^20–24. Nevertheless, an independent association between periodontitis and comorbidities persists even upon adjusting known confounding factors ^25–33. Consistent with this, the activity of certain comorbid conditions (or surrogate markers thereof) and markers of systemic inflammation (e.g., IL-1, IL-6, C-reactive protein, fibrinogen and neutrophil counts in the blood) are improved following successful local periodontal treatment ^13,34–48. These clinical studies together with emerging collective evidence from preclinical models suggest that periodontitis exerts a systemic effect on overall health above and beyond the local effect in the affected periodontal tissue ⁴⁹.

In this chapter, we review and discuss emerging evidence in support of potential causal mechanisms that may explain how periodontitis affects and is affected by comorbidities and why the susceptibility to periodontitis and linked comorbidities increases with aging. Specifically, we focus on mechanisms involving maladaptive adaptation of bone marrow hematopoietic stem and progenitor cells (HSPCs) in the context of trained immunity and clonal hematopoiesis ^50–52. These concepts will be expounded upon after a brief review of evidence establishing the capacity of periodontitis to cause systemic inflammation.

2. Periodontitis-associated systemic inflammation

Studies from independent clinical research groups show that periodontitis is associated with low-grade systemic inflammation (Figure 1). Indeed, as compared with healthy individuals, periodontitis patients display higher levels of IL-1, IL-6, C-reactive protein, and fibrinogen, as well as increased numbers of neutrophils ^{43–45,53–64}. Other reports document increased serum levels of TNF, IFNγ, IL-17, and oncostatin M in patients with periodontitis ^65–68. Interestingly, there is significant correlation between the levels of certain inflammatory cytokines (such as TNF, IL-6, oncostatin) in local gingival crevicular fluid samples and corresponding systemic (serum) samples ^46,68,69. In both GCF and serum, inflammatory cytokines are found in higher levels in periodontitis relative to health and generally decrease after successful non-surgical periodontal treatment ^46,68. The notion that local, tooth-related lesions contribute to the systemic inflammatory burden is also supported by the conclusions of a meta-analysis that apical periodontitis is linked with higher serum levels of C-reactive protein, IL-6, and the central complement component C3, the concentration of which is significantly reduced after the treatment and resolution of apical periodontitis ⁷⁰.

Figure 1. Periodontitis-related systemic inflammation and inflammatory modulation of bone marrow progenitor cells.

In periodontitis, bacteria from the tooth-associated dysbiotic biofilm may translocate through the ulcerated periodontal pockets into the blood circulation, resulting in bacteremias and systemic inflammation. Systemic inflammation, in turn, may cause inflammatory modulation of hematopoietic stem and progenitor cells promoting increased myelopoiesis. HSC, hematopoietic stem cell; MPP, multipotent progenitor; GMP, granulocyte-monocyte progenitor.

The periodontitis potential to trigger systemic inflammation is also in keeping with the beneficial actions of local periodontitis treatment on improving systemic inflammatory markers discussed above, ^13,34–48 as well as with the results of a prospective study of 11,869 individuals (mean age 50) ⁷¹. This report demonstrated that poor maintenance of oral hygiene (never or rarely brushing teeth) correlated with enhanced systemic concentrations of fibrinogen and C-reactive protein and increased risk for cardiovascular disease events ⁷¹. Although periodontitis is related to elevated risk of common systemic comorbidities ^{7,9,29,30,72–77}, the reverse is also true, i.e., periodontitis becomes more prevalent in those patients with systemic comorbidities, such as, RA, cardiometabolic conditions, and IBD ^{9,26,75,78–86}. Consistent with this, systemic inflammation (using fibrinogen and white blood cell counts as markers) has been dose-dependently and longitudinally linked with periodontitis in an 11-year prospective study of 1784 participants ⁸⁷. Therefore, periodontitis and linked comorbidities appear to be bidirectionally associated.

Regarding how periodontitis could affect systemic health, two major mechanisms have been suggested that are consistent with clinical findings. First, periodontal bacteria or their products (such as, LPS and proteases), may reach the blood circulation via the ulcerated epithelium of the periodontal pockets causing systemic complications ^6,55,88–92. The surface area covered by the epithelium of the periodontal pockets in periodontitis patients is large and estimated at 8 to 20 cm^{2 93}. Consistently, periodontitis is often linked with bacteremias (Figure 1), the occurrence of which may correlate with the severity of clinical periodontal inflammation ^55,94–98. Second, in severe periodontitis, innate and adaptive immune cells within the connective tissues subjacent to periodontal pockets secrete abundant levels of pro-inflammatory cytokines (e.g., IL-1β, IL-6, G-CSF ^99,100), which may thus enter the bloodstream. In this regard, viral infections in the oral cavity that affect the periodontium may modify the periodontal host response in ways that amplify local inflammatory responses and hence increase the systemic inflammatory burden ^101,102. Thus, at least in principle, periodontitis-associated systemic inflammation could arise from the spillover of inflammatory cytokines derived from the periodontium to the circulation or induced by the hematogenous translocation of periodontal bacteria and products thereof. The periodontitis-associated systemic Inflammatory response, and particularly IL-6, can additionally cause an acute phase response in the liver, leading to elevated C-reactive protein and fibrinogen that can contribute to systemic complications, such as atherogenesis ^55,58,63,64.

Given that chronic inflammatory diseases are, in large part, driven by the action of inflammatory immune cells, it can be reasoned that systemic inflammation-induced alterations in their bone marrow progenitors are likely to reciprocally influence multiple chronic inflammatory disorders that may emerge as comorbidities ⁵¹. Alterations in HSPCs in the bone marrow leading to the generation of progeny with augmented proinflammatory potential can occur as a result of trained innate immunity (TII) ^50,103 or somatic mutations occurring in the context of the aging-related clonal hematopoiesis of indeterminate potential (CHIP) ^104–106 (Figure 2). TII and CHIP are not mutually exclusive and, although distinct phenomena, share a bias for enhanced myelopoiesis (over other lineages) resulting in the production of mature myeloid cells with enhanced proinflammatory capacity ^51,52. These events, in turn, can influence the initiation and/or the progression of periodontitis and comorbid inflammatory conditions, as discussed in the following sections. Before presenting evidence and engaging in discussions of how TII and CHIP affect periodontitis and associated comorbidities, we provide a brief background on these two important concepts in the context of bone marrow myelopoiesis.

Figure 2. A vicious feed-forward loop linking periodontitis to inflammation-adapted HSPCs in the context of trained immunity and clonal hematopoiesis.

The ability of bone marrow HSPCs to not only sense but also adapt to pro-inflammatory stimuli underlies the induction of trained immunity, leading to sustained enhanced myelopoiesis and generation of myeloid cells with heightened immune preparedness and responsiveness. Clonal hematopoiesis of indeterminate potential (CHIP) results from age-related somatic mutations in HSPCs that endow the mutant HSPCs with a proliferative advantage, especially under inflammatory conditions, thereby generating an outsized fraction of hyper-inflammatory mutant myeloid cells. Periodontitis-associated systemic inflammation may thus contribute to induction of trained innate immunity and shaping of an inflammatory bone marrow microenvironment conducive for the selective expansion of CHIP-mutant HSC clones. The generated hyper-responsive or hyper-inflammatory myeloid cells are recruited to sites of infection and inflammation and can thus exacerbate periodontitis, which in turn can exert increased inflammatory influence on the bone marrow. Thus, a self-sustained feed-forward loop may be generated between inflammation-adapted HSPCs and periodontitis that could contribute to the chronicity of this oral disorder.

3. Trained innate immunity

The bone marrow is the epicenter of hematopoiesis where the self-renewable hematopoietic stem cell (HSC), also known as long-term (LT)-HSC, has the ability to produce all types of mature blood cells ¹⁰⁷. LT-HSCs can differentiate via short-term HSCs (ST-HSCs) to multi-potent progenitors (MPPs). LT-HSCs, ST-HSCs and MPPs comprise the so-called ‘hematopoietic stem and progenitor cells’ (HSPCs) ^107–109. HSPCs express TLRs and growth factor and cytokine receptors (e.g., receptors for IL-1β, IL-6, M-CSF) ^107,109 and can thus respond to systemic inflammation (or infection) by initiating increased production of myeloid cells ^51,110 (Figure 1). The standard differentiation pathway for myelopoiesis downstream of MPP cells includes common myeloid progenitors (CMP) and granulocyte macrophage progenitors (GMP), the precursors of monocytes/macrophages and neutrophils.

Under the influence of inflammation, HSPCs may become adapted for long-term enhanced proliferation and skewed differentiation toward the myeloid lineage. This sustained inflammatory modulation of HSPCs is based on enduring cell-autonomous (metabolic, transcriptional, and epigenetic) mechanisms that underlie the induction and persistence of TII, a relatively recent concept ^51,111–113. Indeed, historically, immunological memory has been viewed as an exclusive ‘privilege’ of the adaptive immune system. However, owing to advances in the past decade, it is now appreciated that innate immune cells can also build and maintain ‘memory’ of earlier inflammatory events, such as those associated with infectious stimuli (e.g., the major fungal cell wall constituent β-glucan) or vaccination (e.g., the bacillus Calmette–Guérin [BCG] vaccine). In this manner, innate immune cells exhibit hyper-responsiveness upon re-challenge; in other words, are in a position to respond faster and stronger to future challenges. However, the anamnestic response is not specific for the original stimulus and thus can be elicited against the same or even unrelated stimulus ^114–116 (Figure 3). This elevated state of inflammatory activation that is based on non-specific innate immune memory is designated ‘trained innate immunity’ (TII) ⁵⁰. Accordingly, TII is defined as a non-specific memory elicited irrespective of adaptive immunity upon earlier encounters, and which promotes augmented responsiveness to ensuing homologous or heterologous challenges ^50,51,103.

Figure 3. Trained innate immunity.

Trained innate immunity represents non-specific immune memory which can be induced, independent of adaptive immunity, upon an earlier microbial challenge and can be recalled at later time points in response to a new challenge that is not necessarily the same with the primary one. The induction of the memory or trained phenotype can be initiated in the bone marrow via long-lived metabolic, epigenetic, and transcriptional adaptations in hematopoietic stem and progenitor cells, which in turn give rise to trained mature myeloid progeny with augmented immune and inflammatory responsiveness.

The long-term effects of TII on blood myeloid cells that have relatively limited lifespan (only a few days) in the circulation ¹¹⁷ are explained by the demonstration that TII can be initiated at the level of the bone marrow through long-lived metabolic and transcriptional alterations in HSPCs, the precursors of differentiated innate immune cells ^111,112. Specifically, TII induces long-lasting alterations in HSPCs, which, through enhanced myeloid-biased differentiation, can give rise to increased numbers of ‘trained’ myeloid cells ^51,112.

At least in preclinical models where trained myelopoiesis is induced by β-glucan, the underlying mechanism involves inflammatory IL-1β-signaling in HSPCs associated with higher IL-1β levels in the bone marrow. Moreover, the activation of HSPCs by β-glucan–induced TII involves their metabolic reprogramming and enhanced GM-CSF/CD131 signaling. Together, these immunometabolic alterations in HSPCs drive myelopoiesis ¹¹². Integral to the long-lasting effects of TII in the bone marrow are inflammation-induced epigenetic modifications in LT-HSCs. Indeed, LT-HSCs in mice trained with LPS can retain epigenetic memory (increased chromatin openness and accessibility) of earlier infections or inflammatory challenges for at least 12 weeks, thereby potentiating myeloid differentiation and innate immune responses following a future challenge ¹¹³. The initiation of TII with persistent transcriptomic myeloid skewing of HSPCs in the bone marrow leading to long-lived and increased responsiveness of peripheral myeloid cells to heterologous stimuli was also shown in healthy human individuals upon BCG vaccination¹¹⁸. Overall, therefore, the induction of innate immune memory, upon exposure to microbial and/or inflammatory stimuli, entails metabolic, epigenetic and transcriptional reprogramming which can persist for months ^{103,119–123} (Figure 3).

TII initiated by modulation of LT-HSCs can give rise to trained myeloid cell populations (neutrophils, monocytes/macrophages) that can replenish and fortify the innate immune system upon stress conditions, such as those related to infections or with inflammatory, tumorigenic or chemotherapeutic challenges ^{111–113,124}. Consequently, TII is thought to represent an evolutionarily maintained beneficial response that promotes host survival upon re-infection or other future challenges that can be potentially detrimental to the host ^{103,125–128}. However, TII may also promote aberrant immune responses that aggravate chronic inflammatory diseases ^51,129–131. In this regard, TII in the bone marrow can contribute to perpetuation of inflammation both quantitatively and qualitatively, i.e., by increasing the production of myeloid cells which at the same time have enhanced inflammatory responsiveness ^51,132,133 (Figure 2).

4. Clonal hematopoiesis of indeterminate potential

In common with other chronic inflammatory diseases, an individual’s susceptibility to periodontitis increases with aging ^134–138. The term “inflamm-aging” was coined to describe the aging-related elevation of low-grade chronic systemic inflammation (e.g., IL-1β- and IL-6), which coincides with an increase in co-morbidities ^139,140. Although the concept of inflamm-aging is well documented epidemiologically, its etiology has been largely unknown ^140–145. A potential mechanistic explanation for the association of aging with inflammation is CHIP ^104–106.

CHIP defines an aging-related condition, whereby somatic mutations that are usually related to myeloid malignancies accrue in HSPCs; these mutations confer a survival/proliferation advantage to the mutant HSPC, thereby enabling its clonal expansion and the generation of mutant progeny cells. The latter represent a separate fraction of leukocytes in the peripheral blood. Importantly, however, individuals with CHIP do not have clinical signs of a hematologic disorder, for instance cytopenias ^106,146 (Figure 4). The increased mutation accumulation by HSCs with aging may, at least in part, result from the fact that aged HSCs are more resistant to DNA damage-induced apoptosis than young HSCs, owing to decreased DNA damage repair pathways in the former ¹⁴⁷. Therefore, aged HSCs may not die despite suffering DNA damage, hence accumulating mutations ¹⁴⁷.

Figure 4. CHIP may potentially affect multiple inflammatory disorders.

With advancing age, HSPCs progressively acquire somatic mutations that confer increased self-renewal capacity and myeloid differentiation bias, giving rise to mutant myeloid progeny with enhanced pro-inflammatory potential, as compared to their counterparts from normal HSPC clones. This condition, which - in the absence of apparent hematological malignancy - is designated clonal hematopoiesis of indeterminate potential (CHIP) has been epidemiologically and experimentally linked with atherosclerotic cardiovascular disease. However, the generation and release of hyper-inflammatory myeloid cells in the context of CHIP may contribute to inflamm-aging and elevate systemic inflammation, which in turn could contribute to the pathogenesis of additional inflammatory pathologies, such as periodontitis and rheumatoid arthritis.

Although the formal definition of CHIP is the presence of expanded blood cell clones harboring a somatic mutation related to a myeloid malignancy without overt hematological symptoms or malignancy ¹⁰⁶, individuals with CHIP have an increased risk of developing hematologic malignant disorders, as well as atherosclerosis and diabetes mellitus ^{146,148–152}. More recently, CHIP in postmenopausal women was associated with natural (that is, non-surgical) premature menopause ¹⁵³. Moreover, CHIP is suspected to also contribute to aging-related inflammatory disorders ⁵¹ (Figure 4). In fact, the “indeterminate potential” part of the term denotes the uncertainty of how CHIP may develop clinically, in part because distinct environmental stress factors (e.g., an inflammatory bone marrow microenvironment) may be needed to cooperate with the underlying somatic mutations for the emergence of pathological phenotypes ^51,154–156.

By definition, CHIP mutations are detected at a variant allele frequency (VAF) of 2% or greater. At this consensus cutoff value, CHIP occurs in >10% of individuals older than 65 with progressively increased prevalence with further aging ^52,106. CHIP-related mutations mostly involve disruptive somatic lesions (nonsynonymous, nonsense, frameshift, or splice-site disruption) in the affected genes ¹⁴⁸. Using an ultra-sensitive sequencing method, a recent study detected CHIP in >20% of individuals 60 to 69 years of age and in 3% of individuals aged 20 to 29 years ¹⁵⁷. Although CHIP prevalence is minimal in young ages, the risk it confers to young individuals for progression of hematologic malignancies and cardiovascular disease is similar to that in the elderly ¹⁰⁶.

The most common CHIP-related mutations affect three genes that encode epigenetic modifiers, namely, TET2 (Ten-eleven translocation methylcytosine dioxygenase 2), DNMT3A (DNA methyltransferase 3A), and ASXL1 (Associated sex combs-like 1) ^{105,106,146,148,158}. Together, mutations in TET2 and DNMT3A, which mediate cytosine methylation and demethylation, respectively, represent approximately two thirds of acquired CHIP mutations. The mutant alleles usually present with loss of function ^{52,106,146,148,158,159}.

As TET2 functions as a demethylase^160–162, loss-of-function mutations cause hypermethylation of DNA at sites involved in gene expression regulation, such as transcription factor–binding sites ^160,161. The epigenetic dysregulation owing to TET2 mutations leads to survival advantage of clonal HSCs, which also are skewed towards the myeloid lineage ^163–168. When the function of DNMT3A is impaired by inactivating CHIP-related mutations, all hematopoietic lineages appear to be affected in humans; consistent with this observation, genetic ablation of Dnmt3a in mice leads to elevated risk of both myeloid and lymphoid malignancies ^168–172. Moreover, hematopoietic-specific deletion of Asxl1 in mice results in cytopenias affecting all lineages and ¹⁷³ loss-of-function mutations in ASXL1 predominantly affect the trimethylation of lysine 27 of histone 3 ¹⁷⁴.

Aging HSPCs with loss-of-function mutations in TET2 display enhanced self-renewal and preferential commitment to the myeloid lineage, giving rise to mature myeloid cells with enhanced proinflammatory activity ^{149,163–166,175}. An inflammatory environment in the bone marrow, due to inflamm-aging or underlying inflammatory comorbidities, favors the expansion of CHIP clones ¹⁵⁵. Specifically, both IL-6 and TNF support the survival of TET2-deficient HSPCs ^176,177. In contrast, under similar inflammatory conditions, TET2-sufficient HSPCs are suppressed and/or are led to exhaustion ^{51,104,176–178}. In other words, TET2-mutant clones appear to have a greater fitness over normal clones in an inflammatory bone marrow environment. Moreover, the increased proliferation of CHIP-mutant HSPCs in an inflammatory environment associated with atherosclerosis accelerates the emergence of somatic CHIP-driver mutations, hence further promoting CHIP ¹⁷⁹. Overall, inflammation in the bone marrow may promote the emergence of CHIP mutant clones, potentially predisposing to the increased risk for pathological conditions in CHIP carriers ^176–178.

5. Maladaptive trained immunity and periodontitis-associated comorbidities

As discussed above, TII is thought to represent an evolutionarily conserved beneficial response that can protect the host from re-infection (with the same pathogen that elicited TII in the first place), as well as from unrelated pathogens that are encountered for the first time ^{50,103,116,125–127}. Consistent with this notion, TII is also present in primitive organisms ^103,180. However, in the setting of modern challenges in western-type societies, TII could also promote aberrant immune responses that may potentially aggravate immune-driven pathologies (‘maladaptive’ TII), thus contributing to the prevalence and severity of chronic inflammatory disorders ^{51,116,129,181,182}. Maladaptive TII may be induced not only by infections or purified microbial molecules but also by an obesogenic diet ¹⁸³. In this regard, oxidized low-density lipoprotein, as well as other unrelated endogenous factors (e.g., uric acid/monosodium urate crystals [inflammasome activators], and the High-Mobility-Group Box 1 protein [activator of Toll-like receptors and the receptor for advanced glycation end products]) can induce TII ¹⁸⁴.

5.1. Implications from cardio-metabolic disease

The concept of maladaptive TII is supported by a preclinical study which modelled metabolic syndrome-induced inflammation by feeding low-density lipoprotein receptor-deficient mice a calorie-rich, high-fat western-type diet. The study showed that the western-type diet induces NLRP3 inflammasome– and IL-1–dependent epigenetic reprogramming of bone marrow progenitor cells (including GMPs), which in turn drive trained myelopoiesis that perpetuates systemic inflammation ¹²⁹ (Figure 5). In line with the long-lasting effects of TII, this maladaptive reprogramming of bone marrow progenitor cells persisted even after the animals were switched back to a healthy (conventional chow) diet ¹²⁹. From a clinical standpoint, chronic metabolic-inflammatory disorders, including T2D, obesity and atherosclerosis can skew hematopoiesis toward inflammatory myelopoiesis, in turn leading to chronic leukocytosis that may promote propagation and chronification of these inflammatory disorders ^{51,132,133,152,185}.

Figure 5. Western diet leads to NLRP3 Inflammasome activation and induction of maladaptive trained immunity.

In mice, western-type diet, in great part through oxidized LDL, induces activation of the NLRP3 inflammasome and caspase 1-dependent secretion of IL-1β, which induces expansion and epigenetic reprogramming of bone marrow progenitor cells (including GMPs), which in turn drive trained myelopoiesis (increased numbers of myeloid cells with enhanced immune responsiveness) that can perpetuate systemic inflammation. These maladaptive alterations persist even after the mice are switched back to a healthy diet.

It can be envisioned that the inflammation-induced epigenetic rewiring of HSPCs towards heightened myelopoiesis might perpetuate inflammation leading to the generation of a feed-forward loop between the bone marrow and cardiometabolic disease, such as atherosclerosis, or a chronic inflammatory disorder, in general, such as periodontitis ^{49,51,132,133,185} (Figure 2). In other words, elevated numbers of circulating inflammatory myeloid cells can augment inflammation in peripheral tissues, which reciprocally can perpetuate HSPC-mediated myelopoiesis ^{133,185–189}.

It also follows that the maladaptive training of HSPCs may constitute mechanistic grounds for comorbidities, such as the increased risk of cardiovascular complications in patients with other types of chronic inflammation, such as periodontitis or rheumatoid arthritis ^190,191. In the latter regard, experimental arthritis-associated systemic inflammation down-regulates cholesterol export genes (Apoe, Abca1, and Abcg1) and hence elevates the content of membrane cholesterol of HSPCs in the bone marrow of mice; this in turn promotes HSPC expansion and augmented myelopoiesis ¹⁹². This is in part because cholesterol accumulation alters the cell membrane properties, thereby leading to enhanced surface abundance of CD131 (the common β-subunit of the IL-3- and GM-CSF-receptor) and increased downstream signaling, which drives myelopoiesis ^112,193. Importantly, apolipoprotein E-deficient (Apoe^−/−) mice subjected to K/BxN-induced arthritis exhibit increased entry of Ly6C^hi monocytes into atherosclerotic lesions (thus elevating the macrophage burden), as compared to non-arthritic Apoe^−/− controls ¹⁹². This study therefore suggests that the arthritis-associated elevation of circulating myeloid cells fuels the increased abundance of macrophages in atheromas ¹⁹². Consistent with these findings, RA patients display systemic inflammation and increased numbers of circulating myeloid cells including CD16⁺ monocytes as well as decreased mRNA expression of cholesterol efflux genes (ABCA1 and ABCG1) in peripheral blood mononuclear cells, as compared to healthy controls ^192,194,195.

In a similar context, a study utilizing positron emission tomography/computed tomography with 2-deoxy-2-[fluorine-18]fluoro-D-glucose (¹⁸F-FDG-PET/CT; more on this technique below) showed that a subset of rheumatoid arthritis patients in clinical remission exhibit elevated metabolic activity in the bone marrow and enhanced inflammation in the arterial wall associated with a promigratory phenotype (as assessed by enhanced expression of activation and pro-adhesive markers) of circulating monocytes ¹⁹⁶. Thus, although 40% of patients with rheumatoid arthritis display remission of the disease, the remission does not necessarily normalize their CVD risk ¹⁹⁶. These findings moreover suggest a common underlying pathophysiological mechanism for RA and CVD that might involve innate immune memory in the bone marrow. Such inflammatory memory may sustain enhanced myelopoiesis in arthritis patients while in remission, increasing their risk for CVD. Interestingly in this regard, rheumatoid arthritis patients have two- to three-fold higher CVD risk, which is only partly attributed to known traditional risk factors for CVD ^197,198. Moreover, in these patients, arthritis activity and duration are correlated with enhanced inflammation in their arterial walls ¹⁹⁶. Conversely, as alluded to above, cardiometabolic conditions can also affect hematopoiesis and potentially TII ^{129,131,133,152,199} and could thus affect the development of (or exacerbation of existing) arthritis.

5.2. Inflammatory periodontitis-bone marrow axis and comorbidities

Although there is currently no direct experimental evidence that periodontitis causes maladaptive training of HSPCs in the bone marrow or, conversely, that periodontitis is affected by the state of the bone marrow, clinical observations are consistent with such notions.

Recent studies have attempted to correlate periodontal inflammation with inflammation in extraoral sites using ¹⁸F-FDG-PET/CT ^200,201, a technique that can identify sites of inflammatory tissue activity ^202,203. ¹⁸F-FDG, a radiolabeled analogue of glucose, is taken up by the cells via cell membrane glucose transporters and subsequently phosphorylated intracellularly by hexokinase. The ability of ¹⁸F-FDG-PET/CT to localize to sites of inflammation is largely attributed to the increased glycolytic activity (high-level expression of glucose transporters and hexokinase activity) of the cells involved in the inflammatory response and particularly neutrophils and monocyte/macrophages ^202,203. Importantly, ¹⁸F-FDG uptake in periodontal tissue correlates not only with periodontal inflammation (as evaluated by magnetic resonance imaging) but also with the severity of PD as inferred indirectly by the extent of radiographic bone loss in the inflamed area ²⁰⁴.

Using ¹⁸F-FDG-PET/CT, another recent study demonstrated a correlation between the metabolic/inflammatory activities of the periodontal tissue and the bone marrow hematopoietic tissue ²⁰⁰. Moreover, by the same technique, periodontal metabolic/inflammatory activity was associated with arterial inflammation, as assessed by ¹⁸F-FDG uptake ^200,201 or histologically by evaluating macrophage infiltration in excised carotid artery plaques ²⁰¹. High-dose atorvastatin therapy reduced periodontal inflammation after a 12-week treatment and the changes in periodontal inflammation correlated with changes in carotid inflammation, as evaluated (in both conditions) by ¹⁸F-FDG-PET/CT ²⁰⁵. Importantly, a more recent study using the same cohort as the above-discussed report²⁰⁰ showed that ¹⁸F-FDG-PET/CT-determined periodontal inflammation was independently associated with both increased arterial inflammation and risk of subsequent cardiovascular events ²⁰⁶. Bone marrow hematopoietic activity was further associated with C-reactive protein, white blood cell count and absolute monocyte count (through not with erythrocyte-related parameters) ²⁰⁰. Together, these studies suggest associations between bone marrow hematopoietic tissue activity and distinct inflammatory comorbidities, although causality and directionality are uncertain regarding these parameters.

It could be argued that sustained chronic inflammation (owing to periodontitis or other systemic disease) may not provide a TII-inducing stimulus for the bone marrow analogous to that induced by defined agonists of TII such as β-glucan or the BCG vaccine, which are administered in an ‘acute’ rather than a ‘chronic’ manner. It should be noted, however, that the chronic nature of periodontitis does not necessarily represent a continual pathologic process but may rather constitute a persistent series of short-lived acute insults (bursts) separated by periods of remission ^207–210. Similarly, a key characteristic of periodontitis-associated inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel disease, are flares and exacerbations that are also separated by intervals of remission ^211–213. Therefore, short-term periods of increased inflammation (‘inflammatory attacks’), such as those involved in the induction of TII, are consistent with this burst-like model of chronic inflammatory diseases.

5.3. Trained myeloid cells and periodontitis-associated comorbidities

Monocytes or neutrophils deriving from the blood of periodontal disease patients display an inherent hyper-reactivity in that they elicit higher levels of proinflammatory cytokines when they are activated ex vivo by inflammatory stimuli, such as LPS or whole bacteria, as compared to the same cell types isolated from the peripheral blood of healthy individuals ^214,215. Interestingly, this hyper-reactivity can persist even after the patients undergo successful non-surgical periodontal therapy; specifically, the enhanced cytokine-inducing capacity of peripheral blood myeloid cells from periodontitis patients could be observed for at least 2 months (time interval for post-treatment review) ^214,215. These findings resemble recent data from individuals treated with TII-inducing agents, such as the BCG vaccine ^118,127.

In this regard, BCG vaccination in healthy volunteers resulted not only in an ‘immediate’ (2 weeks post-vaccination) hyper-reactivity of circulating monocytes in terms of proinflammatory cytokine production upon ex vivo stimulation with unrelated bacterial and fungal pathogens, but also in long-term hyper-reactivity. Indeed, the enhanced cytokine-inducing capacity (upon ex vivo stimulation) of circulating monocytes from BCG vaccinated individuals persisted for at least 3 months after vaccination and was accompanied by higher surface expression of activation markers, such as Toll-like receptor 4 and the Mac-1 integrin ¹²⁷. This enhanced hyper-reactivity could be associated with the fact that BCG vaccination imprinted persistent epigenetic, transcriptional, and functional adaptations in bone marrow HSPCs toward enhanced myelopoiesis ¹¹⁸. Consistent with the notion that the TII phenotype is initiated at the level of progenitors and is passed on to their progeny, similar epigenetic changes in inflammation-associated loci were also observed in peripheral blood CD14⁺ monocytes of the vaccinated individuals ¹¹⁸. Moreover, peripheral blood mononuclear cells from patients with coronary artery disease exhibit heightened cytokine production (as compared with the same cells from healthy individuals) after ex vivo stimulation; importantly, transcriptome analysis of HSPCs and GMPs from these patients revealed enrichment for neutrophil- and monocyte-related pathways, indicating that the progenitors are skewed to differentiate into the myeloid lineage ²¹⁶. In general, peripheral blood monocytes isolated from patients with different autoimmune or inflammatory diseases display characteristics consistent with a trained phenotype, such as enhanced responsiveness to ex vivo stimulation, enhanced glycolysis, and epigenetic rewiring ²¹⁷.

On the premise that periodontal disease may influence hematopoietic tissue inflammation ²⁰⁰, it could be reasoned that, in periodontitis patients, peripheral blood myeloid cells are in a ‘trained’ state, which endows them with hyper-reactivity (compared to cells from healthy controls) to inflammatory stimuli. This trained state of peripheral myeloid cells of periodontitis patients, which may be maintained even after periodontal therapy, might represent a form of long-lasting innate immune memory imprinted in HSPCs in an inflammatory bone marrow environment ^50,112,118 (Figure 2). In fact, the recent association of periodontitis with the severity of COVID-19 disease (increased risk for admission to intensive care unit, necessity of assisted ventilation and death) ²¹⁸ might, in part, be due to a trained/hyper-responsive state of periodontal patients’ myeloid cells. This notion is in keeping with the observation that, in comparison with COVID-19 patients without periodontitis, COVID-19 patients with periodontitis exhibited increased levels of systemic inflammatory markers and white blood cell counts ²¹⁸. The latter may imply increased myelopoiesis, but further tests would be required to implicate trained myelopoiesis.

In addition to monocytes/macrophages, neutrophils and granulocytes in general are also targets of TII ^118,124. Ligature-induced periodontitis in mice increases the neutrophil counts in the bone marrow and enhances the neutrophil response to subsequent acute infectious peritonitis, both quantitatively and qualitatively (enhanced proinflammatory potential), suggesting that periodontitis can enhance the severity of other inflammatory conditions ²¹⁹. Whether the enhanced responsiveness of the neutrophils involves TII in the bone marrow was not addressed. However, consistent with this preclinical observation, experimental gingivitis in human volunteers for 3 weeks increased the ex vivo inflammatory responsiveness of blood neutrophils ²¹⁹. By comparing the HSPC transcriptome of individuals before and 3 months after BCG vaccination, a recent study demonstrated upregulation of genes associated with granulocytic lineage priming, thus suggesting that BCG immunization skews the myeloid cell lineage toward enhanced granulopoiesis ¹¹⁸. Interestingly, peripheral blood neutrophils from periodontitis patients display a Type I IFN signature (elevated expression of IFN-stimulated genes relative to neutrophils from healthy individuals), associated with increased plasma concentrations of IFNα, as compared to those of periodontally healthy controls ²²⁰. Moreover, the periodontitis-associated peripheral blood neutrophils exhibit a hyper-responsive phenotype ex vivo with regards to ROS production when primed with type-I IFNs (albeit not by LPS), which induce an mRNA expression profile in neutrophils similar to that observed in vivo ²²⁰. The authors concluded that type-I IFNs may be a factor determining the hyper-reactive phenotype of periodontitis patients’ peripheral blood neutrophils. It is intriguing to speculate that this type-I IFN-associated hyper-responsiveness of peripheral blood neutrophils from periodontitis patients might arise from trained granulopoiesis in the bone marrow.

The aforementioned notion is supported indirectly by studies in mice. Specifically, mice systemically treated with fungal-derived β-glucan exhibit type I IFN-dependent transcriptomic and epigenetic rewiring of bone marrow granulopoietic progenitors (also designated as trained granulopoiesis), giving rise to neutrophils with augmented ROS-dependent tumor-killing capacity ¹²⁴. Single-cell epigenetic analysis demonstrated that the neutrophils generated from trained granulopoiesis exhibited both enhanced Type I IFN-signaling and increased chromatin accessibility in the genes encoding the ROS-producing molecules neutrophil cytosolic factors 1 and 2, suggesting that this type I IFN signature is sustained at the epigenetic level and passed on to the progeny ¹²⁴. Thus, it is possible that trained granulopoiesis induced by different inflammatory TII agonists (as documented in BCG-vaccinated humans ²²¹ or upon β-glucan administration in mice ¹²⁴) or by systemic inflammation (owing to periodontitis or other inflammatory condition) might promote the generation of a hyper-reactive neutrophil phenotype with Type I IFN signature and ability for enhanced production of ROS upon rechallenge. Such recall responses can be either protective (e.g., in tumor immunity or in acute infections) or destructive (e.g., in periodontitis and inflammatory diseases in general). It is noteworthy that feeding mice with western-type diet also leads to a strong Type I IFN signature in bone marrow granulopoietic progenitors (GMPs) ¹²⁹. Given that different inflammatory stimuli induce bone marrow progenitor training via Type I IFN signaling, this pathway merits further investigation for its role in mediating maladaptive training of bone marrow progenitors towards perpetuation of inflammation that could contribute to the emergence of periodontitis-associated comorbidities.

From the above, it can be reasoned that systemic inflammation associated with a certain chronic inflammatory disorder, such as periodontitis, may cause maladaptive training of hematopoietic progenitors, resulting in augmented myelopoiesis and production of ‘trained’ inflammatory myeloid cells, which in turn can populate remote organs and tissues, including the periodontium, where they can enhance local inflammation (Figure 2). This adaptation can be epigenetically regulated and thus can be sustained for a long time, during which HSPCs can influence (through their myeloid progeny) the development or course of different inflammatory disorders that emerge as comorbidities.

6. CHIP and chronic inflammation in periodontitis and other conditions

The concept that CHIP-related loss-of-function mutations are associated with human disease is not adequately explored with the exception of myeloid malignancies and cardiometabolic conditions ^146,149,175. Conceivably, CHIP may promote increased susceptibility to different chronic inflammatory pathologies, such as periodontitis and rheumatoid arthritis (Figure 4). To substantiate this notion, it would be instructive to first discuss the mechanisms by which CHIP promotes cardiometabolic inflammation.

Most investigations on CHIP-associated cardiovascular risk in humans have primarily focused on mutations in DNMT3A and TET2; however, only TET2 has been examined thoroughly in preclinical animal models. These studies offered causal evidence linking TET2 loss-of-function and exacerbated atherosclerosis ^{149,155,175,222}. Low-density lipoprotein receptor–deficient (Ldlr^–/–) mice that were transplanted with bone marrow cells bearing homozygous or heterozygous null mutations in the Tet2 gene exhibited preferential expansion of the mutant clones and accelerated atherosclerotic lesions ^149,175. As with human TET2-associated CHIP ^168,223, even if only a fraction (10%) of transplanted HSCs bore the TET2 deficiency, the recipient mice still exhibited preferential clonal expansion of Tet2^–/– hematopoietic cells and accelerated atherosclerosis ¹⁴⁹.

In the aforementioned preclinical studies, the atherosclerotic lesions contained macrophages with increased production of proinflammatory cytokines, including IL-1β and IL-6 (as compared to Ldlr^–/– controls receiving wild-type bone marrow cells) ^149,175. Consistently, activated TET2-deficient macrophages produce and release increased levels of IL-1β owing to both increased Il1b transcription and augmented expression and activity of the NLRP3 inflammasome ^149,224. Similarly, activated TET2-deficient macrophages produce elevated amounts of IL-6. The TET2 deficiency-associated unrestrained IL-1β and IL-6 production are irrespective of the catalytic activity of TET2 but may rather involve histone deacetylation (TET2 associates with certain histone deacetylases and facilitates their function) ^149,225. Furthermore, TET2-associated CHIP in mice results in elevated macrophage-derived IL-1β in white adipose tissue and worsens insulin resistance in obesogenic diet-fed mice or in old mice ²²⁶, in line with the observational association between CHIP and Type 2 diabetes in humans ¹⁵¹ These findings provide proof-of-concept for a causal role of CHIP in human coronary heart disease and diabetes.

Cancer-free individuals with CHIP (due to mutations in TET2, DNMT3A or ASXL1) have normal total and differential leukocyte counts ^158,175. Consistently, Ldlr^–/– mice transplanted with TET2-deficient bone marrow cells have normal blood cell counts despite a mild myeloid skewing ^149,175. Thus, what appears to cause pathology in CHIP-related atherosclerosis (and likely in other conditions associated with CHIP) is not a quantitative but a qualitative alteration, that is, the dominance of clones; which give rise to myeloid progeny with heightened proinflammatory potential. As these hyper-inflammatory myeloid cells circulate and populate different tissues including the periodontium, CHIP is likely to be associated with different inflammatory conditions, including periodontitis (Figure 4). Therefore, future epidemiologic and mechanistic studies are warranted to explore the full spectrum of pathologies that can be influenced by CHIP.

The therapeutic targeting of inflammatory cytokines, such as IL-6 and IL-1β, may inhibit the detrimental effects of CHIP on cardiometabolic and other chronic inflammatory disorders. IL-1β, in particular, is involved in multiple processes, including the innate immune training of HSPCs in the bone marrow ^112,227, augmented myelopoiesis in CVD ^152,228,229, and in CHIP-associated exacerbated atherosclerotic inflammation ^149,175. The successful application of IL-1β blockade (using canakinumab) in the CANTOS trial for the treatment of atherosclerosis is consistent with the above-mentioned effects of IL-1β ²³⁰. Importantly, patients with TET2 mutant clones respond more favorably to the IL-1β-blocking treatment than patients with non-TET2 mutant CHIP clones ²³¹. This is likely because TET2-related CHIP is significantly associated with elevated IL-1β, although in general CHIP is linked with increased IL-6 concentrations ¹⁵⁹. That IL-6 may mediate CHIP-related pathology is further suggested by a longitudinal cohort study. Patients with DNMT3A- or TET2- associated CHIP clones were shown to have elevated risk of incident CVD; however, a subset of patients who additionally carry a mutation in the IL-6 receptor (which affects its surface expression; p.Asp358Ala) presented with fewer CVD events ²³².

7. Summary and perspective

Periodontitis-associated systemic inflammation may potentially contribute to the inflammatory adaptation of HSPCs, a process that underlies the induction of TII, a non-specific epigenetic memory that promotes amplified responses by HSPCs and their progeny to future infectious or inflammatory challenges ^51,114 (Figure 1). In this manner, TII and the generated trained/hyper-reactive myeloid cells could play a role in the chronicity of periodontitis by setting off a vicious cycle of reciprocally reinforced interactions between the periodontium and the bone marrow (Figure 2). As the trained monocytes/neutrophils are recruited to different tissues or organs and have increased propensity for activation in response to local challenges, TII is likely to be a common mechanistic basis for the development of comorbidities. In a similar vein, the ability of CHIP-mutant HSCs to give rise to hyper-inflammatory myeloid cells should elevate systemic and peripheral inflammation that may contribute not only to the pathogenesis of atherosclerotic CVD ^149,175 but perhaps also to other inflammatory comorbid disorders, such as periodontitis and arthritis (Figure 2).

Future research should strive for a better patho-mechanistic understanding of the intertwining between periodontitis and linked comorbidities in the context of TII (Figure 3) and CHIP (Figure 4), two non-mutually exclusive states associated with elevated inflammatory potential in innate immune cells. Inhibiting key cytokines involved in these processes, such as IL-1β and IL-6, may mitigate the risk of multiple comorbid pathologies. For instance, blocking IL-1β may potentially interfere with maladaptive training of bone marrow HSPCs and thereby disrupt an inflammatory vicious cycle perpetuating different inflammatory comorbidities ^51,112, as well as the detrimental effects of hyperinflammatory myeloid cells derived from CHIP-mutant HSPCs ^149,175. Blocking potent inflammatory cytokines requires caution, as such treatments may interfere with the ability of the host to respond effectively to potentially life-threatening infections. However, for treatments that aim to manipulate TII in the bone marrow, the specificity of the treatment and mitigation of potential side effects could be achieved by using nanoparticles with high avidity for the bone marrow. This technology may facilitate targeted delivery of therapeutic compounds (inhibitors or agonists) to the appropriate cell type for modulating TII in a manner that is protective for the patient ^233,234.