Myeloid-derived Suppressor Cells in Obesity-associated Periodontal Disease: A Conceptual Model

Abstract

Periodontitis is a common chronic inflammatory disease characterized by destruction of the supporting structures of the teeth. Severe periodontitis is highly prevalent—affecting 10–15% of adults—and carries several negative comorbidities reducing the quality of life. Although a clear relationship between obesity severity and periodontal disease incidence exists, mechanisms that support this linkage are incompletely understood. In this conceptual appraisal, a new “two-hit” model is presented to explain obesity-exacerbated periodontal bone loss. This proposed model recognizes a previously unappreciated aspect of myeloid-derived suppressor cell population expansion, differentiation, and activity that can directly participate in periodontal bone loss, providing new mechanistic and translational perspectives.

Introduction

The most recent epidemiological data indicates that the adult obesity rate in the United States stands at 42.4%, the first time the national rate has surpassed the 40% mark, providing further evidence of the obesity crisis (1). This rate has steadily continued to rise since the turn of the century where the rate stood at 30.5% of American adults. Worldwide, obesity has nearly tripled since 1975 (2). Most of the world’s population live in countries where being overweight and obesity kills more people than being underweight. The fundamental cause of obesity and being overweight is an energy imbalance between calories consumed and calories expended. Globally, there has been an increased intake of energy-dense foods that are high in fat and sugar along with a decrease in physical activity due to the increasingly sedentary nature of many forms of work, changing modes of transportation, and increasing urbanization. Changes in dietary and physical activity patterns are often a consequence of environmental and societal changes that can affect special populations (3). People who are considered obese (with a body mass index (BMI) over 30, based upon the person’s weight and height), are at significantly higher risk for many noncommunicable diseases, including cardiovascular diseases, diabetes, musculoskeletal disorders (mainly osteoarthritis), and several cancers (1–4).

Obesity is a major risk factor that can act to enhance periodontal disease progression. However, studies to date have not identified the precise mechanisms whereby these obesity-related factors directly influence periodontal disease pathophysiology. This creates new challenges and therapeutic opportunities in this era of precision medicine, especially in special populations, where many groups present with obesity (5).

The development of obesity and metabolic diseases are complex processes involving genetic and environmental interactions that connect metabolism with the immune system. Chronic low-grade systemic inflammation in response to obesity is a consequence of immune dysregulation that results from the continuous exposure to bacterial lipopolysaccharide and saturated free fatty acids under hyperglycemic conditions. This low-grade circulating inflammation generated by increased circulating cytokines/adipokines may influence mucosal immunity in the oral cavity, and thereby affect oral microbial colonization. Although inflammation is an immunologically proper response to various pathogens and tissue damage, excessive inflammation secondary to obesity and hyperglycemia results in excess production of inflammatory cytokines, as well as several chemokines, and interferons that can ultimately damage several organs/tissues, including alveolar bone. Thus, as a direct or indirect consequence of this excessive immune response, several chronic inflammatory bone-related diseases develop concurrently with obesity, including osteoporosis, arthritis, and periodontal disease (6–8).

Myeloid progenitor cells are the precursors of red blood cells, platelets, granulocytes (polymorphonuclear leukocytes: neutrophils, eosinophils, and basophils), monocyte-macrophage lineage cells, dendritic cells, and mast cells and osteoclasts. Myeloid-derived suppressor cells represent one of these myeloid populations of cells that are known to expand during obesity (9–11). Since this immature cell population is not fully committed towards becoming a macrophage or other myeloid-derived lineages, we propose that these myeloid cell subpopulations can leave the bone marrow and traffic to a site of chronic infection and differentiate into osteoclasts. Thus, myeloid-derived suppressor cells can potentially participate in obesity-associated bone loss in periodontal disease. This conceptual perspective considers mechanisms of the obesity-induced changes with myeloid-derived lineages as it relates to osteoclastogenesis with consideration of how metainflammation can contribute to bone microarchitecture differences and alveolar bone quality, thereby increasing periodontal disease susceptibility.

Metainflammation and the Relationship with Periodontitis

Periodontitis is a common chronic inflammatory disease characterized by destruction of the supporting structures of the teeth, including alveolar bone. Severe periodontitis is highly prevalent and associated with several negative comorbidities that diminish the quality of life (12–14). Although there is a clear relationship between the degree of obesity and periodontal disease incidence, severity, and complications (6, 15–18), early studies focused on aspects of change in immune functioning due to systemic inflammation with simultaneous increased bacterial susceptibility (19, 20). Obesity-associated changes in innate immune responses have largely been attributed to chronic effects of hyperglycemia and free fatty acid elevated cytokines, chemokine expression. Indeed, this dysregulation of the immune system appears to be a crucial component of the pathogenesis of obesity-associated periodontal disease progression (reviewed in (21)).

Western diet has greatly contributed to the surge in development of obesity in the US with concomitant comorbidities, including metabolic diseases, that can influence chronic periodontitis. A diet rich in fats not only causes elevated levels of circulating fatty acids, but it also shifts the bacterial population within the gut to a more Gram-negative state (22). Since lipopolysaccharides are a constitutive part of the cell wall of Gram-negative bacteria, this creates a 2–3-fold increase in circulating lipopolysaccharide (22). The mechanism for the leakage of LPS from inside the gut lumen into epithelial cells or the blood stream is thought to be provoked through leakage via weakened tight junctions between endothelial cells or by chylomicron-facilitated transport (23). Mice fed a high fat diet show a decrease in the expression of proteins associated with the formation of the tight junctions in the gut (24). High fat diets have also been shown to cause an increase in glucose tolerance, increased levels of macrophage infiltration in adipose tissue and markedly higher levels of pro-inflammatory markers (24). Additionally, obesity-associated changes in adipose tissues produce increased release of proinflammatory cytokines and hormones globally referred to as adipocytokines, which induce inflammatory processes and oxidative stress disorders, generating a similar pathophysiologic feature that unites both diseases.

During the past few decades, adipose tissue has become an area of intense investigation. This is in part due to the growing worldwide obesity epidemic, and in part to the identification of leptin as an adipokine secreted from adipose tissues. Adipocytes from fat tissue show the ability to secrete adipokines, which are critical in controlling appetite and body weight. Adipocytes can secrete other cytokines, such as adiponectin and resistin and many other adipokines, including visfatin and omentin, which may have roles in periodontal disease (reviewed in (25). In addition to adipocytes, immune cells, such as macrophages and T lymphocytes, also reside in adipose tissues, and these cells may induce insulin resistance by promoting a proinflammatory milieu within the adipose tissue. Whereas normal adipose tissues have only 5–10% macrophages, obesity creates ~60% macrophage infiltrate (26). As a consequence of this increased macrophage infiltrate, obese individuals typically have elevated levels of interleukin-6, tumor necrosis factor-α, and prostaglandin E2 in circulation. In addition, the heightened production of inflammatory mediators is also facilitated by the adipokine leptin that induces additional tumor necrosis factor-α, interleukin-1β, and interleukin-6, creating a sustained feed-back loop supporting a proinflammatory environment in adipose tissues. This newer appreciation of adipose has prompted the recognition that adipose acts as an endocrine organ. Collectively, these factors all contribute to low-grade metabolic-associated inflammation, collectively referred to as metainflammation.

Periodontal disease is also known to cause low-grade inflammation (27). Although the same mediators, namely tumor necrosis factor-α, interleukin-6, and interleukin −1β, are engaged during both metainflammation and periodontitis, the nature of inflammation the occurs with metainflammation is distinct from classical acute inflammation, since it does not produce the cardinal signs of inflammation, including fever, redness, or pain (28). Metainflammation primarily involves adipose tissue, liver, and pancreas, while infectious inflammation can occur in any tissue (reviewed in (29)). Metainflammation elicits immune cells, such as macrophages, monocytes, dendritic cells, monocytes, T- and B-lymphocytic cells, adipocytes, hepatocytes, and pancreatic beta cells, while periodontitis engages the same immune cells, but distinctly employs epithelial cells, fibroblasts, osteoblasts, and osteoclasts. Thus, a complex interrelationship between obesity and periodontal disease exists where the common thread of metainflammation appears to be a major component contributing towards pathophysiology of both conditions intersecting bone with the immune system.

Obesity and Impact on Bone Quality

The maintenance of bone homeostasis is dependent on the balance of activity of bone-resorbing osteoclasts and bone-forming osteoblasts (30, 31). Abnormal bone resorption by osteoclasts results in bone destruction and is characteristic of bone-related diseases, such as osteoporosis, rheumatoid arthritis, and periodontal disease (32, 33). Osteoclasts are derived from monocyte/macrophage lineage cells. Formation of functional osteoclasts is dependent on macrophage colony-stimulating factor and receptor activator of nuclear factor κB ligand (34–36). macrophage colony-stimulating factor permits abundance and survival of the osteoclasts precursor cells by acting through its receptor c-Fms to activate chiefly specific intracellular protein kinases (37). Receptor activator of nuclear factor κB ligand (RANKL) promotes osteoclast differentiation, referred to as osteoclastogenesis, via its receptor (receptor activator of nuclear factor κB) leading to recruitment of tumor necrosis factor receptor-associated factor-6 and, in turn, activation of multiple downstream targets, including mitogen-activated protein kinases, activator protein-1 and nuclear factor κB (38). In addition, receptor activator of nuclear factor κB-driven osteoclastogenesis is also dependent on the generation of a calcium signal through the activation of the immunoreceptor tyrosine-based activation motifs of DNAX-activation protein 12 and Fc-receptor common γ subunit (38). This enables tumor necrosis factor receptor-associated factor-6-mediated and immunoreceptor tyrosine-based activation motifs-mediated signals to interact cooperatively in transcriptional upregulation of nuclear factor of activated T cells c1, the master osteoclast transcription factor. The key element here is that the osteoclasts are derived from the myeloid cell lineage in the bone marrow.

Obesity produces changes in bone health (39, 40). Although early studies supported a positive correlation between body mass and bone density (41), more recent data supports an inverse relationship between bone quality and obesity (39, 40). Related to oral bone health, the extent and character of alveolar bone changes associated with diet-induced obesity (DIO) has been evaluated using computed tomography. Alveolar bone quality of the maxillary trabecular bone was determined where the region of interest was defined between the maxillary first molar roots (see Figure 1). Since there is no standard way dictated in the maxilla, new methodology was developed to understand how obesity affects oral bone quality. From the analysis, only trabecular bone integrity was altered in diet-induced obesity mice at 32 weeks. These data agree with data from tibia in mice, where high fat diet (HFD) decreased only trabecular bone mass without affecting cortical bone mass (42). These studies are also in agreement with other evidence where the alveolar bone density and microarchitecture are negatively affected during obesity in rats and young growing mice (43, 44). Together, these data support the concept that oral bone integrity is diminished during obesity potentially increasing the risk of periodontal bone loss in a disease state.

Figure 1. HFD reduces alveolar bone quality.

Micro-computed tomography analyses of mouse maxillae were conducted after being fed high-fat diet (HFD) or low-fat diet (LFD) for 8, 16, and 32 weeks. The trabecular bone region of interest (ROI) was defined between the maxillary first molar drawn in the transverse plane, from coronal to apical regions. The cortical maxillae ROI was defined based upon landmarks on the palatal aspect of the maxillary first molar. Trabecular thickness (Tb.Th) was the only parameter that reached significant difference at 32 weeks of HFD. Trabecular number, separation, and area did not show significant differences. Cortical area, thickness and area parameters also did not show significant differences. **P<0.01 by Two-way ANOVA Tukey’s multiple comparisons test (n=4 mice/group/time point).

Many factors, such as estrogens, cytokines, growth and transcription factors, vitamin D, total caloric intake, type of nutrients, alcohol consumption, oxygen tension, and cellular oxidation–reduction pathways can influence bone marrow adipogenesis despite osteoblastogenesis (bone formation) to modulate fat–bone interplay (45). However, the mechanisms by which all these events occur remain unclear, and this molecular control could be crucial to understanding the pathogenesis of both obesity and its impact on bone pathophysiology that occurs in periodontitis. Although it is well established that bacterial constituents, including lipopolysaccharide, can initiate inflammatory alveolar bone loss in the periodontal microenvironment, metabolic stress associated with obesity supports exuberant production of proinflammatory cytokines leading to more severe alveolar bone loss in preclinical models (46–49). There are limited data to understand how obesity alters extrinsic factors that influence obesity-associated oral bone loss and there are no studies to support how cell intrinsic differences contribute towards oral bone loss in obese states. Clearly, additional studies in this area are needed to understand the osteoimmunological aspects of obesity and oral bone during periodontal disease.

Myeloid derived suppressor cells: Evidence for context-dependent activity

Myeloid lineage progenitors generated in the bone marrow classically differentiate into macrophages, dendritic cells, and granulocytes. While myeloid-derived suppressor cells are a distinct lineage of cells and differ from monocytes, granulocytes and dendritic cells, this heterogeneous population is composed of activated myeloid cells with suppressive functions derived from a common myeloid precursor found in the bone marrow of both mice and humans (Figure 2). Although suppressive myeloid cells were described more than 30 years ago, the diverse phenotypes of myeloid-derived suppressor cells and their biological roles have only recently begun to be characterized in detail (10, 50, 51). These cells are defined by having myeloid markers and potent immunosuppressive activity. In mice, there are two relatively distinct subsets of myeloid-derived suppressor cells: monocytic MDSC (M-MDSC = CD11b⁺ LY6G⁻ LY6C^high) and granulocytic myeloid-derived suppressor cells (PMN-MDSC = CD11b⁺ LY6G⁺ LY6C^low) (52). Human myeloid-derived suppressor cells are less easily categorized into monocytic vs. granulocytic because of the lack of a Ly-6G (Gr-1) gene homolog in humans. However, human myeloid-derived suppressor cells have been defined as CD11b⁺ CD33⁺ HLA-DR⁻, with monocytic myeloid-derived suppressor cells being CD14^± CD15^low/− and granulocytic myeloid-derived suppressor cells being CD14⁻ CD15⁺ CD66b⁺, which appears consistent with hematologic morphology (52). See Figure 3 for a summary of the cell surface markers currently being employed to define myeloid-derived suppressor cells populations in humans and mice. These definitions and classifications are somewhat contentious, especially in humans, since there is significant heterogeneity of myeloid-derived suppressor cells populations and the variability in cell surface markers used by different groups to define myeloid-derived suppressor cells, as well as the possibility of overlap between myeloid-derived suppressor cell phenotypes (52, 53). As a consequence, newer recommendations in the field mandate that myeloid-derived suppressor cell populations require not only cell surface markers but also require functional studies, including the inhibition of T cell proliferation and activity, to be a bona fined myeloid-derived suppressor cell (52).

Figure 2. Monocytic- myeloid-derived suppressor cells (M-MDSCs) can differentiate directly into osteoclasts.

(A) Representative images of isolated bone marrow M-MDSC post macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) stimulation stained with tartrate resistant acid phosphatase (TRAP) (B) Osteoclast number (n=10–14 in each group); *** P<0.001, by Two-way ANOVA Tukey’s multiple comparisons test.

Figure 3. Myeloid cell differentiation.

Myeloid cells originate from bone marrow-derived hematopoietic stem cells (HSCs) that differentiate into common myeloid progenitors (CMPs). During normal myelopoiesis, CMPs differentiate into granulocytes including eosinophils, basophils, and neutrophils, as well as monocytes, macrophages, and dendritic cells. MDSCs also differentiate from CMPs and are categorized as M-MDSCs or PMN-MDSCs. HSC, hematopoietic stem cell; CMP, common myeloid progenitor; DC, dendritic cell; macrophage; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells (adapted from (50)).

Obesity has clearly been shown to enhance myeloid-derived suppressor cell expansion (9–11). Myeloid-derived suppressor cells expand and become activated in response to a variety of factors, including some inflammatory cytokines, other pro-inflammatory factors e.g. lipopolysaccharide and prostaglandins, and others (54). Although increased frequency of myeloid-derived suppressor cells has been widely reported in the context of cancer immunology, myeloid-derived suppressor cells have also been shown to contribute to chronic and acute inflammatory processes associated with obesity (9–11) and are recurrently detected in different inflammatory-based pathological disorders. In acute infections, myeloid-derived suppressor cells may have a beneficial role to limit the inflammatory process when the infectious stimuli has been cleared to limit tissue damage (55, 56). In contrast, during chronic inflammation, expansion and activation of myeloid-derived suppressor cells contributes to immunosuppression and oxidative stress.

Differentiation and function of myeloid-derived suppressor cells are influenced by the inflammatory microenvironment generated, signifying disease-specific functions of myeloid-derived suppressor cells as well as cellular plasticity. Several reports support myeloid-derived suppressor cell plasticity as osteoclast progenitors in various pathological conditions associated with bone destruction (57–59). However, only one study addressed myeloid-derived suppressor cells in the context of periodontal disease (58). P. gingivalis was shown to increase myeloid-derived suppressor cell expansion in a chamber model of infection. The same group did show myeloid-derived suppressor cells can differentiate into osteoclasts. However, they did not show this in the context of a periodontal disease model. Thus, it is plausible that M-myeloid-derived suppressor cell expansion and homing to alveolar bone can contribute towards periodontal disease progression through osteoclastogenesis. Indeed, we have shown that myeloid-derived suppressor cells (CD11b⁺ LY6G⁻ LY6C^high) isolated from 12-week-old mice can differentiate directly into osteoclasts in response to osteoclast-specific cytokines (Figure 4). Following cell sorting and functional assays to define M- myeloid-derived suppressor cells (data not shown), bone marrow-derived M-myeloid-derived suppressor cells were plated in vitro with M-CSF and RANKL to induce myeloid-derived suppressor cells cellular differentiation into multinucleated osteoclasts. Quantitative analysis of osteoclast formation using tartrate-resistant acid phosphatase (TRAP) staining indicated that osteoclast number is significantly higher in myeloid-derived suppressor cells isolated from high fat diet fed mice compared to M-MDSCs from low fat diet fed mice. To date, no mechanistic data are available to understand the cell intrinsic differences to understand how obesity drives M-MDSC differentiation into osteoclasts. However, there is some evidence from other studies suggesting that chemokines are in play, but this has only been evaluated in the context of bone metastasis (57). Our group has recently shown that mice deficient in the RNA binding protein, tristetraprolin, where there are significant amounts of tumor necrosis factor-α being expressed, have an increased expansion of myeloid-derived suppressor cell population and increased bone loss in the alveolar bone (60). Given that obesity is linked to periodontitis and elevated levels of myeloid-derived suppressor cells, we postulate that myeloid-derived suppressor cell expansion and metabolic priming in obese individuals may be an important new mechanism to explain how obesity facilitates periodontal disease progression.

Figure 4. Myeloid-derived suppressor cell populations cell surface markers in mice and humans.

Each box contains the markers for the identification of monocytic (M)- and granulocytic (PMN)-myeloid-derived suppressor cell (MDSC) populations. These cells are occasionally detected during health but are elevated when there are infections, inflammation, or cancer. M-MDSC are expanded during obesity (see text for details).

A common mechanistic thread that may explain why myeloid-derived suppressor cells can directly differentiate into osteoclasts is oxidative stress. Studies have indicated that reactive oxygen species, including superoxide and hydrogen peroxide, are crucial components that regulate the differentiation process of osteoclasts (61). Both nitic oxide and reactive oxygen species, are well-known mediators of osteoclast differentiation, nitic oxide can induce osteoclast differentiation of macrophages. An inhibitor of inducible nitric oxide synthase (iNOS) and iNOS knock-out mice show reduced bone loss due to impaired osteoclast function. Stimulation of macrophages via receptor activator of nuclear factor κB ligand (RANKL) transiently increases reactive oxygen species production through TNF receptor-associated factor (TRAF) 6, Rac1 and NADPH oxidase (Nox) 1. Inhibitors that block Nox1 or a deficiency in TRAF6 inhibit response of macrophages to RANKL, thus resulting in reduced osteoclastogenesis (57). In addition, nitric oxide synthesis was required for the generation of myeloid-derived suppressor cell mediated osteoclastogenesis where inhibition of nitric oxide production reduced osteolysis both in vitro and in vivo. Since M-MDSCs are major producers of nitric oxide, M-MDSCs may be the major source of myeloid-derived suppressor cells-generated osteoclasts. Indeed, we have observed many more osteoclasts formed from M-MDSC cultures compared to PMN-MDSC cultures (unpublished data).

While epidemiological data supports a positive association between periodontal diseases and obesity, there is a considerable gap in knowledge regarding the mechanistic link that governs obesity-associated remodeling of alveolar bone. As discussed above, M-MDSCs are progenitors of the monocytes lineage that can give rise to osteoclasts. Studies from cancer bone metastasis indicate enhanced osteoclastogenesis from MDSCs (Gr1⁺CD11b⁺), indicating that they are primed to be osteoclast precursors and that the bone microenvironment in vivo triggers their differentiation into functional osteoclasts (57). Importantly, it was established that only myeloid-derived suppressor cells isolated from bones bearing metastatic tumors and not MDSC derived from non-metastatic tumors underwent osteoclast differentiation. These data imply that for myeloid-derived suppressor cells to differentiate into osteoclasts, signals from both bone marrow cells and bone metastases are needed. Based upon these data, we believe that it is reasonable to conceptualize that signals arising from obese bone marrow will alter the fate of the myeloid-derived suppressor cell populations in the periodontal microenvironment to become an osteoclast.

Current Perspective

We know that MDSC population of cells are expanded in patients as a consequence of obesity-associated inflammation. Although their specific role in periodontitis is difficult to propose given the broad heterogeneity and plasticity of M-MDSCs reported in different pathologies, we postulate that M-MDSCs traffic to the periodontal microenvironment to promote alveolar bone loss. Since obesity is known to enhance M-MDSC expansion, coupled with increased numbers of studies suggesting that M-MDSCs can function as osteoclast progenitors in pathological conditions (57, 58, 62), implies that a similar previously unrecognized mechanism may be operative in obesity-associated chronic periodontitis. Currently, there are no reports of myeloid-derived suppressor cells expansion in periodontitis patients with or without concomitant metabolic disease. Nor are there studies showing the capacity of M-MDSCs from human peripheral blood to degrade bone matrix that could contribute towards periodontal disease progression. We provided the first evidence that suggests when myeloid-derived suppressor cells populations are expanded with simultaneous suppression of T-cell and B-cell populations, there is spontaneous alveolar bone loss (60). In addition, we have shown that M-MDSCs (CD11b⁺Ly6C^hi Ly6G⁻) possess cellular plasticity by virtue of their ability to efficiently form mature osteoclasts (62). In this conceptual perspective, we offer a novel “two-hit” model to explain obesity-exacerbated alveolar bone destruction. We believe that specific myeloid progenitor subpopulations are expanded and reprogrammed during obesity or a high body mass index (first hit) and then traffic to the site of the periodontal infection to become osteoclasts that contribute towards obesity-associated alveolar bone loss in a context dependent manner during periodontal bone loss (second hit; Figure 5). This conceptual model represents a previously unappreciated aspect of M-MDSC biology that maybe operative in obesity-associated periodontal disease progression.

Figure 5: Obesity-associated M-MDSC expansion and osteoclastogenesis model for a mechanistic understanding of obesity-associated periodontal bone loss.

BMI=body mass index. Refer to text for additional details.