Keywords: Dendritic cells, periodontitis, diabetes mellitus, hyperglycemia, immunometabolism
Abstract :
Introduction: Periodontitis in patients with diabetes mellitus results in chronic inflammation, which is the central issue in developing an efficient and consistent treatment plan. Dendritic cells (DC) are antigen presenting cells that initiate the immune inflammatory responses and contribute to the pathogenesis of both diseases. In this study, we investigated the impact of hyperglycemic microenvironment on DC immunometabolism, the cell phenotypes and immunogenic functions.
Methodology: Human monocyte differentiated DC and mice bone marrow derived DC were cultured in the presence of 5.5-, 11-, and 25- mM glucose to simulate diabetic microenvironment. Cells were activated with advanced-glycation-end product (AGE) and lipopolysaccharides (LPS) fromPorphyromanas gingivalis for 24 hours and processed for transcription, metabolic and microscopic analysis. Expression of activation markers (CD80, CD83, CD86, HLA-DR) and proteins involved in glycolysis (HK2, LDHA, GLUT1) in DC were calculated by qRT-PCR. Lactic acid production and OXPHOS assays, including Seahorse metabolic flux analyzer were utilized to determine the effects on metabolism. Impact on the phagocytic capacity was analyzed using fluorescent microspheres uptake. Cytokine expressions for tumor necrosis factor alpha [TNF-\(\alpha\)], interleukin [IL]-1\(\beta\), IL-6, IL-10, and Interferon gamma [IFN-\(\gamma\)] were evaluated in cell supernatants from DC and DC-T cell coculture.
Results: Under simulated hyperglycemic microenvironment an increase in cell dendrite extensions, and activation markers were upregulated in both monocytes differentiated DC and BMDC. There was a significant increase in glycolysis as evident from the gene expression, cell metabolic flux, and lactic acid production. Cell OXPHOS activities was reduced to compensate for the increase in glycolysis. Pro-inflammatory cytokines (TNF-\(\alpha\) and IL-1\(\beta\)) were significantly increased and this increase was directly proportional to the glucose concentrations. Whereas, phagocytic capability of DC, and their ability to activate T cells decreased with hyperglycemia.
Conclusions: Hyperglycemic microenvironment resulted in DC changes with increased expressions of activation markers, glycolytic metabolism, and increased pro-inflammatory cytokines, while impairing phagocytosis and adaptive immunity induction. BMDC and human monocyte differentiated-DC exhibit similar responses toward hyperglycemia, AGE, and LPS. This work emphasizes that diabetes mellitus has an inflammatory impact on DC immunometabolism and immunogenic functions.
Introduction :
Periodontitis and diabetes mellitus (DM) are both chronic diseases with a high prevalence and have a strong inter-relationship (1). Periodontitis affects approximately 25 – 50% of the adult population worldwide, and is considered as the main cause of tooth loss (2). On the other hand, DM is a multifactorial metabolic disease affecting at least 422 million people globally (3). DM is characterized by hyperglycemia due to impairment in either insulin secretion or its action, or both (4). Previous clinical studies have reported that DM is a major risk factor for periodontitis and its treatment outcome (2). DM can increase the severity/prevalence of periodontitis by three folds (1). Interestingly, there is a significant likelihood for periodontitis patients to have uncontrolled DM. So it is not surprising that a strict glycemic control and treating periodontitis can improve the treatment outcome of periodontitis and DM (1,5).
Chronic inflammation and impaired immune cell responses are the key linkers between the two diseases (6). Pro-inflammatory cytokines such as TNF-\(\alpha\), IL-1\(\beta\) and IL-6, and inflammatory molecules such as advanced glycation end products (AGE), adipokines, RANKL, and reactive oxygen species (ROS) generated by hyperactivated immune cells in each disease condition could exacerbate the other and react with the immune cells to damage their respective immunogenic responses. If this persists for long, it could result in a negative feedback on local tissue inflammation and bone resorption in patients with periodontitis and DM (6–8).
Recent studies have emphasized that metabolic programming of immune cells tightly controls their expression, differentiation, and functions (9–11). A Warburg effect is commonly adopted by both innate and adaptive immune cells to fulfill their respective immunogenic functions, where glycolysis is upregulated ; and this dysregulation could directly promote pathogenesis of both periodontitis and DM (12). Innate immune cells such as neutrophils use glycolysis for adhesion and microbicidal activities, but systemic glucose or infectious microenvironment could reduce this metabolic reprogramming, thereby promote bacterial infections and insulin resistance (13,14). It is possible to determine if adaptive T and B cells will transition to glycolysis or remains on mitochondrial metabolism based on their effector or memory subsets. In DM and periodontitis, the metabolism of T and B cells were altered with attenuated effector functions via mTORC1 and AMPK related pathway (10,15,16). Such metabolic reprograming from mitochondrial respiration to glycolysis is also required for the activation and function of dendritic cell (DC), the most efficient antigen presenting cells (APC) that connect the innate and adaptive immunity (17–19).
Pathogen associated molecular patterns (PAMPs), or danger associated molecular patterns (DAMPs) signals initiates phenotypic and functional changes in DCs upon interacting with its surface receptors. DCs upregulate surface co-stimulatory activation markers upon phagocytosis of the antigens, which upregulates cytokine secretions, and migrations to lymph node for T cell activations (20). For oral immune homeostasis, DCs play a crucial role as their absence or dysfunction directly promotes periodontal inflammation via uncontrolled bacterial infection (21,22). Recent studies have also emphasized on the essential role of DC in DM autoimmune responses and suggested its potential to be good a therapeutic target in DM (23,24). There is enough evidence to point at DCs in the pathogenesis of periodontitis complicated with DM. Conflicting DC phenotypes and functions were reported in these two diseases, with a combination of upregulated and suppressed activation markers CD80 and CD86 and an overall upregulated pro-inflammatory cytokine profile. However, the specific effect on DC immunometabolism in response to periodontal pathogen complicated by diabetic microenvironment is vague. To date, there are no reports on how the underlying immunometabolism may govern these dysregulated phenotypes and immunogenic functions of DC in periodontitis and DM.
In this study we investigated the potential alterations of DC immunometabolism induced by periodontal bacterial LPS, complicated with hyperglycemia. DCs were exposed to an in vitro microenvironment simulating periodontal infections and diabetic hyperglycemia by using LPS from oral bacteria Porphyromonas gingivalis (P. gingivalis ), different glucose concentrations, and AGE. We hypothesized that these stressors could dysregulate the DC immunometabolism, specifically the glycolysis pathway and its downstream mitochondrial respiration, therefore impairing the cell phenotypes and functions.