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.