Discussion
In this study, we have demonstrated that the stimulation of AGE, hyperglycemia, and LPS from oral bacterial P. gingivalis can synergistically dysregulate the DC phenotype, immunometabolism, and effector functions. Hyperglycemia and infection-activated immune cells together induce inflammation, which is the key to connect periodontitis and DM, two chronic inflammatory diseases with a bidirectional relationship to each other. The Warburg effect-like transition from mitochondrial oxidative phosphorylation to aerobic glycolysis as the main pathway for ATP generation is one critical step for DC activation upon interaction between antigen and TLR (26). Interaction between LPS and surface TLR on DCs could activate the glycolysis transition via Akt-induced signaling, initiated by TLR-associated TBK1 and IKK\(\varepsilon\) proteins, thus the Akt downstream protein mTORC1 is also determined to be involve in the glucose-sensitive transduction circuit for glycolysis metabolism transition (27,28). PFKFB3, an major driver of glycolysis pathway, is identified as a direct target mTOR under LPS stimulation (29). Our data showed a significant increase in gene expression of enzymes such as HK2, LDHA, and GLUT that catalyzes different steps of glycolysis. In the presence of LPS, levels of L-lactate, which is the final product of glycolysis, increased in DC. The induction of glycolytic activity by LPS has been well reported in other immune cells such as macrophages, monocytes, and neutrophils (30–32). LPS can attenuate mitochondrial OXPHOS activity. In macrophages LPS suppresses isocitrate dehydrogenase and succinate dehydrogenase in the citric acid cycle, and blocks the mitochondrial respiration while promoting glycolysis (33,34). Excessive citrate and succinate, both upregulate inflammatory molecules such as NO and IL-1\(\beta\) (33). On the other hand, cells under hyperglycemia shunt glycolytic intermediates into pentose phosphate pathway as a protective mechanism, which limits the glycolytic activity (35,36). As macrophages and DC are closely inter-related in pro- and anti-inflammatory phenotypes, similar pathways could be involved in DC. Moreover, AGE can also reduce glycolysis and mitochondrial respiration through HIF-1\(\alpha\)/PDK4 pathway, thereby reducing ECAR and OCR (37,38). This may explain the observed inhibition of glycolysis pathway in the LPS and AGE-stimulated group under hyperglycemia in the cell metabolic efflux assay. An inhibitory effect of hyperglycemia and AGE on glycolysis could reduce the stimulating effect from LPS. Under diabetic microenvironment, glycolysis is not only disrupted by hyperglycemia, AGE, and LPS, rather other DM-associated metabolites such as ROS, free fatty acid, and cholesterol could also impact cell immunometabolism transitions by regulating different signaling pathways (31).
The relative expression of DC surface markers can reflect the cell activation status. Upregulation of protein CD80, CD86, and MHC-II complex is a key evidence of DC activation. Here we have shown that depending on glucose concentration these three co-stimulatory molecules can polarize the cells toward a hyperactivated status in both inactivated and activated DC. Similar results were reported in DC obtained from various lineages under in vitro and in vivostimulations (27,39,40). Thomas et al demonstrated that brief exposure of hyperglycemic culture could significantly elevate co-stimulatory surface markers in all immature, mature, and tolerogenic DC (39). BMDC retrieved from streptozotocin induced-diabetic mice showed comparable phenotypes as well (27,39). Such increase in CD80, CD86, CD1\(\alpha\), and MHC-II was also observed when comparing the hyperglycemia treated-human monocyte derived DC to the normoglycemia cell culture (40). Glucose and LPS activated-Akt pathway that initiate glycolysis transition may also upregulate the expression of these DC surface markers. The transcription factor NF-\(\kappa\)B directly regulates the receptor genes that are involved in the cell immunogenic functions (41,42). Note that the HIF-1\(\alpha\) activated by Akt pathway during DC metabolic reprogramming is also a transcription factor that regulate CD80 and CD86 expression (43,44).
The metabolic reprogramming in innate immune cells is a TLR induced event paired with phagocytosis, which is a critical for rapid infection clearance by APCs. Our data demonstrated that hyperglycemia significantly impairs the phagocytosis by immature DC in a glucose-dose-dependent manner. Similar results have been reported in DC differentiated from human PBMC obtained from T2DM patients, where cells were unable to take up the FITC-dextran fluorescein, or this uptake was significantly reduced in hyperglycemic media (45). Similarly, in other phagocytes such as macrophages and monocytes the phagocytotic abilities were reduced in both in vitro and in vivo hyperglycemic environment. High glucose locks the cell in their pro-inflammatory phenotype that is characterized by dysregulated activation markers and upregulated pro-inflammatory cytokines (46–48). Similar results were seen in the present study where hyperglycemia induced a pro-inflammatory DC with compromised phagocytic effect. Reduced phagocytosis in these innate immune cells collectively under hyperglycemia can completely impair the infection clearance; thus leading to the chronic immune-inflammatory responses seen in periodontitis and DM patients (6). Such lowered phagocytotic capability could also result from hyperglycemia induced altered cell surface receptors. Under hyperglycemia and hypoxia conditions, in the expression of genes encoding CD36 and SCARB-1 in human monocyte-differentiated macrophages is reduced. CD36 and SCARB-1 are proteins responsible for phagocytosis (47). Expression of FC\(\gamma\) receptor CD32 and CD64 that mediate phagocytosis with antigen recognition receptor CR3 in monocytes were also reported to be reduced with increasing HbA1c level and body mass index in DM patients (48). Interestingly, no significant differences were observed in phagocytosis protein CD36 on DC from DM patient but MFGE8, another phagocytosis protein was significantly reduced (49). MFGE8 is frequently associated with impaired phagocytosis of various phagocytes generally and under DM conditions (50–52). However, there is limited knowledge to associate the reduced MFGE8 levels with the aerobic glycolysis transition upon DC activation. Either way, correcting the expression levels of this protein can result in profound wound healing potential by promoting DC and macrophages into immune suppressive phenotypes, which favors mitochondrial respiration rather than glycolysis (53,54). The antigen presentation ability beyond phagocytosis mechanism could also be impacted by hyperglycemia. High glucose evidently delays antigen processing and impairs antigen presentation in human monocyte-derived macrophages via repressed Rab and Cathepsin, leading to reduced T cell activation (55). Defect in antigen presentation has been reported in splenic macrophages from NOD mice (56).
Exaggerated cytokine secretion is a characterized functional change upon DC activation. Significant levels of pro-inflammatory cytokine, TNF-\(\alpha\) and IL-1\(\beta\) from the activated DC indicates that hyperglycemia, AGE, and LPS could together stress DC to exert a more immunogenic state. Similar cytokine profiles were reported in other studies from human and mice DC as each of the three stressors are known to be pro-inflammatory (40,57,58). Besides their role in interconnecting the chronic inflammatory states in periodontitis and DM, such increase in these pro-cytokines may help in developing other autoimmunity in innate immune cells as well. Recent studies have shown that TNF-\(\alpha\) could stimulate the synovial fibroblast cells to increase the glycolysis related-proton efflux and promote rheumatoid arthritis, which is another risk factors associated with both periodontitis and DM (59). Similarly, in autoimmune disease Sjögren syndrome as well the inflammatory response between TNF-\(\alpha\) and DC via TLR-7 and TLR-9 has been confirmed (60). IL-1\(\beta\) can induce autocrine responses during bacterial infection and reprogram the immunometabolism through glycolysis (61). If this immunometabolism transition to aerobic glycolysis can play a role in the trained immunity of DC under hyperglycemic stress remains unclear, yet it is an important question worth investigating.
DC have a unique capacity to induce activation and proliferation of antigen specific T cell responses as its APC function in adaptive immunity. The presence of co-stimulatory molecules CD80, CD86, and MHC-II complex play an important role in T-cell activation; wherein, signals from DC synergistically interact with both the T cell receptor and CD28 on T cells for complete activation (62). On the other hand, lack of any one of the co-stimulatory signals can result in immune tolerance or ignorance of activation to prevent aberrant response of T cells. Increase in the expression of DC co-stimulatory markers should lead to hyperactivation of the T cell lymphocytes (63); however, contradictory results were observed in our study. Although DC stimulated with hyperglycemia, AGE, and LPS had high expression of co-stimulatory markers , which increases with increase in glucose levels. T cells cocultured with DC in higher glucose showed attenuated proliferation and Th-1 type cytokine secretions, indicating a lowered T cell priming capability. This was expected as the DC cultured in high glucose concentration were more activated and matured while the phagocytosis ability of immature DC is only optimal (64). Samuel et al had also indicated that Th1 is only secondary predominant while Th17 is the predominant pathway in DM mice (65). Such impairment in the DC function could also be due to compromised phagocytosis and antigen presentation capability in hyperglycemia. Antigen presentation on DC is required for the effective physical interaction and T cell activation in vivo , whereas unpulsed DC fail to induce lymphocyte responses as shown by Ingulli et al (66). Also, a minimum threshold antigen dose is required for a stable DC-T cell priming (67). With attenuated phagocytosis ability and therefore the lowered antigen presentation on DC in hyperglycemia, the T cell activation is weak, and this may lead to a prolonged inflammation in DM and periodontitis. However, the antigen presentation using LPS model is a non-specific or bystander response from T cells, as presence of LPS could induce inflammatory responses from a wide array of cells. Extension of the adaptive immune responses using OT-II mice may could help in further understand such compromised antigen presentation from DC by pulsing DC and T cells co-culture with specific antigens like ovalbumin (67).
Periodontitis is a chronic disease caused by a multispecies bacterial biofilm, and DM is a systemic autoimmune disease that involves numerous inflammatory molecules and immune cells. Therefore, the simple use of bacterial LPS and hyperglycemia may not comprehensively mimic the complicated disease microenvironment. In vivo studies utilizing diabetic mice with induced periodontitis could provide better understanding of the diseased state and immune responses. While we performed the transcription analysis of DC surface markers in this study, it is possible that the hyperglycemia could affect the cell translation mechanism. Furthermore, the intracellular localization of the phagocytosed fluorescent microspheres would be an enticing next step to understand the DC phagocytosis mechanism.
In conclusion, this study highlights that hyperglycemia with AGE and LPS can polarize DC toward a more pro-inflammatory phenotype and cytokine profile by upregulating glycolysis, but its phagocytosis and T cell priming is compromised. Together, this DC function dysregulation may explain the conflicting adaptive immune cell profiles observed in DM and periodontitis.