The role of hyperglycemia in mechanisms of exacerbated inflammatory responses within the oral cavity

Abstract

Immune modulating factors are necessary for pathogen clearance, but also contribute to host tissues damage, as those seen in periodontal diseases. Many of these responses can be exacerbated by host conditions including type 2 diabetes [T2D], where toll-like receptor 4 [TLR4] and the receptor for advanced glycated end products [RAGE] play a significant role. Here we investigate causality associated with the increase in inflammatory markers observed in periodontally diseased patients with T2D using multi-variant correlation analysis. Inflammation associated with periodontal diseases, characterized by elevated pro-inflammatory cytokines, innate immune receptor expression, and cellular infiltrate was exacerbated in patients with T2D. In addition, a feed forward loop regulated by poor glycemic control was associated with a loss of mucosal barrier integrity and accumulation of innate immune receptor ligands resulting in an exacerbation of ongoing inflammation, where RAGE and TLR4 cooperated to induce responses in oral epithelial cells, which were exacerbated by hyperglycemia.

Figure 1. Inflammation in periodontally diseased tissues is characterized by elevated pro-inflammatory cytokines and innate immune receptor expression

Periodontal tissues, GCF and plasma were collected from 20 periodontally healthy [PH] and 20 periodontally diseased [PD] participants. (A) Tissue mRNA copy number for RAGE and TLR4 was determined. (B) Linear regression and Pearson correlation analysis was performed between tlr4 and rage. (C) Tissue mRNA levels and (D) GCF protein content was determine for IL8, IL1α, and TNFα. (E) Plasma LPS levels were determined. (F) Tissue mRNA levels of MPO (neut), CD14 (mac), CD3 (T cell), and CD19 (B cell) were determined and the fold change over PH calculated. *P value <0.05 PH vs. PD one-way ANOVA with Bonferroni’s multiple comparisons correction. ^P value <0.0001 PH vs. PD Student’s t test.

Figure 2. Increased inflammatory markers are present in periodontally diseased tissues of patients with Type 2 Diabetes

Periodontal tissues, GCF and plasma were collected from 20 periodontally diseased [PD] and 20 periodontally diseased participants with T2D [PDD]. (A) Tissue mRNA copy number for RAGE and TLR4 was determined. (B) Tissue mRNA levels of IL8, IL1α, and TNFα were determined and the fold change over PD calculated. (C) GCF protein content was determine for IL8, IL1α, and TNFα. (D) Tissue mRNA levels of MPO (neut), CD14 (mac), CD3 (T cell), and CD19 (B cell) were determined and the fold change over PD calculated. Plasma (E) CML and (F) LPS levels were determined. *P value <0.05 PDD vs. PD or PH one-way ANOVA with Bonferroni’s multiple comparisons correction. ^P value <0.0001 PH vs. PD Student’s t test.

Figure 3. Responsiveness of primary oral epithelial cells to LPS, CML and glucose stimulation

1×10⁶ primary epithelial cells [HOK] from periodontally healthy participants were left unstimulated or were stimulated for 24hrs with 1ng, 10ng, 100ng or 1000ng of either (A, C) ultra-pure TLR4 agonists [E. coli LPS], (B, C) the advanced glycated end product [AGE] N-carboxyl (methyl-lysine) [CML] or (D) 5mM (100mg/dl), 10mM, (150mg/dl), 12.5mM (200mg/dl), 15mM (300mg/dl), or 20mM (250m/dl) of D-glucose. After which the (A, B) supernatants were used to quantify IL8, IL1α, and TNFα secretion and (C, D) cell viability evaluated. All assays were performed on 5 separate donors.

Figure 4. Cooperation of RAGE and TLR4 in the innate immune responses of oral epithelial cells is exacerbated by hyperglycemia

1×10⁶ primary epithelial cells [HOK] were stimulated for 24hrs with the indicated ligands in the (A–B, E–G) absence or (C–D, H–J) presence of 12.5mM glucose pre-treatment after which qPCR was used to quantify the copy number of (A, C) rage and (B, D) tlr4 and ELISA was used to quantify (E, H) IL8, (F, I) IL1α, and (G, J) TNFα in the supernatants. *P value <0.05 unstim vs. LPS, CML and CML+LPS. ^P value <0.05 stimulations in the absence of neutralization vs. the presence of neutralizations. ^† P value <0.01 stimulations in 5mM glucose vs. 12.5mM glucose. one-way ANOVA with Bonferroni’s multiple comparisons correction.