Epithelial TLR4 Levels Correlate With Cytokine and Antimicrobial Peptide Production

Objectives: Experimental gingivitis studies indicate variation in response to microbial plaque accumulation. The innate immune system and in particular Toll-like receptor (TLR) -4 is one of the crucial pattern recognition receptors involved in the host response to Gram negative bacterial infection, relevant to the human gingival epithelial response to microbial plaque. Pro-inflammatory cytokines and antimicrobial molecules (such as beta defensin -2) are produced in response to P. gingivalis challenge of human gingival epithelial cells (HGECs). Methods: 6 primary human gingival epithelial cell cultures (HGECs), three with normal expression of TLR4 and three with significantly reduced TLR4 levels were established from gingival tissue biopsies derived from young patients undergoing surgical removal of third molars. HGECs at the 4th passage were cultured until confluent and then exposed to heat-killed P. gingivalis. Cells from these challenge assays were processed to produce mRNA for qPCR array analysis and supernatants were assayed by Luminex-100 (Upstate) for the pro-inflammatory cytokines, TNFa, GM-CSF, IL-6 and IL-8. Results: There were remarkably consistent differences whereby HGECs with normal TLR4 levels, both at the mRNA and protein level, exhibited strong pro-inflammatory cytokine production and low anti-microbial peptide production (for example human beta defensin -2) whereas low TLR4 expressing cells gave the opposite result, with low pro-inflammatory cytokine production and high AMP levels (as much as fifty fold increases). Conclusions: There appears to be either compensation or mutual antagonism between the pro-inflammatory cytokine response and the antimicrobial peptide production of human gingival epithelial cells and this relates strongly to TLR4 levels. Supported by grants HRSA-C76HF01199-01, CDC-PA04189, CDC H7S/CCU424835-01.