Altered microRNA expression correlates with reduced TLR2/4-dependent periodontal inflammation and bone resorption...

Periodontitis (PD) is a polymicrobial dysbiotic immuno-inflammatory disease. Toll-like receptors (TLRs) are present on gingival epithelial cells and recognize pathogen-associated molecular patterns on pathogenic bacteria, inducing the secretion of proinflammatory cytokines and initiating innate and adaptive antigen-specific immune responses to eradicate the invading microbes. Since PD is a chronic inflammatory disease, TLR2/TLR4 play a vital role in disease pathogenesis and in maintaining the periodontium during health. Many factors modulate the TLR-mediated signaling pathway, including specific microRNAs (miRNAs). The present study was designed to characterize the function of TLR2/4 signaling in the miRNA profile after polybacterial infection with Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in C57BL6/J wild-type, global knockout strains of TLR2^-/-, and TLR4^-/- mice (n = 16/group) using RT-qPCR. The selection of 15 dominant miRNAs for RT-qPCR analysis was based on prior NanoString global miRNA expression profiling in response to polymicrobial and monobacterial infection. Polybacterial infections established gingival colonization in wild-type, TLR2^-/-, and TLR4^-/- mice with the induction of bacterial-specific IgG. A significant reduction in alveolar bone resorption and gingival inflammation was observed in the mandibles of TLR2/4^-/- mice compared to C57BL6/J wild-type mice (P < 0.0001). Periodontal bacteria disseminated from gingival tissue to multiple organs in wild-type and TLR2^-/- mice (heart, lungs, brain, and kidneys) and were limited to the heart (F. nucleatum), lungs (P. gingivalis), and kidneys (T. forsythia) in TLR4^-/- mice. The diagnostic potential of miRNAs was assessed by receiver operating characteristic curves. Among the 15 miRNAs, three were upregulated in C57BL6/J wild-type mice, two in TLR2^-/-, and seven in TLR4^-/- mice. Notably, the anti-inflammatory miR-146a-5p was consistently upregulated in wild-type and TLR2^-/- mice but not in TLR4^-/- mice. Additionally, miR-15a-5p was upregulated in wild-type and TLR2^-/- mice. let-7c-5p was upregulated in TLR4^-/- mice and downregulated in wild-type mice. The upregulated miRNAs (miR-146a, miR-15a-5p, and let-7c-5p) and downregulated miRNAs could be candidate biomarkers for oral bacteria and TLRs-mediated induction of periodontitis if they are confirmed in large-scale human studies. Multi-species oral bacterial infection alters the TLR2/4 signaling pathways by modulating the expression of several potential biomarker miRNAs in the periodontium.

Periodontitis is the most prevalent chronic immuno-infectious multispecies dysbiotic disease of the oral cavity. The Toll-like receptors (TLRs) provide the first line of defense, one of the best-characterized pathogen detection systems, and play a vital role in recognizing multiple microbial products. Multispecies infection with periodontal bacteria Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia induced gingival inflammation, alveolar bone resorption (ABR), and microRNA (miRNA) expression in the C57BL6/J wild-type mice, whereas infection did not significantly increase ABR in the TLR2/4 deficient mice. Among the 15 miRNAs investigated, miR-146a-5p and miR-15a-5p were upregulated in wild-type and TLR2^-/- mice, and miR-146a-5p, miR-30c-5p, and let-7c-5p were upregulated in the TLR4^-/- mice compared to sham-infected controls. Notably, the inflammatory miRNA miR-146a-5p was uniquely upregulated among the wild-type and TLR2^-/- mice but not in the TLR4^-/- mice. The upregulated miRNAs (miR-146a, miR-15-a-5p, let-7c-5p) and downregulated miRNAs could be candidate biomarkers for oral bacteria and TLRs-mediated induction of periodontitis if they are confirmed in large-scale human studies.