Altered microRNA Expression Correlates with Reduced TLR2/4-Dependent Periodontal Inflammation and Bone Resorption Induced by Polymicrobial Infection.

Unlabelled: 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 (PAMPs) on pathogenic bacteria, induce the secretion of proinflammatory cytokines, and initiate innate and adaptive antigen-specific immune responses to eradicate the invading microbes. Since PD is a chronic inflammatory disease, TLR2/TLR4 plays a vital role in disease pathogenesis and maintaining the periodontium during health. Many factors modulate the TLR-mediated signaling pathway, including specific miRNAs. The present study was designed to characterize the function of TLR2/4 signaling to the miRNA profile after polybacterial infection with and in C57BL6/J wild-type, 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 induction of bacterial-specific IgG. A significant reduction in alveolar bone resorption (ABR) and gingival inflammation was observed in the mandibles of TLR2/4 mice compared to C57BL6/J wild-type mice ( <0.0001). Periodontal bacteria disseminated from gingival tissue to the multiple organs in wild-type and TLR2 mice (heart, lungs, brain, kidney) and limited to heart ( ), lungs ( ), kidney ( ) in TLR4 mice. The diagnostic potential of miRNAs was assessed by receiver operating characteristic (ROC) curves. Among 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 all the mice. Additionally, miR-15a-5p was upregulated in wild-type and TLR2 mice. let-7c-5p was upregulated in TLR4 mice and downregulated in the wild-type mice. Multi-species oral bacterial infection alters the TLR2/4 signaling pathways by modulating the expression of several potential biomarker miRNAs in periodontium.

Importance: Periodontitis is the most prevalent chronic immuno-infectious multispecies dysbiotic disease of the oral cavity. The Toll-like receptors (TLR) provide the first line of defense, one of the best-characterized pathogens-detection systems and play a vital role in recognizing multiple microbial products. Multispecies infection with periodontal bacteria and induced gingival inflammation, alveolar bone resorption (ABR) and miRNA expression in the C57BL6/J wild-type mice and whereas infection did not increase significant ABR in the TLR2/4 deficient mice. Among the 15 miRNAs investigated, miR-146a 5p, miR-15a-5p were upregulated in wild-type and TLR2 mice and miR-146a-5p, miR-30c-5p, let-7c-5p were upregulated in the TLR4 mice compared to sham-infected controls. Notably, inflammatory miRNA miR-146a-5p was upregulated uniquely among the three different infection groups. The upregulated miRNAs (miR-146a, miR-15-a-5p, let-7c-5p) and downregulated miRNAs could be markers for TLRs-mediated induction of periodontitis.