Altered microRNA Expression Correlates with Reduced TLR2/4-Dependent Periodontal Inflammation and Bone Resorption Induced by Polymicrobial Infection
Authors: Jeepipalli Syam , Gurusamy Parvathi , Luz Martins Ana Rafaela , Colella Eduardo , Nadakuditi Sandhya R , Desaraju Tushar , Yada Ashitha , Onime Jennifer , William John , Bhattacharyya Indraneel , Chan Edward K. L. , Kesavalu L. 
Published: January 2025

ABSTRACTPeriodontitis (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 withStreptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola,andTannerella forsythiain 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 (p<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 (F. nucleatum), lungs (P. gingivalis), kidney (T. forsythia) 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.IMPORTANCEPeriodontitis 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 bacteriaS. gordonii, F. nucleatum, P. gingivalis, T. denticola,andT. forsythiainduced 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.