MicroRNAs (miRNAs) are small, non-coding RNAs of ~18-25 nucleotides that have gained extensive attention as critical regulators in complex gene networks including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly downregulate the expression of factors of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment. In this study, we examined the expression profiles of three previously identified viral miRNAs (v-miRs) from the human herpesvirus (HHV) family, HSV-1 (miR-H1), KSHV (miR-K12-3-3p), and HCMV (miR-US4) in healthy and diseased periodontal tissues and observed increased levels of v-miRs in diseased tissues. To understand the significance of this increase, we overexpressed v-miRs in human oral keratinocytes (HOK), a common target for various HHV, and analyzed the impact of miR-H1 and miR-K12-3-3p on the host transcriptome. More than 1300 genes were altered in HOK overexpressing miR-H1 and miR-K12-3-3p. Global pathway analysis of deregulated genes identified several key cellular pathways that may favor viral persistence. Using bioinformatic analysis, we predicted hundreds of potential v-miR binding sites on genes downregulated by miR-H1 and miR-K12-3-3p and validated three novel target v-miR sites suggesting widespread direct and indirect modulation of numerous host genes/pathways by a single v-miR. Finally, in vitro HSV-1 infection assays showed that miR-H1 can regulate viral entry and infection in human oral keratinocytes (HOK). Overall, our results demonstrate clinical and functional relevance of pathogenic viral molecules viz., v-miRs that regulate both host and viral functions and may contribute to the pathogenesis of inflammatory oral diseases.

Aim: To evaluate human and herpesvirus-encoded microRNA (miRNA) expression in healthy and diseased gingiva of obese and non-obese subjects and compare the impact of localized and systemic inflammation on human miRNA profiles. Material and methods: Healthy and inflamed gingival biopsies were collected from obese and non-obese subjects. Human and herpesvirus miRNA expression was quantified using quantitative PCR. Predicted targets of dysregulated miRNAs were identified using bioinformatics analysis, validated by dual luciferase assays and their expression assessed in healthy and diseased tissues. Results: Our results show differential expression of miRNAs in both diseased groups compared to healthy counterparts. MMP-16 is identified as a novel target of miRNAs altered in disease. Expression analysis of genes predicted as target of differentially expressed miRNAs show significant changes in disease compared with healthy tissues. Finally, quantitation of four herpesvirus derived viral miRNAs show that expression and prevalence of herpesvirus miRNAs in diseased gingiva of obese subjects. Conclusion: Our findings show that miRNA (both cellular and virus) expression are differentially responsive to local and systemic inflammation. Some of these miRNAs can modulate key cellular genes with direct consequences on inflammatory pathways suggesting their impact on oral tissue transcriptome and functions.

Aim: To evaluate the potential role of miR-26 family members in periodontal pathogenesis by assessing innate immune responses to periopathic bacteria and regulation of cytoskeletal organization.Materials and Methods: Expression of miR-26a-5p and miR-26b-5p was quantified in gingival biopsies derived from healthy and periodontally diseased subjects before and after non-surgical (scaling and root planing) therapy by RT-qPCR. Global pathway analysis and luciferase assays were performed for target identification and validation.Cytokine expression was assessed in miR-26a-5p transfected human oral keratinocytes upon stimulation with either live Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans or Pg lipopolysaccharide (LPS). Wound closure assays were performed in cells transfected with miR-26a-5p, while the impact on cytoskeletal organization was assessed by F-actin staining.Results: miR-26a-5p and miR-26b-5p were downregulated in diseased gingiva and restored 4-6 weeks post-therapy to levels comparable with healthy subjects. Target validation assays identified phospholipase C beta 1 as a bona fide novel target exhibiting antagonistic expression pattern in disease and post-therapy cohorts. miR-26a-5p transfected cells secreted higher levels of cytokine/chemokines upon stimulation with periopathogens and demonstrated impaired cell migration and cytoskeletal rearrangement.Conclusions: Downregulated miR-26a-5p levels in periodontal inflammation may interfere with key cellular functions that may have significant implications for host defence and wound healing.

MicroRNA (miR) play a critical role in the pathogenesis of immune-mediated diseases. We hypothesize that periopathogens (P. gingivalis [Pg] and A. actinomycemtemcomitans [Aa]) impair host immune response by perturbing miR expression. Periopathogen burden, cytokine and miR expression was quantified by qPCR in healthy and inflamed human gingival biopsies. Human primary CD14+ monocytes were differentiated to M1 or M2 macrophages (MΦ) and challenged with live Pg or Aa to monitor expression profiles of pro- and anti-inflammatory miRs by qPCR. Supernatant levels of cytokines were assessed by multiplex bead assay while, protein levels were quantified by western blot. Murine gingival miRNA and mRNA profiles were validated during disease progression in a liguture-induced periodontitis model and bone loss measured by microCT. Higher periopathogen burden and inflammatory cytokine expression was observed in inflamed gingiva with a higher expression of pro-inflammatory miR-155 and reduced levels of anti-inflammatory miR-142-3p. This corroborates with NFκB activation and downregulation of PU.1 suggesting a distinct role of a miR and transcription factor (TF) axis. miR-155-mediated downregulation of PU.1, a TF of miR-142, suppressed transcription of miR-142, thereby alleviating silencing of IL-6 mediated by miR-142-3p. Our murine ligature model showed progressive bone loss in the presence of Pg and higher expression of cytokine profiles. Local delivery of miR-142-3p showed reduction in IL-6. Periopathogen-mediated activation of miR-155/NFκB axis augments inflammatory signaling by decreasing levels of PU.1 and expression of its target miR-142. This mechanism may be critical in the pathogenesis of periodontal disease. Supported by R01 DE027980, R21 DE026259, R03 DE027147