Human and herpesvirus microRNAs in periodontal disease

Abstract

Periodontitis is a multi-etiologic infection characterized clinically by pathologic loss of the periodontal ligament and alveolar bone. Herpesviruses and specific bacterial species are major periodontal pathogens that cooperate synergistically in producing severe periodontitis. Cellular immunity against herpesviruses and humoral immunity against bacteria are key periodontal host defenses. Genetic, epigenetic, and environmental factors are modifiers of periodontal disease severity. MicroRNAs are a class of noncoding, gene expression-based, posttranscriptional regulatory RNAs of great importance for maintaining tissue homeostasis. Aberrant expression of microRNAs has been associated with several medical diseases. Periodontal tissue cells and herpesviruses elaborate several microRNAs that are of current research interest. This review attempts to conceptualize the role of periodontal microRNAs in the pathogenesis of periodontitis. The diagnostic potential of salivary microRNAs is also addressed. Employment of microRNA technology in periodontics represents an interesting new preventive and therapeutic possibility.

Keywords: herpesviruses; inflammation; microRNAs; periodontal disease; post-transcriptional regulation; saliva.

Figure 1:. Model of host and viral-encoded miRNA biogenesis.

Human primary miRNAs (pri-miRNA) are generally transcribed RNA polymerase II (Pol II), while viral miRNAs are transcribed by RNA pol II and in some instances RNA pol III or tRNase Z. Subsequent to pri-miRNAs, both human and viral miRNA biogenesis is largely similar. Pri-miRNA are processed to precursor- miRNAs (pre-miRNA) in the nucleus by the “microprocessor complex” composed of Drosha and DGCR8. Pre-miRNA are exported to cytoplasm by Exportin 5 and Ran-GTPase complex. In the cytoplasm, pre-miRNAs are processed to duplex miRNA by RNA endonuclease Dicer and its co-factor TRBP. Dicer and TRBP relay the short dsRNA to an Argonaute homolog (Ago1-4 isoforms in mammals) and GW182 (TNRC6A). The mature miRNA guided protein machinery targets respective mRNAs that eventually affects target’s structural stability or interferes with translation. DGCR8- DiGeorge Syndrome Critical Region Gene 8; TRBP: Transactivation response element RNA-Binding Protein; TNRC6A- Trinucleotide Repeat Containing Adaptor 6A.

Figure 2.. Regulation of innate immune responses by cellular and viral miRNAs.

Tissue homeostasis at the gingival sulcus is disrupted due to the dysbiotic colonization of “keystone” periodontal pathogens elevating the pathogenicity of the whole community. Microbial products (PAMPs/DAMPs) induce innate immune signaling once bound to their ligand. TLRs (except TLR3) recruit the adaptor protein MyD88, thereby activating activate NFκB and MAP kinases by acting on their upstream activators IRAK, TRAF6 and TAK1. Activated NFκB and MAPK translocate to the nucleus and bind their respective motifs in the promoters of target genes to induce transcription. This includes early response and inflammatory genes like TNFα, IL1β, IL6, IL12, IL23, causing increased cytokine/chemokine production. Various cellular miRNAs including miR-9, miR-132 and miR-146a are shown to regulate innate immunity in response to periodontopathogenic challenge. Viral miRNAs also impair innate immunity by suppressing bacterial phagocytosis and cytokine secretion that may create favorable microenvironment for bacteria. HSV-1 miR-H1 and KSHV miR-K12-3 are two viral miRNAs that are enriched in inflamed gingiva and exhibit immunomodulatory functions. TLR- Toll-like Receptor; PAMP- Pathogen Associated Molecular Pattern; DAMP- Damage Associated Molecular Pattern; MyD88- Myeloid Differentiation Primary Response Gene 88; NFκB- Nuclear Factor Kappa-light-chain-enhancer of activated B cells; MAPK- Mitogen-Activated Protein Kinase; IRAK- Interleukin 1 Receptor Associated Kinase; TRAF6- TNF Receptor Associated Factor 6; TAK1- Transforming growth factor-β-Activated Kinase 1; TNFα- Tumor Necrosis Factor α; IL- Interleukin; PLCγ- Phospholipase γ; JNK- c-Jun N-terminal kinases; HSV-1- Herpes Simplex Virus-1; KSHV- Kaposi Sarcoma-Associated Herpesvirus; TIRAP: TIR Domain Containing Adaptor Protein; TRAF- TNF receptor Associated Factor; TRIF- TIR-domain-containing adapter-inducing interferon-β; ROS- Reactive Oxygen Species; NFATn- Nuclear Factor of Activated T-Cells n; IP3- Inositol 1,4,5-tris Phosphate

Figure 3.. microRNA regulates unique function and phenotype of various immune cell relevant to periodontal disease pathogenesis.

A diverse set of immune cells contribute to immunity against periodontopathogens. To elicit adept immune response in a dynamic manner, rapid changes in transcriptional profiles can occur. miRNAs can play important role in swift polarization of immune cells necessary to mount robust immune activation. A few miRNAs have been characterized that may be critical for the acquisition of various immune cell and osteoclasts relevant to periodontal disease. Th- T helper; Treg- T regulatory

Figure 4.. microRNA regulate inflammatory and pro-resolving macrophage phenotype.

A unique subset of pro- (red) and anti-inflammatory (blue) miRNAs have been characterized in the context of periodontal disease or periodontopathogen challenge. Dysregulation in the expression of specific miRNAs could be an important strategy by perioddontopathogens to alter the functional phenotype of macrophages (Mφ), a critical microbicidal innate immune cell. For instance, in periodontopathogen challenged myeloid cells miR-24 favors M2 over M1 phenotype by inhibiting proinflammatory cytokine production and inducing M2-associated surface markers

Figure 5.. Genome organization of viral microRNAs in 5 herpesviruses associated with oral diseases.

EBV- Epstein-Barr Virus; HHV6B- Human Herpesvirus 6B; HSV-1- Herpes Simplex Virus-1; KSHV- Kaposi Sarcoma-Associated Herpesvirus; HCMV- Human Cytomegalovirus

Figure 6.. Herpesvirus miRNA and mRNA are enriched in inflamed gingival biopsies.

Total RNA isolated from periodontally healthy and disease gingiva was used for viral miRNA and mRNA quantification by reverse transcription PCR (RT-qPCR). Dot plots show mean Ct values of (A) hsv1-miR-H1, (B) hcmv-miR-US4 and (C) kshv-miR-K12-3, and (D) ICP0 mRNA of HSV-1 in gingiva of healthy and periodontitis subjects (n=6/group). Ct value ≥35 was considered as not detected. Lower Ct values indicate higher expression. Numbers of positive samples are mentioned for each group. Student’s t-test was used to calculate p-values. **p<0.01; ***p<0.001. HSV-1- Herpes Simplex Virus 1; HCMV- Human Cytomegalovirus; KSHV- Kaposi Sarcoma-Associated Herpesvirus; ICP0- Infected Cell Protein 0

Figure 7.. Salivary detection of viral transcript for periodontal health monitoring.

Total RNA was isolated from saliva of periodontally healthy and diseased subjects (n=4/group) and the expression of HSV-1 mRNA (gB) and miRNA (miR-H6) was examined by quantitative reverse transcription PCR (RT-qPCR). As a control, RNA, U6B Small Nuclear (RNU6B) was used. Student’s t-test was used to calculate p-values. *p<0.05; ***p<0.001.