Abstract
Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as an etiological factor. However, the underlying mechanisms remain obscure. In our recent miRNA profiling of healthy and diseased human tooth pulps, elevated expression of human herpesvirus encoded viral microRNAs (v-miRs) were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p [Kaposi sarcoma-associated virus (KSHV)], miR-H1 [herpes simplex virus 1 (HSV1)], and miR-UL-70-3p [human cytomegalovirus (HCMV)] to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR-transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the KSHV-infected cell lines. We show that HOK-derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Concurrent expression analysis of precursor (pre)-miRNA and mature miRNA suggest transcriptional or posttranscriptional impact of v-miRs on the cellular miRNAs. Employing bioinformatics, we predicted several pathways targeted by deregulated cellular miRNAs that include cytoskeletal organization, endocytosis, and cellular signaling. We validated three novel targets of miR-K12-3-3p and miR-H1 that are involved in endocytic and intracellular trafficking pathways. To evaluate the functional consequence of this regulation, we performed phagocytic uptake of labeled bacteria and noticed significant attenuation in miR-H1 and miR-K12-3-3p but not miR-UL70-3p transfected primary human Mφ. Multiple cytokine analysis of E. coli challenged Mφ revealed marked reduction of secreted cytokine levels with important roles in innate and adaptive immune responses suggesting a role of v-miRs in immune subversion. Our findings reveal that oral disease associated v-miRs can dysregulate functions of key host cells that shape oral mucosal immunity thus exacerbating disease severity and progression.
Highlights
Viruses often infect the oral cavity and multiple studies indicate an association of one or more human herpesvirus (HHV) in oral inflammatory diseases [1, 2]
Since oral keratinocytes are the primary sites of infection for various HHV, we questioned whether induced expression of viral microRNAs (v-miRs) observed in clinical samples of pulpitis [24] and periodontitis (Naqvi et al, submitted) can impact cellular miRNA profiles
We selected three different v-miRs identified in our previous studies namely miR-K12-3-3p, miR-H1, and miR-UL-70-3p that belong to Kaposi sarcoma-associated virus (KSHV), herpes simplex virus 1 (HSV1), and human cytomegalovirus (HCMV), respectively [(24); Naqvi et al, submitted]
Summary
Viruses often infect the oral cavity and multiple studies indicate an association of one or more human herpesvirus (HHV) in oral inflammatory diseases [1, 2]. Several v-miRs target viral genes to which they are antisense (e.g., EBV miR-BART2 targeting of BALF5), while others control transcripts emanating from other genomic locations (e.g., HSV1 miR-H6 targeting ICP4) [17, 18]. Given their significant capacity to regulate hundreds of host transcripts, viral miRNAs can influence and potentially regulate the host transcriptome. The impact of v-miRs on the host miRnome has not been examined This is significant as a single miRNA can simultaneously fine tune expression of hundreds of transcripts. Examining the contribution of v-miRs on the host miRnome would provide a deeper understanding of their contribution in host–pathogen interactions
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