Human Cytomegalovirus and Human Herpesvirus 6 Coinfection of Dermal Fibroblasts Enhances the Pro-Inflammatory Pathway Predisposing to Fibrosis: The Possible Impact on Systemic Sclerosis

Abstract

Systemic sclerosis (SSc) is a severe autoimmune disease likely triggered by genetic and environmental factors, including viral infections. Human cytomegalovirus (HCMV) and human herpesvirus 6A species (HHV-6A) have been associated with SSc, based on in vivo and in vitro evidence, but the data are still inconclusive. Furthermore, despite both viruses being highly prevalent in humans and able to exacerbate each other’s effects, no data are available on their joint effects. Hence, we aimed to study their simultaneous impact on the expression of cell factors correlated with fibrosis and apoptosis in in vitro coinfected fibroblasts, representing the main target cell type in SSc. The results, obtained by a microarray detecting 84 fibrosis/apoptosis-associated factors, indicated that coinfected cells underwent higher and more sustained expression of fibrosis-associated parameters compared with single-infected cells. Thus, the data, for the first time, suggest that HCMV and HHV-6A may cooperate in inducing alterations potentially leading to cell fibrosis, thus further supporting their joint role in SSc. However, further work is required to definitively answer whether β-herpesviruses are causally linked to the disease and to enable the possible use of targeted antiviral treatments to improve clinical outcomes.

Coinfection of Dermal Fibroblasts by Human Cytomegalovirus and Human Herpesvirus 6 Can Boost the Expression of Fibrosis-Associated MicroRNAs

“…Primary human dermal fibroblasts derived from the adult skin of a single donor (NHDF-Ad, CC-2511; Lonza, Basel, Switzerland) were cultured in complete fibroblast cell medium (Fibroblast Cell Basal Medium, FCBM), supplemented with 2% fetal bovine serum (FBS), 0.1% r-human fibroblast growth factor-B, 0.1% insulin, 0.1% gentamicin sulphate/amphotericin-B (Clonetics™ FGM™-2 Bullet Kit™; Lonza, Basel, Switzerland), as previously described [ 13 , 14 , 34 ]. Following the manufacturer’s instructions, fibroblasts were sub-cultivated at around 80% confluence, using a “ReagentPack Subculture Reagent Kit” (Lonza, Basel, Switzerland).…”

“…The virus presence in infected cells was assessed and quantified by specific quantitative real-time PCR (qPCR), using 100 ng of total extracted DNA as a template. Specifically, the HCMV DNA amount was quantified by the qPCR CMV ELITe MGB ® Kit (ELITechGroup, Turin, Italy), designed to detect the HCMV DNA exon 4 region of the immediate-early (IE)1 gene, following the manufacturer’s instructions, as already described [ 8 , 14 ]. HHV-6A presence was assessed by a specific qPCR targeting the U94 viral gene, as previously described [ 17 , 35 ].…”

“…To date, the etiological agents of SSc are still unclear, but it is widely accepted that the disease occurs as a result of a multistep and multifactorial process, involving genetic predisposition of the host and environmental factors, the latter including both physicochemical agents and infectious agents [ 6 , 7 ]. Among environmental factors, viral infections have been consistently suggested as possible etiological factors, mainly those which persist in the host and can reactivate in susceptible subjects, such as Human Cytomegalovirus (HCMV) and Human Herpesvirus 6 (HHV-6) [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Both viruses belong to the Herpesviridae family, Beta-herpesvirinae subfamily, and are ubiquitous and highly prevalent in the human population.…”

“…Both viruses can establish a productive infection in fibroblasts and endothelial cells [ 8 , 13 , 14 , 25 , 26 , 27 , 28 ]. The in vitro infection of human primary dermal fibroblasts with HCMV and HHV6 has been shown to have a strong impact on the expression of pro-fibrotic factors [ 8 ].…”

“…The in vitro infection of human primary dermal fibroblasts with HCMV and HHV6 has been shown to have a strong impact on the expression of pro-fibrotic factors [ 8 ]. Interestingly, we recently observed that the simultaneous presence of HCMV and HHV-6 determines a more prominent and sustained expression of transcriptional factors associated with fibrosis and apoptosis pathways, compared to that observed in singly infected cells, highlighting for the first time the potential cooperation of these beta-herpesviruses in sclerodermic disease [ 14 ].…”