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.

Keywords: HCMV; HHV-6; systemic sclerosis; tissue fibrosis factors.

Figure 1

Primary human dermal fibroblasts infected with HCMV and HHV-6A. Images were acquired with a phase-contrast microscope at 4X original magnification, and are representative of sample duplicates in two independent experiments.

Figure 2

Expression of fibrosis-associated factors in response to HCMV/HHV-6A coinfection of human dermal fibroblasts. Cell samples were collected at the indicated days post infection (d.p.i.) and analyzed by qPCR microarray. (a) Scatterplot representation: the threshold was 2-fold change in infected vs. control cells; red and blue dots represent up-regulated and down-regulated factors, respectively; results are expressed as mean values of duplicate samples in two independent experiments. (b) Detailed values of down- and up-regulated factors: dark blue, down-regulation >10-fold; light blue, down-regulation between 2- and 9.9-fold; light red, up-regulation between 2- and 9.9-fold; red, up-regulation between 10- and 99.9-fold; dark red, up-regulation >100-fold. All values above the 2-fold threshold are indicated in bold.

Figure 3

Expression of apoptosis-associated factors in response to HCMV/HHV-6A coinfection of human dermal fibroblasts. Cell samples were collected at the indicated days post-infection (d.p.i.) and analyzed by qPCR microarray. (a) Scatterplot representation: the threshold was at 2-fold change in infected vs. control cells; red and blue dots represent up-regulated and down-regulated factors, respectively; results are expressed as mean values of duplicate samples in two independent experiments. (b) Detailed values of down- and up-regulated factors: dark blue, down-regulation >10-fold; light blue, down-regulation between 2- and 9.9-fold; light red, up-regulation between 2- and 9.9-fold; red, up-regulation between 10- and 99.9-fold; dark red, up-regulation >100-fold. All values above the 2-fold threshold are indicated in bold.

Figure 4

Factors uniquely or mostly affected by viruses’ coinfection. (a) Fibrosis-associated factors; (b) apoptosis-associated factors. Results are expressed as mean values ± S.D. of duplicate samples in two independent experiments.