Effect of cytomegalovirus and Epstein-Barr virus replication on intestinal mucosal gene expression and microbiome composition of HIV-infected and uninfected individuals

Abstract

Background: HIV-infection is associated with dramatic changes in the intestinal mucosa. The impact of other viral pathogens is unclear.

Methods: One hundred and eight (108) biopsies from left and right colon (n = 79) and terminal ileum (n = 29) were collected from 19 HIV-infected and 22 HIV-uninfected participants. Levels of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA were measured by droplet digital PCR. Mucosal gene expression was measured via multiplex-assay. Microbiome analysis was performed using bacterial 16S-rDNA-pyrosequencing. The effect of CMV and EBV replication on the microbiome composition and mRNA-expression of selected cytokines (IL-6, IFN-γ, IL-1β, CCL2, IL-8, and IFN-β1) was evaluated.

Results: Overall, CMV and EBV were detected in at least one intestinal site in 60.5 and 78.9% of participants, respectively. HIV-infected individuals demonstrated less detectable CMV (PB = 0.02); CMV was more frequently detected in terminal ileum than colon (PB = 0.05). Detectable EBV was more frequent among HIV-infected (P B= 0.04) without differences by intestinal site. The number of operational taxonomic units did not differ by CMV or EBV detection status. Among HIV-infected participants, higher CMV was only associated with lower relative abundance of Actinobacteria in the ileum (P = 0.03). Presence of CMV was associated with upregulated expression of all selected cytokines in the ileum (all P < 0.02) and higher expression of IL-8 and IFN-β1 in the colon (all P < 0.05) of HIV-uninfected participants, but not among HIV-infected. EBV had no effect on cytokine expression or microbiome composition whatsoever.

Conclusion: These results illustrate a complex interplay among HIV-infection, intestinal CMV replication, and mucosal gut environment, and highlight a possible modulatory effect of CMV on the microbial and immune homeostasis.

Figure 1. Posterior medians and 95% intervals for probabilities of detecting CMV (pCMV, Panel A) and EBV (pEBV, Panel B) DNA, derived from Bayesian hierarchical regression models

Y-axis represents individual study participants. Small dots represent observed data with colors indicating sample sites (red = colon, blue = ileum). Large grey dots, vertical grey lines, and the horizontal grey line indicate estimated random intercepts for subjects, 95% intervals, and the group intercepts, respectively. Observed values are jittered to avoid overlap.

Figure 2. Cytokine levels for six selected cytokines (CCL-2, INFβ1, IFNγ, IL1β, IL-6 and IL-8) for HIV positive (in red) and HIV uninfected controls (in blue) divided by intestinal site (colon versus ileum)

X-axis shows probability of detecting CMV DNA (low versus high); Y-axis shows cytokine levels (normalized to the average of the two housekeeping genes). The lines and grey areas represent model-based estimates and their 95% confidence intervals

Figure 3. Principal coordinates analyses (PCA) of gut microbiome beta diversity among HIV-infected and uninfected control subjects, by CMV and EBV replication status

Panel A shows PCA applied to the Unifrac metric colored by HIV status and shaped by CMV status. Similarly, Panel B shows PCA of all samples colored by HIV status and shaped by EBV status; x and y-axes represent first and second principal coordinates, respectively.

Figure 4. Relative abundance for Actinobacteria for HIV positive (in red) and HIV uninfected controls (in blue) divided by intestinal site (colon versus ileum) and by CMV detection status

X-axis shows probability of detecting CMV DNA (low versus high); Y-axis shows relative abundance (log transformed) of Actinobacteria phylum. The large dots and vertical lines represent means and their 95% confidence intervals