Effect of HIV suppression on the cytokine network in blood and seminal plasma

Abstract

Objective: HIV infection disrupts the cytokine network and this disruption is not completely reversed by antiretroviral therapy (ART). Characterization of cytokine changes in blood and genital secretions is important for understanding HIV pathogenesis and the mechanisms of HIV sexual transmission. Here, we characterized the cytokine network in individuals longitudinally sampled before they began ART and after achieving suppression of HIV RNA.

Methods: We measured concentrations of 34 cytokine/chemokines using multiplex bead-based assay in blood and seminal plasma of 19 men with HIV-1 prior to and after viral suppression. We used Partial Least Squares Discriminant Analysis (PLS-DA) to visualize the difference in cytokine pattern between the time points. Any cytokines with VIP scores exceeding 1 were deemed important in predicting suppression status and were subsequently tested using Wilcoxon Signed Rank Tests.

Results: PLS-DA projections in blood were fairly similar before and after viral suppression. In contrast, the difference in PLS-DA projection observed in semen emphasizes that the immunological landscape and immunological needs are very different before and after ART in the male genital compartment. When tested individually, four cytokines were significantly different across time points in semen (MIG, IL-15, IL-7, I-TAC), and two in blood (MIG and IP-10).

Conclusion: Viral suppression with ART impacts the inflammatory milieu in seminal plasma. In contrast, the overall effect on the network of cytokines in blood was modest but consistent with prior analyses. These results identify specific changes in the cytokine networks in semen and blood as the immune system acclimates to chronic, suppressed HIV infection.

Figure 1:. Effect of ART on chemokine/cytokine in blood and seminal plasma

Shown is the difference of the concentrations of 34 chemokine/cytokines (log10 normalized) in PWH before and after antiretroviral therapy. Chemokine/cytokine concentrations in blood plasma (A) or seminal plasma (B) were log10-transformed and plotted as boxplots. For each cytokine and each boxplot, the box represent the interquartile range (IQR), the middle line represents the median, while the dots are outliers. A negative boxplot value reflects a decrease of the concentration of a given chemokine/cytokine following ART initiation, while a positive value reflects an upregulation of a given chemokine/cytokine.

Figure 2:. Visual comparison of blood cytokine level and time before/after viral suppression

Shown are graphical representations of log-transformed cytokine levels before (orange) and after (blue) HIV suppression. X-axis is shown without numbers, but is in weeks.

Figure 3:. Two-dimensional PLS projections in blood and seminal plasma

Shown are PLS-DA projections in two LVs with ellipses representing Hotelling’s 2-samples T2 with 95% confidence intervals in blood plasma (A) and in seminal plasma (B). The E-statistic was used to test the statistical differences in the separation between the cytokine profiles of the pre- and post-suppression groups. The multivariate distance between pre-suppression and post-suppression observations was significant in semen (p = 0.04) but not in blood (p = 0.54).

Figure 4:. Latent variables LV1, LV2, and VIP scores of each cytokine in PLS-DA projections

Shown are variable importance in projection (VIP) score plots for all cytokines in blood plasma (A) and in seminal plasma (B) colored by importance (red = higher VIP, grey = lower). The VIP score gives an estimate of the contribution of each cytokine to the PLS-DA regression model. Only the names of those cytokines with VIP above 1 are shown. The VIP scores derived by the latent variable loadings showed that more cytokines in semen (n = 9) were important in determining group classification than in blood (n = 7).

Figure 5:. Visual comparison of VIP scores between blood and seminal plasma

Shown is a graphical representation of the VIP scores for each cytokine in blood (blue) and semen (orange). Cytokines with the highest VIP scores were the best predictors of suppression status. Only three cytokines (IP-10, MIG, and I-TAC) had VIP>1 both in blood and semen.