Effects of viremia and CD4 recovery on gut "microbiome-immunity" axis in treatment-naïve HIV-1-infected patients undergoing antiretroviral therapy

Abstract

Background: Human immunodeficiency virus type 1 (HIV-1) infection is characterized by persistent systemic inflammation and immune activation, even in patients receiving effective antiretroviral therapy (ART). Converging data from many cross-sectional studies suggest that gut microbiota (GM) changes can occur throughout including human immunodeficiency virus (HIV) infection, treated by ART; however, the results are contrasting. For the first time, we compared the fecal microbial composition, serum and fecal microbial metabolites, and serum cytokine profile of treatment-naïve patients before starting ART and after reaching virological suppression, after 24 wk of ART therapy. In addition, we compared the microbiota composition, microbial metabolites, and cytokine profile of patients with CD4/CD8 ratio < 1 (immunological non-responders [INRs]) and CD4/CD8 > 1 (immunological responders [IRs]), after 24 wk of ART therapy.

Aim: To compare for the first time the fecal microbial composition, serum and fecal microbial metabolites, and serum cytokine profile of treatment-naïve patients before starting ART and after reaching virological suppression (HIV RNA < 50 copies/mL) after 24 wk of ART.

Methods: We enrolled 12 treatment-naïve HIV-infected patients receiving ART (mainly based on integrase inhibitors). Fecal microbiota composition was assessed through next generation sequencing. In addition, a comprehensive analysis of a blood broad-spectrum cytokine panel was performed through a multiplex approach. At the same time, serum free fatty acid (FFA) and fecal short chain fatty acid levels were obtained through gas chromatography-mass spectrometry.

Results: We first compared microbiota signatures, FFA levels, and cytokine profile before starting ART and after reaching virological suppression. Modest alterations were observed in microbiota composition, in particular in the viral suppression condition, we detected an increase of Ruminococcus and Succinivibrio and a decrease of Intestinibacter. Moreover, in the same condition, we also observed augmented levels of serum propionic and butyric acids. Contemporarily, a reduction of serum IP-10 and an increase of IL-8 levels were detected in the viral suppression condition. In addition, the same components were compared between IRs and INRs. Concerning the microflora population, we detected a reduction of Faecalibacterium and an increase of Alistipes in INRs. Simultaneously, fecal isobutyric, isovaleric, and 2-methylbutyric acids were also increased in INRs.

Conclusion: Our results provided an additional perspective about the impact of HIV infection, ART, and immune recovery on the "microbiome-immunity axis" at the metabolism level. These factors can act as indicators of the active processes occurring in the gastrointestinal tract. Individuals with HIV-1 infection, before ART and after reaching virological suppression with 24 wk of ART, displayed a microbiota with unchanged overall bacterial diversity; moreover, their systemic inflammatory status seems not to be completely restored. In addition, we confirmed the role of the GM metabolites in immune reconstitution.

Keywords: Antiretroviral therapy; Cytokines; HIV; Immunological responders; Inflammation; Microbiome-immunity axis; Microbiota; Short chain fatty acid; Viremia.

Figure 1

Box-plots showing alpha diversity indices (Chao1, Shannon, and evenness indices) in samples. Statistical differences were evaluated using paired Wilcoxon signed-rank test for Chao, Shannon, and evenness indices. P value less than 0.05 were considered statistically significant.

Figure 2

Cluster analysis (A) and principal coordinate analysis showing that samples do not separate into two groups depending on their condition (0-24 wk) (B).

Figure 3

Segment plots depicting taxa with significantly differences between high viremia (time point 0) and viral suppression (time point 24) conditions. Lines connect paired samples and highlight the differences in normalized abundance for the indicated rank. Orange or blue colors highlight decrease or increase, respectively. Numbers in the top-left corner represent counts of increased (orange) and decreased (blue) measurement for paired samples.

Figure 4

Boxplots showing statistically different levels of serum short-chain fatty acids between high viremia and viral suppressor patients, assessed by the Wilcoxon test. P value < 0.05 was considered statistically significant.

Figure 5

Boxplots showing statistically different levels of serum cytokines between high viremia and viral suppressor patients, assessed by the Wilcoxon test. A P value < 0.05 was considered statistically significant.

Figure 6

Boxplots showing the results of taxa-level differential abundance analysis between immunological responders and immunological non-responders at 24 wk. Plot titles report the shrunk Log2 fold change (according to the DESeq2 function lfcShrink). All results have a P value < 0.05. NR = INRs, R = IRs. IRs: Immunological responders; INRs: Immunological non-responders.

Figure 7

Boxplots showing statistically different fecal short-chain fatty acid abundances between immunological responders and immunological non-responders, assessed by the Mann-Whitney test. ^a P value < 0.05 was considered statistically significant.