Gut Bacteria Metabolism Impacts Immune Recovery in HIV-infected Individuals

Abstract

While changes in gut microbial populations have been described in human immuno-deficiency virus (HIV)-infected patients undergoing antiretroviral therapy (ART), the mechanisms underlying the contributions of gut bacteria and their molecular agents (metabolites and proteins) to immune recovery remain unexplored. To study this, we examined the active fraction of the gut microbiome, through examining protein synthesis and accumulation of metabolites inside gut bacteria and in the bloodstream, in 8 healthy controls and 29 HIV-infected individuals (6 being longitudinally studied). We found that HIV infection is associated to dramatic changes in the active set of gut bacteria simultaneously altering the metabolic outcomes. Effects were accentuated among immunological ART responders, regardless diet, subject characteristics, clinical variables other than immune recovery, the duration and type of ART and sexual preferences. The effect was found at quantitative levels of several molecular agents and active bacteria which were herein identified and whose abundance correlated with HIV immune pathogenesis markers. Although, we cannot rule out the possibility that some changes are partially a random consequence of the disease status, our data suggest that most likely reduced inflammation and immune recovery is a joint solution orchestrated by both the active fraction of the gut microbiota and the host.

Keywords: Antiretroviral therapy; Gut microbiota; HIV; Immune recovery; Metabolome; Metaproteome.

Graphical abstract

Fig. 1

Changes in metabolite profiles. Principal Component Analysis (PCA) plots for the models were built using the set of LC-MS (−) filtered data that were present in at least 50% of the samples of each group and i) present in at least 80% of the Quality Controls (QCs) that obtained a coefficient of variation < 30% or ii) present in < 20% of the QCs (for details see Materials and Methods). (a) Gut microbiota (left panel): 5 principal components, R²: 0.310, Q2: 0.084. (b) Plasma (right panel): 5 principal components, R²: 0.483, Q2: 0.308. The empty circle labels the placebo cohort.

Fig. 2

Metabolite biomarkers associated to clinical variables. The relative abundance of C16 ceramide that is linked with innate immune activation marker %CD8 + HLA-DR + CD38 + T in each of the 4 group of individuals investigated is shown (P < 0.0239). Horizontal bars represent median. Inset represents the abundance of C16 ceramide versus %CD8 + HLA-DR + CD38 + T (Rho = 0.95).

Fig. 3

Changes in protein expression profiles. The metaproteomic analysis PCO plot shows the similarity in the abundance of quantified proteins in each individual (Bray Curtis similarity; Bray and Curtis, 1957). An Analysis of Similarities (ANOSIM) revealed significant differences in protein abundance between groups with a global R of 0.668 at a significance level of 0.01%. Compared to the uninfected group, the Significant Percentages (SIMPER) analyses yielded an average dissimilarity of 82.22% in comparison to the VU group, 74.22% in comparison to the IR group and 72.31% in comparison to the INR group, with a cut off for low contributions at 90%. Symbol codes are included in the figure. The 6-weeks longitudinal samples, label as “_T” (for time), are incorporated in the PCA with empty symbols.

Fig. 4

Taxonomic distribution of proteins that are expressed by gut bacteria at the family level. The taxonomy bar-chart was built with Unipept and is based on the peptides that we identified with the Mascot-Server. In the upper row, the number of totally identified peptides with the Mascot-Server are shown for each individual. The second row shows the id (#) of the individuals within groups, which are shown in the third row. The 6-weeks longitudinal samples, label as “_T” (for time), are incorporated right to the same initial sample. Note: we are aware that the classification based on family level allows a gross interpretation of the active members and their functions. Unfortunately, the peptide information commonly obtained by metaproteomics does not allow a deeper taxonomic characterization. Analyzing the phylogeny of all peptides (after quality check) already enabled the affiliation down to family level of only approximately 50% of the peptides in each sample; the others could be either assigned to levels of phyla or order.

Fig. 5

Diversity parameters of potentially active microbiota based on the analysis of proteins identified and quantified using MaxQuant. (a) Species richness. (b) Pielou's evenness. (c) Shannon index. P-values are given per each of the comparisons.

Fig. 6

Linear discriminative analysis (LDA) effect size (LEfSe) analyses of statistically significant active families. (a) Negative LDA scores (red) are enriched in HIV-infected patients, while positive LDA scores (blue) are enriched in healthy controls. (b) LDA scores for active families are enriched in each of the examined groups.

Fig. 7

Proposed mechanisms for the contribution of gut bacteria and human cell products to the recovery of mucosal and systemic immunity during HIV infection. (a) The intestinal epithelium in a healthy individual. A continuous enterocyte lining with intact tight junctions promotes barrier integrity and prevents the translocation of commensal bacteria from the intestinal lumen into the intestinal wall. The microbiota comprises active microbiota species that yield a defined repertoire of expressed proteins and metabolites that interact with the gut mucosa. Human cells promote immune-tolerance mechanisms. No inflammation occurs in this healthy state. (b) The intestinal epithelium in an HIV-infected individual not receiving ART. These individuals are characterized by progressive immunodeficiency, immune hyper-activation and excess mortality. The microbiota comprises a distinct set of active microbiota species that yield a different repertoire of expressed proteins and metabolites than those documented in healthy individuals. The cleavage of sialic and dolichol components from epithelial cells by gut bacteria causes abnormal glycan composition in enterocytes; this leads to the destruction of tight junctions and increased microbial translocation. Human cells increase the production of lipotoxins, which accumulate in the bloodstream. The biosynthesis of leukotrienes also increases, and they accumulate in the gut environment (faecal fluid), promoting inflammation and toxicity. (c) The intestinal epithelium in an HIV-infected individual who is an immunological ART responder. The individual is characterized by the variable recovery of the mucosal architecture and immune functions, low-level inflammation and subtle health vulnerability. Distinct active species (including Bifidobacteria), proteins and metabolites characterize this individual. The cleavage of epithelial cell components by gut bacteria is limited to the cleavage of dolichol-glycans, which lowers the destruction of enterocytes and limits microbial translocation. Gut bacteria (e.g. Succinivibrionaceae family) accumulate AA and its inflammatory mediator LTB₄ in addition to the cannabinoid oleamide and biliverdin (a viral inhibitor), which may contribute to health recovery by inhibiting viral replication, stimulating the immune system, and ultimately reducing inflammation. Human cells reciprocally increase the biosynthesis of anti-inflammatory and pro-solving lipid mediators (the resolving intermediate 5,6-epoxy-18-HEPE) and lipotoxins, which circulate in the bloodstream. (d) The intestinal epithelium in an HIV-infected ART non-responder. This individual is characterized by chronic mucosal impairment, systemic immune impairment, chronic inflammation and excess mortality. The set of microbial products in this individual are most similar to those in the ART-untreated patients. N-glycan metabolism occurs as in (c). Lipotoxins and leukotrienes are also produced as in (b). Gut bacteria accumulate bilirubin.