Butyrate administration is not sufficient to improve immune reconstitution in antiretroviral-treated SIV-infected macaques

Abstract

Defective gastrointestinal barrier function and, in turn, microbial translocation have been identified as significant contributors to persistent inflammation in antiretroviral (ARV)-treated people living with HIV. Metabolic supplementation of short-chain fatty acids (SCFAs), generally produced by the commensal microbiome, may improve these outcomes. Butyrate is a SCFA that is essential for the development and maintenance of intestinal immunity and has a known role in supporting epithelial integrity. Herein we assessed whether supplementation with the dietary supplement sodium butyrate would improve immune reconstitution and reduce inflammation in ARV-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques. We demonstrate that butyrate supplementation does not significantly improve immune reconstitution, with no differences observed in systemic CD4+ T-cell frequencies, T-cell functionality or immune activation, microbial translocation, or transcriptional regulation. Our findings demonstrate that oral administration of sodium butyrate is insufficient to reduce persistent inflammation and microbial translocation in ARV-treated, SIV-infected macaques, suggesting that this therapeutic may not reduce co-morbidities and co-mortalities in treated people living with HIV.

Figure 1

Butyrate supplementation of ARV treatment does not alter SIV suppression or target cell frequencies. (a) Longitudinal plasma SIV viral load in ARV-only (black) and ARV + butyrate (blue) animals, with individual animals denoted by individual symbols as identified in Table 1. (b) Circulating CD4+ T-cell count. (c–e) PBMC, RB, MLN, Jej, and BAL % CD4+ T-cells (c) and %CCR5 + CD4+ (d) and CD8 + (e) TM. (f, g) PBMC, RB, and MLN %Ki-67 + CD4+ (f) and CD8 + (g) TM. All timepoints are relative to the day of ARV initiation. Lines among datapoints represent means. Lines among p-values span timepoints considered significantly different, color-coded by treatment status. Significance methods as follows: Two-way ANOVA (a), unpaired and paired two-way t-tests as appropriate (b–g).

Figure 2

T-cell markers of intestinal homing and tissue residency remain unchanged with butyrate supplementation of ARV treatment. (a–f) PBMC, RB, MLN, Jej, and BAL %CD103 + (a, b) and CD69 + (c, d), CD69 + CD103 + (e, f) CD4+ (a, c, e) CD8 + (b, d, f) TM in ARV-only (black) and ARV + butyrate (blue) animals, with individual animals denoted by individual symbols as identified in Table 1. All timepoints are relative to the day of ARV initiation. Lines among datapoints represent means. Lines among p-values span timepoints considered significantly different, color-coded by treatment status. Asterisks denote timepoints where groups significantly differed. Significance assessed by unpaired and paired two-way t tests as appropriate.

Figure 3

T-cell function does not improve with butyrate supplementation of ARV treatment. (a–h) PBMC, RB, and MLN %IL-2⁺ (a, b), IFNγ⁺ (c, d), TNFα⁺ (e, f), and IL-17 ⁺ (g, h) CD4⁺ (a, c, e, g) and CD8 ⁺ (b, d, f, h) TM in ARV-only (black) and ARV + butyrate (blue) animals, with individual animals denoted by individual symbols as identified in Table 1. (i) %FOXP3 + RB and MLN CD4+ TM. All timepoints are relative to the day of ARV initiation. Lines among datapoints represent means. Significance assessed by unpaired two-way t tests.

Figure 4

Butyrate supplementation of ARV treatment neither promotes intestinal repair nor reduces microbial translocation. (a) Plasma iFABP2 (left) and zonulin (right) concentrations in ARV-only (black) and ARV + butyrate (blue) animals, with individual animals denoted by individual symbols as identified in Table 1. (b) Representative IHC images of day 180 colons stained for E. coli from an ARV-only (left) or ARV + butyrate (right) animal. (c) Translocation index of Colon sections stained for E. coli as in (b). Dashed line represents the colonic translocation index of a single, healthy macaque stained as a batch control. (d) Plasma sCD14 concentrations. (e) Heatmap depicting relative colonic transcript abundance (columns) in macaques (rows) at day 180 as assessed by NanoString. Animal treatment status (left, as in a) and relative expression values (right), as indicated. Data clustered by Euclidian distance with Ward's minimum distance linkage. (f) PCA considering treatment-group and within-animal relative frequency of Colonic transcripts at day 180 as assessed in (e). (g) Relative expression of PPARG and NOS2 in the Colon and Jej, at day 180 as assessed in (e). (h) Normalized histone H3 acetylation expression as measured by ELISA in the Colon and Jej at day 180. (i) Fold-change frequencies of ASVs identified as differentially abundant in response to butyrate supplementation. Data show ARV + butyrate:ARV-only frequency fold-change, color-grouped by phylum and individually shaded by family as indicated. (j, k) Alpha (j) and Beta (k) diversity estimates at days 0 and 180. Alpha diversity estimates include observed (left) and Shannon (right) richness. Beta diversity assessed by unweighted UniFrac. All timepoints are relative to the day of ARV initiation. Lines among p values span timepoints considered significantly different, color-coded by treatment status. Solid lines among datapoints represent means. Dashed lines among datapoints represent a SIV-batch control. Significance methods as follows: unpaired and paired two-way t-tests as appropriate (a, c–d, g–h, and j), MetaLonDa (i), and adonis (k).