Fecal Microbiota Composition Drives Immune Activation in HIV-infected Individuals

Abstract

The inflammatory properties of the enteric microbiota of Human Immunodeficiency Virus (HIV)-infected individuals are of considerable interest because of strong evidence that bacterial translocation contributes to chronic immune activation and disease progression. Altered enteric microbiota composition occurs with HIV infection but whether altered microbiota composition or increased intestinal permeability alone drives peripheral immune activation is controversial. To comprehensively assess the inflammatory properties of HIV-associated enteric microbiota and relate these to systemic immune activation, we developed methods to purify whole fecal bacterial communities (FBCs) from stool for use in in vitro immune stimulation assays with human cells. We show that the enteric microbiota of untreated HIV-infected subjects induce significantly higher levels of activated monocytes and T cells compared to seronegative subjects. FBCs from anti-retroviral therapy (ART)-treated HIV-infected individuals induced intermediate T cell activation, indicating an only partial correction of adaptive immune cell activation capacity of the microbiome with ART. In vitro activation levels correlated with activation levels and viral load in blood and were particularly high in individuals harboring specific gram-positive opportunistic pathogens. Blockade experiments implicated Tumor Necrosis Factor (TNF)-α and Toll-Like Receptor-2 (TLR2), which recognizes peptidoglycan, as strong mediators of T cell activation; This may contradict a previous focus on lipopolysaccharide as a primary mediator of chronic immune activation. These data support that increased inflammatory properties of the enteric microbiota and not increased permeability alone drives chronic inflammation in HIV.

Keywords: Chronic inflammation; Fecal bacteria; HIV; Immune activation; Microbiome; TLR2.

Fig. 1

Levels of sCD14, LPS Binding Protein (LBP) and CD8+ T cell activation in plasma and peripheral blood. (A) Plasma levels of soluble CD14. (B) Plasma levels of LBP. (C) PBMC was stained directly ex vivo for CD8 + HLA-DR + CD38+ activated T cells. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig. 2

Principal Coordinates Analysis (PCoA) of 16S rRNA data from whole stool and associated FBCs. The composition of the bacterial communities in stool and fecal bacterial communities (FBC) from females, men who have sex with men (MSM) and men who have sex with women (MSW) with and without HIV were compared. (A) Unweighted and (B) Weighted UniFrac PCoA plots. The large spheres represent FBCs and the smaller spheres whole stool. FBCs are joined to their source stool sample with a line. HIV-infected subjects are indicated with a “+”. (C) Genus level taxa bar charts of 16S rRNA data from whole stool and FBCs. Genera representing <2% of the community are collapsed into the “Other” category (red). Samples are grouped by HIV and treatment status with stool (right) and associated FBCs (left) adjacent to each other and joined with a bridge.

Fig. 3

FBCs from HIV-infected MSM induce monocyte activation and inflammatory cytokines. Healthy PBMC was stimulated for 24 h with FBC isolated from 6 HIV negative MSW, 13 HIV negative MSM, 8 HIV positive MSM on ART and 11 HIV positive untreated MSM. (A) Representative dot plots and accumulative data showing percent CD14 shedding of CD33+ cells after stimulation with FBCs. Percent shedding was calculated by subtracting the percent of CD14+ of CD33+ cells in the unstimulated control by the percent of CD14+ of CD33+ cells in the FBC stimulated cells. (B) Percent CD80+ of CD33+ cells after stimulation with FBC. (C) Levels of soluble CD14 (sCD14) and (D) cytokines in the supernatant. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig. 4

FBCs from HIV-infected MSM induce high levels of T cell activation in PBMC. Healthy PBMC was stimulated with FBC from 6 SN MSW, 13 SN MSM, 8 HIV⁺ART⁺MSM or 11 HIV⁺ART⁻MSM subjects for 4 days. (A) Representative dot plots and accumulative data showing HLA-DR+ CD38+ CD4+ activated T cells. (B) Levels of cytokines in the supernatant. Each dot represents the median of 6 different PBMC stimulated with the same FBC. (C) Whole PBMC or isolated CD4+ T cells were stimulated with FBC isolated from 10 HIV⁺ART⁻MSM for 4 days. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig. 5

TLR, MHC-II and TNF-α blockade ameliorate T cell activation induced by FBCs from HIV positive untreated MSM and CD4+ T cell activation is correlated to monocyte activation. Healthy PBMC was pretreated with (A) TLR2 or TLR4 or (B) HLA-DR (MHC-II) blockade for 30 min prior to stimulation with FBC isolated from 10 HIV positive untreated MSM for 4 days. Frequency of CD4 + HLA-DR + CD38+ T cells is shown for TLR blockades while frequency of CD38+ CD4+ T cells is shown for HLA-DR blockade. (C) FBC stimulated PBMC was treated with IL-6, IFN-γ or TNF-α blockade for 4 days. (D) Correlation between CD14 shedding of CD33+ cells and CD4 + HLA-DR + CD38+ T cells after stimulation with FBCs. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig. 6

FBCs isolated from HIV-infected females induce monocyte and T cell activation. Healthy PBMC was stimulated with FBCs isolated from 5 HIV-negative, and 7 HIV-infected treated females. (A) Percent change of CD14+ and CD80+ CD33+ cells after 24 h stimulation. (B) Frequency of CD4 + HLA-DR + CD38+ T cells after 4 days of culture. (C) Levels of IFN-γ and IL-17 in PBMC after 4 days of culture in the supernatant. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig. 7

Immune cell activation induced by FBCs in vitro is correlated to ex vivo peripheral sCD14 and T cell activation levels and HIV viral load. (A) Correlation of sCD14 induced in vitro by FBC to ex vivo sCD14 levels in the plasma of the fecal donor. (B) The frequency of CD8 + HLA-DR + CD38+ activated T cells induced by FBCs in vitro was correlated to ex vivo levels from the same subjects who donated sample. (C) Correlation of peripheral viral load in HIV⁺ART⁻ subjects with CD4 and CD8 activated T cells induced by FBCs from the same donor in vitro. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative.