Enhancement of HIV-1 infection and intestinal CD4+ T cell depletion ex vivo by gut microbes altered during chronic HIV-1 infection

Abstract

Background: Early HIV-1 infection is characterized by high levels of HIV-1 replication and substantial CD4 T cell depletion in the intestinal mucosa, intestinal epithelial barrier breakdown, and microbial translocation. HIV-1-induced disruption of intestinal homeostasis has also been associated with changes in the intestinal microbiome that are linked to mucosal and systemic immune activation. In this study, we investigated the impact of representative bacterial species that were altered in the colonic mucosa of viremic HIV-1 infected individuals (HIV-altered mucosal bacteria; HAMB) on intestinal CD4 T cell function, infection by HIV-1, and survival in vitro. Lamina propria (LP) mononuclear cells were infected with CCR5-tropic HIV-1BaL or mock infected, exposed to high (3 gram-negative) or low (2 gram-positive) abundance HAMB or control gram-negative Escherichia coli and levels of productive HIV-1 infection and CD4 T cell depletion assessed. HAMB-associated changes in LP CD4 T cell activation, proliferation and HIV-1 co-receptor expression were also evaluated.

Results: The majority of HAMB increased HIV-1 infection and depletion of LP CD4 T cells, but gram-negative HAMB enhanced CD4 T cell infection to a greater degree than gram-positive HAMB. Most gram-negative HAMB enhanced T cell infection to levels similar to that induced by gram-negative E. coli despite lower induction of T cell activation and proliferation by HAMB. Both gram-negative HAMB and E. coli significantly increased expression of HIV-1 co-receptor CCR5 on LP CD4 T cells. Lipopolysaccharide, a gram-negative bacteria cell wall component, up-regulated CCR5 expression on LP CD4 T cells whereas gram-positive cell wall lipoteichoic acid did not. Upregulation of CCR5 by gram-negative HAMB was largely abrogated in CD4 T cell-enriched cultures suggesting an indirect mode of stimulation.

Conclusions: Gram-negative commensal bacteria that are altered in abundance in the colonic mucosa of HIV-1 infected individuals have the capacity to enhance CCR5-tropic HIV-1 productive infection and depletion of LP CD4 T cells in vitro. Enhanced infection appears to be primarily mediated indirectly through increased expression of CCR5 on LP CD4 T cells without concomitant large scale T cell activation. This represents a novel mechanism potentially linking intestinal dysbiosis to HIV-1 mucosal pathogenesis.

Fig. 1

Abundances of HIV-altered mucosal bacteria (HAMB) species in HIV-infected and uninfected study participants. Bacterial taxa were identified in colon biopsies from 17 participants with chronic, untreated HIV-1 infection and 14 uninfected control participants using bacterial 16S ribosomal DNA sequencing. Of the 21 species that were significantly over (6) or under (15) represented in HIV-1-infected participants (Additional file 1: Table S1), 7 species that represented each of the 3 phyla (Bacteroides, Proteobacteria and Firmicutes) were evaluated in the LPAC model. a Phylogenetic tree illustrating taxonomic hierarchy for each HAMB species (GN gram-negative; GP gram-positive). b, c Plots showing individual relative abundance of each species that were (b) increased or (c) decreased in HIV-1 infected participants (squares) compared to uninfected participants (circles). Values are shown as a fraction of all classified species detected within each individual. Lines represent the median value. Statistical analysis was performed using the Mann–Whitney test

Fig. 2

HAMB enhance HIV-1 infection and depletion of LP CD4 T cells. LPMC (n = 7) were spinoculated with CCR5-tropic HIV-1_BAL or mock control and exposed to High abundance or Low abundance HAMB species or to control bacteria (E. coli, B. infantis) (2.5 bacteria: 1 LPMC) for 4 days. LPMC were harvested and levels of productive infection and depletion of LP CD4 T cells determined. a Representative gating strategy illustrating intracellular p24 levels in viable CD4 T cells (gated as CD3+ CD8−) in presence or absence of bacteria with gates established on matched mock controls. b Percentages of intracellular p24-expressing (p24+) CD4 T cells in presence or absence of bacteria with background p24 (mock) values removed. c Levels of CD4 depletion in presence or absence of bacteria relative to depletion in matched mock conditions. Values are shown as ‘box and whisker’ with the box extending from the 25th to 75th percentile, the line in the box indicating the median value and the whiskers indicating maximum and minimum values. White box indicates no bacteria, light gray boxes indicate HAMB species and dark gray boxes indicate control bacteria. Statistical analysis was performed using the Wilcoxon matched–pairs signed rank test comparing frequencies of p24 + CD4 T cells or levels of depletion induced in response to bacteria to HIV-1 only. *p < 0.05, ^#p = 0.08. Legend details the abbreviations used for each bacteria (x axis)

Fig. 3

HAMB induce low levels of LP CD4 T cell activation and proliferation. LPMC (n = 6) were pre-labeled with CFSE and exposed to High abundance or Low abundance HAMB species or to control bacteria (E. coli, B. infantis) (2.5 bacteria: 1 LPMC) for 5 days. LPMC were harvested and frequencies of activated (CD38⁺HLA-DR⁺, CD25⁺) and proliferating (CFSE^lo) LP CD4 T cells were evaluated using flow cytometry. a Representative gating strategy illustrating CD38⁺HLA-DR⁺, CD25⁺ and CFSE profiles of viable LP CD4 T cells in presence or absence of bacteria with gates established on media (CD25⁺, CFSE) or FMO controls (CD38/HLA-DR). b Percentages of LP CD4 T cells co-expressing CD38 and HLA-DR. FMO control values have been subtracted. c Percentages of CD4 LP T cells expressing CD25. d Percentages of CFSE^lo LP CD4 T cells. Values are shown as symbols representing each individual donor to highlight the 1 donor that exhibited unusually high responses to HAMB. Line indicates the median value. Statistical analysis was performed using the Wilcoxon matched–pairs signed rank test comparing percentages of activated or proliferating LP CD4 T cells induced in response to bacteria to no bacteria. *p < 0.05, ^#p = 0.06. Legend details the abbreviations used for each bacteria (x axis)

Fig. 4

HAMB increase CCR5 expression on LP CD4 T cells. LPMC (n = 6) were exposed to High abundance or Low abundance HAMB species or to control bacteria (E. coli, B. infantis) (2.5 bacteria: 1 LPMC) for 5 days. LPMC were harvested and expression levels of HIV-1 co-receptors CCR5 and CD4 on LP CD4 T cells determined using flow cytometry. a Representative gating strategy illustrating CCR5 and CD4 expression on viable LP CD4 T cells in presence or absence of bacteria. b Expression levels (Mean fluorescence intensity; MFI) of CCR5 on CD4 LP T cells. Isotype control values have been subtracted (net MFI). c CD4 expression levels (MFI) on LP CD4 T cells. Values are shown as ‘box and whisker’ with the box extending from the 25th to 75th percentile, the line in the box indicating the median value and the whiskers indicating maximum and minimum values. White box indicates no bacteria, light gray boxes indicate HAMB species and dark gray boxes indicate control bacteria. Statistical analysis was performed using the Wilcoxon matched–pairs signed rank test comparing CCR5 or CD4 expression levels on LP CD4 T cells induced in response to bacteria to no bacteria. *p < 0.05, ^#p = 0.06. Legend details the abbreviations used for each bacteria (x axis)

Fig. 5

HAMB-mediated increased CCR5 expression is a result of indirect stimulation of LP CD4 T cells. a CCR5 expression in response to individual TLR ligands (TLRL) was measured by exposing LPMC (n = 6–7) to TLR4L (commensal E. coli LPS, 1 μg/ml) or TLR2L (Bacillus subtilis LTA, 1 μg/ml) or cultured without TLRLs (None) for 5 days. LPMC were harvested and CCR5 expression levels (Mean fluorescence intensity; MFI) on LP CD4 T cells evaluated. Isotype control values have been subtracted (net MFI) and values are illustrated as ‘box and whisker’ with the box extending from the 25th to 75th percentile, the line in the box indicating the median value and the whiskers indicating maximum and minimum values. Statistical analysis was performed using the Wilcoxon matched–pairs signed rank test, *p < 0.05. b Levels of CCR5 expression on LP CD4 T cells in response to stimulation of total LPMC or CD4-enriched LP cells with P. stercorea (2.5:1 LPMC/CD4 T cell) or E. coli LPS (1 μg/ml) for 24 h. Values shown are mean ± SEM of stimuli–specific CCR5 expression levels (net; no stimulation values subtracted; n = 3). Percent values indicate the average decrease in levels of CCR5 expression on CD4 T cells stimulated directly relative to expression levels on CD4 T cells from total LPMC stimulated cultures. Statistical analysis was performed using Paired t test, *p < 0.05, #p = 0.07. (C) LPMC (n = 6–7) were exposed to CD3/CD2/CD28 beads (1 bead: 2 LPMC) or cultured without exogenous stimuli (None) for 5 days. LPMC were harvested and frequencies of activated (CD38⁺HLA-DR⁺; CD25⁺) LP CD4 T cells and CCR5 expression levels determined. Values are shown with isotype (CCR5) or FMO control values removed and illustrated as ‘box and whisker’ plots. Statistical analysis was performed using the Wilcoxon matched–pairs signed rank test. *p < 0.05