Metformin facilitates viral reservoir reactivation and their recognition by anti-HIV-1 envelope antibodies

Abstract

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-week supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4⁺ T cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4⁺ T cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4^lowHIV-p24⁺ T cells. These observations coincided with increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T cells by HIV-1 envelope antibodies. Thus, metformin exerts pleiotropic effects on post-integration steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

Keywords: Medication; Molecular microbiology; Virology.

Graphical abstract

Figure 1

Dose-response effects of metformin on HIV-1 replication in memory CD4⁺ T cells in vitro (A) Shown is the flow chart for the HIV-1 infection in vitro. Briefly, memory CD4⁺ T cells from HIV-uninfected donors were stimulated with anti-CD3/CD28 Abs in the absence/presence of metformin (0.5, 1, or 5 mM) or INK128 (50 nM) for 3 days. Then, cells were exposed to the replication-competent NL4.3BaL HIV strain (50 ng HIV-p24/10⁶ cells). Cell-culture supernatants were collected and media containing IL-2 and/or metformin or INK128 was refreshed every 3 days until day 12 post-infection. Shown are statistical analysis for integrated HIV-DNA levels were quantified by real-time nested PCR at day 3 post infection (B) and HIV-1 replication determined as HIV-p24 levels in cell-culture supernatants measured by ELISA at day 9 post-infection (C). Shown is a flow cytometry dot plot analysis of the intracellular HIV-p24 and surface CD4 expression allowing the identification of CD4^lowHIV-p24⁺ cells (productively infected) and CD4^highHIV-p24⁺ cells in one representative donor (D); as well as the statistical analysis of CD4^lowHIV-p24⁺ T cell frequency (E) and their geometric MFI of HIV-p24 expression (F), and of CD4^highHIV-p24⁺ T cell frequency (G) and their geometric MFI of HIV-p24 expression (H). Each symbol represents 1 donor (n = 8; median ± interquartile range). Friedman and uncorrected Dunn’s multiple comparison p-values are indicated on the graphs.

Figure 2

Effects on metformin on single-round HIV-1 infection in vitro (A) Shown is the flow chart for the single-round HIV-1 infection in vitro. Briefly, memory CD4⁺ T cells from HIV-uninfected donors were stimulated by anti-CD3/CD28 Abs in the absence/presence of metformin (1 mM) or INK128 (50 nM) for 3 days. Then, cells were exposed to a replication-incompetent single-round VSV-G-pseudotyped HIV-1 construct (100 ng HIV-p24/10⁶ cells). Cell-culture supernatants and cells were collected at day 3 post-infection. Shown are levels of early (RU5) (B) and late HIV reverse transcripts (Gag) (C), as well as integrated HIV-DNA (D) quantified by real-time nested PCR. Shown are representative flow cytometry dot plots of intracellular HIV-p24 and surface CD4 expression from one donor (E) and statistical analysis of the productively infected CD4^lowHIV-p24⁺ T cells in terms of frequencies (F) and the geometric MFI of HIV-p24 expression (G). Shown are absolute HIV-p24 levels in cell culture supernatants quantified by ELISA (H). Each symbol represents one donor (n = 7; median ± interquartile range). Friedman and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Figure 3

Effects of metformin on viral outgrowth in memory CD4⁺ T cells from ART-treated PWH (A) Shown is the flow chart for the viral outgrowth assay (VOA). Briefly, memory CD4⁺ T cells from ART-treated PWH were stimulated with CD3/CD28 Abs, in the presence (n = 11) or the absence (n = 6) of ARVs and in the presence/absence of metformin (1 mM) or INK128 (50 nM) for 3 days. Supernatants were collected, cells were split in two news wells, and media containing IL-2 and metformin or INK128 was refreshed every 3 days. Experiments were performed in 4–8 original replicates per condition. One original replicate at day 0 generated 8 final replicates at day 12. At the end of the experiment, T cells derived from the same original replicate were merged for PCR and flow cytometry analysis. Shown are (B) integrated HIV-DNA levels quantified by real-time nested PCR and (C) levels of HIV-p24 in cell-culture supernatants quantified by ELISA. Shown are (D) representative flow cytometry dot plot of cell-associatedHIV-p24 and surface CD4 expression; (E) statistical analysis of the frequency of CD4^lowHIV-p24⁺, (F) the frequency of CD4^highHIV-p24⁺ cells; and (G) the geometric MFI (GeoMFI) of HIV-p24 expression in CD4^low HIV-p24⁺ and CD4^high HIV-p24⁺ T cell subsets. Each symbol represents one donor; bars indicate the median ± interquartile range. Kruskal-Wallis test and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Figure 4

Metformin increases BST2 expression on productively infected CD4^lowHIV-p24⁺ T cells (A) Shown are representative flow cytometry dot plots of BST2 and HIV-p24 co-expression on T cells at day12 post-infection with HIV-1_{NL4.3 Bal}in vitro (upper panel) and at day 12 post TCR-mediated VR reactivation in VOA (bottom panel). (B and C) The HIV-1_NL4.3Bal infection in vitro was performed as described in Figure 3. Cells collected at day 12 post-infection were stained on the surface with CD4 and BST2 antibodies and with HIV-p24 antibodies and analyzed by flow cytometry for n = 8. Shown are (B) levels of BST2 expression on uninfected (CD4^highHIV-p24^-) and productively infected (CD4^lowHIV-p24⁺) T cells in one representative donor and (C) statistical analysis of BST2 expression (GeoMFI) relative to the medium condition (considered 1). (D and E) The VOA was performed as described in Figure 1 with cells from n = 6 ART-treated PWH. Cells collected at day 12 post-stimulation were stained on the surface with CD4 and BST2 Abs and intracellularly with HIV-p24 Abs and analyzed by flow cytometry. (D and E) Shown are levels of BST2 expression on CD4^highHIV-p24^- and CD4^lowHIV-p24⁺ T cells in (D) one representative donor and (E) statistical analysis of BST2 expression (absolute GeoMFI). (F) Shown is the BST2 expression relative to the medium condition of HIV-uninfected memory CD4⁺ T cells at day 3 post-TCR stimulation. Each symbol represents one donor (median ± interquartile range). Friedman (C and F), Kuskal-Wallis (E) and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Figure 5

Metformin increases Bcl-2 expression on HIV-infected and uninfected T cells The HIV-1_NL4.3Bal infection in vitro was performed as described in Figure 3. Cells collected at day 12 post-infection were stained with CD4, HIV-p24, and Bcl-2antibodies and analyzed by flow cytometry. Shown are (A) levels of Bcl-2 expression on uninfected (CD4^highHIV-p24^-) and productively infected (CD4^lowHIV-p24⁺) T cells in one representative donor and (B) statistical analysis of Bcl-2 expression (GeoMFI) relative to the medium condition (considered 1). Each symbol represents one donor (n = 4; median ± interquartile range). Friedman and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Figure 6

Metformin facilitates the recognition of reactivated HIV reservoirs by anti-HIV Env antibodies The VOA was performed on memory CD4⁺ T cells from ART-treated PWH, as described in Figure 3A. Cells harvested at day 12 post-TCR triggering and cultured in the presence/absence of metformin (1 mM) were stained on the surface with a set of unconjugated human anti-HIV-1 Env bNAbs (2G12, PGT121, PGT126, PGT151, 3BNC117, 101074, VRC03) and nnAbs (F240, 17b, A32), followed by incubation with anti-human Alexa Fluor 647-conjugated secondary Abs. Further, cells were stained on the surface with CD4 Abs, as well as intracellularly with HIV-p24 Abs. (A) Show are flow cytometry dot plot representations of HIV-Env Ab binding on productively infected CD4^lowHIV-p24⁺ T-cells in one representative donor, gated as depicted in Figure S5A. Statistical analysis were performed to determine the effect of metformin on the frequency of CD4^lowHIV-p24⁺ T cells recognized by anti-HIV-Env Abs (B), as well as the geometric MFI of HIV-Env expression (C). Finally, statistical analysis were performed to determine the effect of metformin on the frequency of T cells co-expressing cell-associated HIV-p24 and surface HIV-Env (D), gated as in Figures S5A and S5B. Wilcoxon test p values are indicated on the graphs. Each symbol represents one donor (n = 8; median ± interquartile range).