Metformin Enhances Antibody-Mediated Recognition of HIV-Infected CD4+ T-Cells by Decreasing Viral Release.

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks 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 CD4lowHIV-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-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people.

IMPORTANCE: Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can control virus replication and prolong the life of people living with HIV (PLWH). However, the virus remains dormant within immune cells in what is called the HIV reservoir. Furthermore, 2.3 million PLWH are also coinfected with hepatitis C virus (HCV) and are at risk of developing chronic liver disease and cancer. HCV treatment with direct acting antivirals (DAA) can completely cure the infection in more than 95% of treated individuals and improve their long-term health outcomes. In this study, we investigated how HCV treatment and cure affect the HIV reservoir. We demonstrate the beneficial impact of DAA treatment as it reduces the HIV reservoirs in particular in people infected with HCV before HIV. These results support the need for early ART and DAA treatment in HIV/HCV coinfections.

Targeting Th17 cells in HIV-1 remission/cure interventions.

Since the discovery of HIV-1, progress has been made in deciphering the viral replication cycle and mechanisms of host-pathogen interactions that has facilitated the implementation of effective antiretroviral therapies (ARTs). Major barriers to HIV-1 remission/cure include the persistence of viral reservoirs (VRs) in long-lived CD4+ T cells, residual viral transcription, and lack of mucosal immunity restoration during ART, which together fuel systemic inflammation. Recently, T helper (Th)17-polarized cells were identified as major contributors to the pool of transcriptionally/translationally competent VRs. In this review, we discuss the functional features of Th17 cells that were elucidated by fundamental immunology studies in the context of autoimmunity. We also highlight recent discoveries supporting the possibility of extrapolating this knowledge toward the identification of new putative Th17-targeted HIV-1 remission/cure strategies.

Flow Cytometry Sorting of Memory CCR6+CD4+ T-Cells for HIV Reservoir Quantification.

Antiretroviral therapy (ART) has transformed the deadly human immunodeficiency virus type I (HIV-1) epidemic into a manageable chronic condition. Current ART is not curative and treatment interruption leads to viral rebound in people living with HIV-1 (PLWH). The main cause of viral rebound is the persistence of HIV reservoirs in long-lived memory CD4+ T cells. Accurate techniques to identify and quantify viral reservoirs are required to monitor therapeutic approaches designed to cure HIV infection. Th17-polarized CD4+ T cells are located at mucosal sites of HIV entry and are preferentially targeted for infection and viral reservoir persistence. They constitute an important reservoir in both blood and colon. In this chapter we describe a step-by-step flow cytometry-based protocol to isolate a fraction of Th17-enriched cells from PBMC based on their expression of the Th17 surface marker CCR6. The isolation of memory CCR6+CD4+ T cells allows subsequent PCR/RT-PCR-based HIV DNA/RNA quantifications, as well as their culture for quantitative viral outgrowth assays (QVOA). This method can be adapted for the isolation of CCR6+CD4+ T cells from peripheral tissues, such as the colon.

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth.

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

LILAC pilot study: Effects of metformin on mTOR activation and HIV reservoir persistence during antiretroviral therapy.

BACKGROUND: Chronic inflammation and residual HIV transcription persist in people living with HIV (PLWH) receiving antiretroviral therapy (ART), thus increasing the risk of developing non-AIDS co-morbidities. The mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism and HIV transcription, and therefore represents an interesting novel therapeutic target. METHODS: The LILAC pilot clinical trial, performed on non-diabetic ART-treated PLWH with CD4+/CD8+ T-cell ratios <0.8, evaluated the effects of metformin (12 weeks oral administration; 500-850 mg twice daily), an indirect mTOR inhibitor, on the dynamics of immunological/virological markers and changes in mTOR activation/phosphorylation in blood collected at Baseline, Week 12, and 12 weeks after metformin discontinuation (Week 24) and sigmoid colon biopsies (SCB) collected at Baseline and Week 12. FINDINGS: CD4+ T-cell counts, CD4+/CD8+ T-cell ratios, plasma markers of inflammation/gut damage, as well as levels of cell-associated integrated HIV-DNA and HIV-RNA, and transcriptionally-inducible HIV reservoirs, underwent minor variations in the blood in response to metformin. The highest levels of mTOR activation/phosphorylation were observed in SCB at Baseline. Consistently, metformin significantly decreased CD4+ T-cell infiltration in the colon, as well as mTOR activation/phosphorylation, especially in CD4+ T-cells expressing the Th17 marker CCR6. Also, metformin decreased the HIV-RNA/HIV-DNA ratios, a surrogate marker of viral transcription, in colon-infiltrating CD4+ T-cells of 8/13 participants. INTERPRETATION: These results are consistent with the fact that metformin preferentially acts on the intestine and that mTOR activation/phosphorylation selectively occurs in colon-infiltrating CCR6+CD4+ T-cells. Future randomized clinical trials should evaluate the benefits of long-term metformin supplementation of ART.

Pharmacological Inhibition of PPARy Boosts HIV Reactivation and Th17 Effector Functions, While Preventing Progeny Virion Release and de novo Infection.

The frequency and functions of Th17-polarized CCR6+RORyt+CD4+ T cells are rapidly compromised upon HIV infection and are not restored with long-term viral suppressive antiretroviral therapy (ART). In line with this, Th17 cells represent selective HIV-1 infection targets mainly at mucosal sites, with long-lived Th17 subsets carrying replication-competent HIV-DNA during ART. Therefore, novel Th17-specific therapeutic interventions are needed as a supplement of ART to reach the goal of HIV remission/cure. Th17 cells express high levels of peroxisome proliferator-activated receptor gamma (PPARy), which acts as a transcriptional repressor of the HIV provirus and the rorc gene, which encodes for the Th17-specific master regulator RORyt. Thus, we hypothesized that the pharmacological inhibition of PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector functions. Consistent with this prediction, the PPARy antagonist T0070907 significantly increased HIV transcription (cell-associated HIV-RNA) and RORyt-mediated Th17 effector functions (IL-17A). Unexpectedly, the PPARy antagonism limited HIV outgrowth from cells of ART-treated people living with HIV (PLWH), as well as HIV replication in vitro. Mechanistically, PPARy inhibition in CCR6+CD4+ T cells induced the upregulation of transcripts linked to Th17-polarisation (RORyt, STAT3, BCL6 IL-17A/F, IL-21) and HIV transcription (NCOA1-3, CDK9, HTATIP2). Interestingly, several transcripts involved in HIV-restriction were upregulated (Caveolin-1, TRIM22, TRIM5α, BST2, miR-29), whereas HIV permissiveness transcripts were downregulated (CCR5, furin), consistent with the decrease in HIV outgrowth/replication. Finally, PPARy inhibition increased intracellular HIV-p24 expression and prevented BST-2 downregulation on infected T cells, suggesting that progeny virion release is restricted by BST-2-dependent mechanisms. These results provide a strong rationale for considering PPARy antagonism as a novel strategy for HIV-reservoir purging and restoring Th17-mediated mucosal immunity in ART-treated PLWH.

Daily variations of gut microbial translocation markers in ART-treated HIV-infected people.

BACKGROUND: Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. METHODS: As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and had a comparable amount of sleep, physical exercise and light exposure. Plasma levels of bacterial lipopolysaccharide (LPS) and fungal (1→3)-ß-D-Glucan (BDG) translocation markers, along with markers of intestinal damage fatty acid binding protein (I-FABP) and regenerating islet-derived protein-3α (REG3α) were assessed by ELISA or the fungitell assay. RESULTS: Participants had a median age of 57 years old (range 50 to 63). Plasma levels of BDG and REG3α did not vary significantly over the course of the study. In contrast, a significant increase of LPS was detected between 12:00 and 16:00 (Z-score: - 1.15 ± 0.18 vs 0.16 ± 0.15, p = 0.02), and between 12:00 and 24:00 (- 1.15 ± 0.18 vs 0.89 ± 0.26, p < 0.001). The plasma levels of I-FABP at 16:00 (- 0.92 ± 0.09) were also significantly lower, compared to 8:00 the first day (0.48 ± 0.26, p = 0.002), 4:00 (0.73 ± 0.27, p < 0.001) or 8:00 on secondary day (0.88 ± 0.27, p < 0.001). CONCLUSIONS: Conversely to the fungal translocation marker BDG and the gut damage marker REG3α, time of blood collection matters for the proper evaluation for LPS and I-FABP as markers for the risk of inflammatory non-AIDS co-morbidities. These insights are instrumental for orienting clinical investigations in PLWH.