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.

INTRODUCTION Antiretroviral therapies (ART) efficiently control HIV-1 replication to undetectable plasma levels and have improved the life expectancy of people living with HIV (PLWH) [1][2][3]. However, ART does not cure HIV, with viral rebound occurring rapidly on treatment interruption [2,[4][5][6]. In addition, immunological dysregulations persist in ART-treated PLWH leading to an increased risk for non-AIDS co-morbidities such as cardiovascular disease [7] and neurocognitive impairment [8]. Therefore, additional therapeutic interventions to purge viral reservoirs and restore immunological competence in ART-treated PLWH are needed [9].
In ART-treated PLWH, HIV reservoirs persist in a small fraction of long-lived memory CD4 + Tcells [3,4,[10][11][12] and likely other cellular/anatomic reservoirs [13]. Studies by our group and others demonstrated that among CD4 + T cells, Th17-polarized cells are strategically located at portal sites of HIV/SIV entry and efficiently support integrative HIV infection [14][15][16]. Subsequently, Th17 cells are depleted from the gut-associated lymphoid tissues during HIV/SIV infection, and their frequency is not restored with ART [14,15]. This leads to dramatic alterations in mucosal barrier integrity, increased microbial translocation from the gut, and systemic immune activation [14,15], all leading to non-AIDS co-morbidities [7,8]. Although the depletion of mucosal Th17 cells is well-documented during HIV/SIV infection, a fraction of Th17 cells is long lived and enriched in HIV-DNA in the blood and colon of ART-treated PLWH [14,15]. The key role played by Th17 cells in mucosal homeostasis, their contribution to HIV persistence, as well as the deleterious consequence of their paucity in ART-treated PLWH, indicate that the design of novel Th17-specific therapeutic strategies is needed for HIV remission/cure [14,15].
Th17 cells are distinguished from the other CD4 + T-cell subsets by a unique transcriptional signature that includes multiple HIV permissiveness factors (eg, CCR5, NF-kB, mTOR, NFATC2IP), the lack of anti-HIV defense mechanisms [14,15], as well as the peroxisome proliferator-activated receptor gamma (PPARy) [17][18][19][20]. PPARy is an intrinsic negative regulator of NF-kB (21) and an inhibitor of HIV transcription [17,[22][23][24]. PPARy is a member of the PPAR subfamily of ligand-dependent non-steroid nuclear receptors; PPARy forms an obligatory heterodimer with retinoic X receptor (RXR) and binds onto PPAR responsive elements (PPREs) expressed on the promoters /regulatory regions of specific genes, thus functioning as a transcriptional repressor or activator [25,26]. PPARy is expressed by multiple immune and non-immune cells and acts as a lipid sensor that controls the expression of numerous genes involved in lipid/glucose metabolism. Natural and synthetic PPARy agonists have been documented to regulate metabolic/inflammatory processes [26][27][28][29], in part via the mTOR activation pathway [30]. It is noteworthy that PPREs are present in the HIV long terminal repeat (LTR) region, indicating that PPARy participates directly in the negative regulation of HIV transcription [31]. Increasing evidence supports a role of PPA-Ry in the regulation of adaptive immunity by acting on T-cell proliferation and differentiation [27,29,[32][33][34]. Of particular importance, it was reported that PPARy inhibits Th17 effector functions by the transcriptional repression of RORyt [32,34], the master regulator of Th17 differentiation [14,15].
Clinical trials were previously performed using PPARy agonists/activators, for example, rosiglitazone (RGZ) for treating the lypodystrophy caused by specific classes of antiretroviral drugs [35], as well as metabolic syndrome and inflammation in HIV-infected individuals [36][37][38][39]. However, to our knowledge, no clinical trials were performed using PPARy targeting drugs in the context of HIV cure/remission strategies. Although the PPARy activation blocks HIV replication in primary T cells [17], with PPARy agonists being expected to promote deep latency, studies in SIV-infected rhesus macaques demonstrated that hematopoietic alterations caused by Nef are dependent on the PPARy activation and are mimicked by the PPARy agonist RGZ [40]. Based on this evidence, Prost et al. proposed that PPARy inhibition may be more appropriate to counteract hematopoietic alterations caused by HIV/SIV infections [40] and emphasized the need for the development of clinically advanced PPARy antagonists [41]. Of particular importance, the pharmacological inhibition of PPARy may promote HIV reservoir reactivation, in a manner similar to that of currently tested latency reversing agents (LRA) [42,43]. This scenario is supported by our previous studies demonstrating that RNA interference against PPARy results in increased viral replication on exposure to wild type and single round VSV-G/HIV [17].
In this study, we investigated the effect of PPARy pharmacological inhibition on HIV reservoir reactivation and immune function restoration in Th17 cells, a subset enriched in PPARy mRNA and protein [17,18]. Our results demonstrate that the PPARy antagonism increased both HIV transcription and RORyt-mediated Th17 effector functions, such as IL-17A and IL-21, in CD4 + T cells from ART-treated PLWH. Of note, IL-21 is a signature-cytokine for follicular helper T-cells (Tfh) [33] that is also key for Th17 survival [14] and has demonstrated antiviral activity in vitro [44] and in non-human primate models [45,46]. Unexpectedly, the PPARy antagonism limited viral outgrowth in CD4 + T cells of ART-treated PLWH ex vivo, as well as on HIV infection in vitro. The unique combination of these immunological and virological features provides a strong rationale for considering the pharmacological inhibition of PPARy for HIV cure/remission strategies.

Ethics statement
This study, using PBMCs from HIV-uninfected and HIV-infected study participants was conducted in compliance with the principles included in the Declaration of Helsinki. This study received approval from the Institutional Review Board (IRB) of the McGill University Health Centre and the IRB of the CHUM-Research Centre, Montreal, Quebec, Canada. All participants signed a written informed consent and agreed with the publication of the results generated using their biological samples.

Flow cytometry analysis
The fluorochrome-conjugated antibodies used for polychromatic flow cytometry are listed in Supplemental Table 3. A viability dye (Molecular Probes® LIVE/DEAD® Fixable Dead Cell Stain Kits, Invitrogen) was used to exclude dead cells. Intracellular staining was performed using Fixation/ Permeabilization Solution Kit (BD). Cells were analyzed using an LSRII cytometer, Diva version 6 (BD Biosciences, San Jose, CA), and FlowJo version 10.0.6 (Tree Star, Inc). Flow cytometry gates were defined using the fluorescence minus one (FMO) strategy [19,20].

Cell sorting
Total and memory CD4 + T cells were enriched from PBMCs by negative selection using magnetic beads (magnetic-activated cell sorting [MACS], Miltenyi), with a purity of >95%, as previously described [19,20]. Highly pure CCR6 + /CCR6 -T cells were sorted by FACS using antibodies listed in Supplemental Table 3, as previously reported by our group [19,20].

Viral outgrow assay
A viral outgrowth assay (VOA) was performed using a protocol previously established by our group [19,20]. Briefly, total memory CD4 + T cells isolated by MACS from PBMCs of PLWH receiving viral-suppressive ART (PLWH+ART) were cultured (RPMI1640, 10% FBS, 1% antibi- otics) at 1x10 6 cells/mL/well in 48-well plates in the presence of immobilized CD3 and soluble CD28 antibodies (1 µg/mL) for up to 12 days. At day 3, cells were washed, split into 2 new wells, and cultured with IL-2 (5 ng/mL). At days 6 and 9, cells from each well were split into 2 new wells, and media was refreshed. Supernatants were collected at days 3, 6, 9, and 12 for HIV-p24 and cytokine quantification by ELISA. At day 12, cells were stimulated with PMA (50 ng/mL) and Ionomycin (1ug/mL) in the presence of Brefeldin A (5 ug/mL) for 5 hours and used for the intracellular detection of HIV-p24, IL-17A, and IFN-y by flow cytometry after staining with specific antibodies (Supplemental Table 3).
Quantification of cell-associated HIV-RNA and HIV-DNA Cell-associated (CA) RNA and DNA was dually extracted from cell pellets (polled 5-6 replicates of 1x10 6 cells/experimental condition) using the AllPrep DNA/RNA Mini Kit (Qiagen), according to the manufacturer's instructions. The quality (260 nm/280 nm ratio) and quantity of RNA/DNA collected were evaluated by Nanodrop.
CA LTR-Gag HIV-RNA (CA HIV-RNA) levels were quantified by 1-step real-time RT-PCR using specific external/internal primers and taqman probes (Supplemental Table 4a) and classical RT-PCR/PCR amplification conditions. The amplified products from the first PCR (ProFlex PCR System 9700; Applied Biosystems) were diluted 10 x in molecular grade water and used as templates in second nested real-time PCR amplifications (RotorGene instrument, Qiagen). For the CA LTR-Gag HIV-RNA (unspliced), standards were generated using plasmid-based transcription in vitro (MEGAscript™ T7 Transcription Kit, ThermoFisher).
To normalize HIV-RNA to HIV-DNA on matched samples, levels of CA Gag HIV-DNA were quantified by ultrasensitive nested real-time PCR using the same primers and Taqman probe used for the CA HIV-RNA quantification (Table 4a). To normalize the HIV-DNA levels per number of cells, the CD3 gene was concomitantly amplified using specific external/internal primers and Taqman probes (Supplemental Table 4b), as previously described [19,20]. ACH2 cells carrying 1 copy of integrated HIV-DNA per cell (The National Institutes of Health AIDS Reagent Program) were used for the standard curve.

Quantification of cell-free HIV RNA
The quantification of cell-free HIV-RNA was performed as previously reported [47]. To enrich in HIV virions, 5 mL aliquots of cell culture supernatants were centrifuged at 25,000g for 90 minutes. Pelleted virions (in 140 µL supernatant) were used for total RNA isolation using the QIAamp Viral RNA Mini Kit (Qiagen; final elution in 60 µL). The extracted RNA was first subjected to DNase (Invitrogen) treatment. HIV-RNA quantification was performed as described above. HIV-RNA quantification was performed in triplicates (using 17 µL eluted total RNA/test), as described above. Results are expressed as the number of HIV-RNA copies per reaction (equivalent of 5 mL cell culture supernatant per test). Standards were generated using RNA extracted from ACH2-culture supernatant. All measures were performed in triplicate.

HIV integration
Integrated HIV-DNA was quantified by ultrasensitive nested real-time PCR in cell lysates (10 5 cells/test in triplicate; detection limit: 3 HIV-DNA copies/test), with normalization relative to CD3 copy numbers (2 CD3 copies per single cell), as previously described [12,19,20], using specific primers and FRET probes (Supplemental Tables c-d).
Real-time RT-PCR for quantification of cellular transcripts Total RNA was isolated using the RNeasy Kit (Qiagen) and quantified using the Pearl nanophotometer (Implen). One step SYBR Green real-time RT-PCR (Qiagen) was carried out in a Light-Cycler 480 II (Roche) according to the manufacturer's recommendations, as we previously reported [17,18]. QuantiTect Primer Assays were purchased from Qiagen.

Statistics
All statistical analyses were performed using the Prism 8 (GraphPad software). Specifications on the statistical test used are included on the graphs and Figure legends. P values are indicated on the graphs with statistical significance as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

RESULTS
PPARy inhibition increases IL-17A and HIV transcription but reduces viral production and release in CD4 + T cells of ART-treated PLWH. We hypothesized that PPARy pharmacological inhibition promotes both HIV reservoir reactivation and immune function restoration in Th17 cells. To test this hypothesis, we characterized the effects of the well-characterized PPARy antagonist T0070907 [53] in memory CD4 + T cells from ART-treated PLWH (Table 1, n=8) ( Figure 1A). Cells were stimulated with CD3/CD28 antibodies for 2 days to induce HIV optimal outgrowth [47] and PPARy expression (Supplemental Figure  1) [17]; cells were further cultured in the presence/absence of T0070907 for 2 additional days.
To study the post-integration steps of viral replication (ie, transcription, virion production and release) while preventing novel infection in vitro, experiments were performed in the presence of the antiretroviral drugs (ARV) Saquinavir and Raltegravir ( Figure 1A). Preliminary experiments allowed the identification of an optimal T0070907 concentration (ie, 10µM) that upregulates IL-17A production without affecting cell viability/proliferation (Supplemental Figure 2A). As expected, exposure to T0070907 resulted in a significant increase of IL-17A mRNA levels ( Figure 1B). Upon this short-term stimulation/culture in vitro, CA HIV-DNA levels remained similar in T cells cultured with or without T0070907 ( Figure 1C), consistent with the well-established stability of HIV-DNA reservoirs [4,10]. Nevertheless, exposure to T0070907 significantly increased absolute CA HIV-RNA levels, as well as CA HIV-RNA:HIV-DNA ratios ( Figure 1D-E), indicating that the drug boosted the TCR-mediated HIV transcription. Unexpectedly, cell-free HIV-RNA levels were significantly reduced by T0070907 in 7 of 8 donors ( Figure 1F), indicative of a post-transcriptional block in virion production/release. Thus, the PPARy antagonism overcomes the PPA-Ry-mediated repression of RORyt and HIV transcription, but also modulates expression of other factors acting at the post transcriptional level, thus resulting in decreased de novo production and release of viral particles.

PPARy antagonism inhibits HIV outgrowth from CD4+ T cells of ART-treated PLWH
Productive HIV replication is regulated at multiple post-transcriptional steps [1]. To further document the effect of PPARy antagonism on de novo HIV production, a VOA that monitors viral reservoir reactivation and cell-to-cell propagation in culture [19,20] was performed ( Figure 2A). To optimally detect replication-competent HIV, memory CD4 + T cells were isolated from PLWH receiving ART for >2 years (#5, #10, #12, and #15) and receiving ART <2 years (#3 and #4) ( Table  1). In a first set of experiments, HIV outgrowth was measured by intracellular HIV-p24 staining at day 12 post-stimulation in cells from 8 splitting replicates merged together (generated from 1 original replicate). Results in Figure 2B-C demonstrate that the HIV outgrowth induced by CD3/CD28 triggering was significantly reduced in the presence of T0070907, with no significant impact on cell viability ( Figure 2D). By merging the cells from the 8 identical replicates, it was possible to stimulate the cells with PMA/Ionomycin and monitor the expression of HIV-p24 in cells production IL-17A and/or IFN-y. Consistent with the well-documented Th17 cell permissiveness to HIV [14,15], when the VOA was performed in the absence of T0070907, the highest frequency of infected cells was detected in Th17 (IL-17A + IFN-y -) and Th1Th17 (IL-17A + IFN-y + ) cells; T0070907 reduced the frequency of HIV-p24 + but not IL-17A + cells (data not shown). These results indicate the ability of T0070907 to limit HIV replication in Th17 cells without altering their effector functions. Considering the stochastic distribution of HIV reservoirs, the VOA was performed again with cells from n=4 ART-treated PLWH (Table 1; ART #3, #4, #12, and #15), but this time using 4 original replicates of 10 6 cells/well ( Figure 2E-F) instead of 1 ( Figure 2B-D). The HIV-p24 ELI-SA quantification was performed in cell culture supernatants collected at days 3, 6, 9, and 12 post-stimulation from all splitting replicates. Results in Figure 2E-F confirmed the capacity of T0070907 to inhibit HIV outgrowth.
Thus, the PPARy antagonism inhibits viral outgrowth by acting on viral replication steps downstream of transcription, steps that are important for de novo viral particle production and/or propagation and spread.

PPARy inhibition reduces HIV replication in vitro
Considering the unexpected antiviral features of T0070907, we further investigated its ability to modulate HIV replication in vitro. For this, we used the transmitted/founder (T/F) strain THRO, documented to exhibit high virulence [55], using the experimental design depicted in Figure  3A. TCR-activated memory CD4 + T cells were infected with HIV THRO and treated with T0070907 (1, 5, 10µM) for up to 9 days, with T0070907 being refreshed in the media every 3 days. Results indicate a dose-dependent effect of T0070907, with a significant increase in IL-17A production and a decrease in HIV replication observed at 10µM ( Figure 3B-C), with no effects on cell viability and proliferation (Supplemental Figure 2A). In parallel, similar experiments were performed with T0070907 being added every 3 days versus once (day 0 post infection) or twice (day 0 and 6 post-infection). Results in Figure 3D clearly demonstrate that the antiviral effect of T0070907 At day 12, cells were stained with a viability dye and then intracellularly with HIV-p24 antibodies. (B-D) In a first set of experiments, the VOA was performed with one original replicate (10 6 cells/well) at day 0 that generated 8 splitting replicates at day 12. Shown is (B) the intracellular HIV-p24 expression in cells pooled from the 8 splitting replicates at day 12 from one representative donor (ART #3), as well as statistical analysis of (C) intracellular HIV-p24 staining and (D) cell viability in n=6 ART-treated PLWH (Table  1; ART #3, #4, #5, #10, #12, and #15). (E-F) In another set of experiments, the VOA was performed in 4 original replicates of 10 6 cells/well cultured at day 0 that each generated 8 splitting replicates at day 12.
Shown are HIV-p24 levels in cell culture supernatant quantified in cell culture supernatant collected from the splitting replicates of each original replicate at days 3 (1 well), 6 (2 wells), 9 (4 wells), and 12 (8 wells) for each donor individually (E) and statistical analysis on n=4 ART-treated PLWH at day 12 (F) (   Figure 2B). This is indicative that PPARy inhibition during the early steps of infection allows a robust control of HIV spread in culture.
To get insights into the mechanisms of T0070907 action, we investigated its effect on the expression of the HIV receptor CD4 and co-receptors CCR5/CXCR4. Although T0070907 did not change CD4 and CXCR4 expression, a significant decrease in CCR5 expression was observed (Supplemental Figure 4A-D). Thus, in addition to reducing viral production/release ( Figure 1F), T0070907 also limits de novo infection in part by limiting CCR5-mediated HIV entry.
PPARy antagonism boosts IL-17A expression and reduces HIV replication in CCR6 + CD4 + T cells IL-17A production and HIV permissiveness are key features of memory CCR6 + CD4 + T-cells [14][15][16]. Thus, we further tested the immunological/virological effects of T0070907 in flow cytometry-sorted memory CCR6 + and CCR6 -T cells on HIV infection in vitro ( Figure 4A). In the absence of T0070907, CCR6 + versus CCR6 -T cells expressed significantly higher levels of IL-17A and CCR5 mRNA ( Figure 4B-C, left panels) and supported a more robust HIV-DNA integration (≈2 log 10 difference) ( Figure 4D, left panel). Similar to results on bulk memory T cells, T0070907 significantly increased IL-17A mRNA expression ( Figure 4B, right panel) and reduced CCR5 mRNA expression as well as HIV-DNA integration in memory CCR6 + T cells ( Figure 4C-D, right panels). Thus, consistent with superior expression of PPARy in CCR6 + Th17/Th1Th17-polarized versus CCR6 -Th1-polarized T cells [17,18,32,34], T0070907 acted on CCR6 + T cells to upregulate IL-17A production and limit HIV de novo infection by mechanisms including CCR5 downregulation.
RNA-Sequencing reveals a complex network of cellular processes positively or negatively regulated by PPARy in memory CCR6+CD4+ T cells To get further insights into the mechanism of action of PPARy antagonism, genome-wide transcriptional profiling was performed in CCR6 + T cells stimulated via the TCR for 3 days and cultured in the presence or absence of T0070907 for an additional 18 hours ( Figure 5A). Differentially expressed genes were classified based on P values (P) or adjusted P values (adj. P) and fold change (FC) gene expression. Profound transcriptional changes were induced by T0070907 in CCR6 + T cells, with 4,002 transcripts upregulated and 1,249 transcripts downregulated (adj. T/F HIV THRO strain (25 ng/10 6 cells) and cultured in the presence of IL-2 (5 ng/ml) and in the presence/ absence of T0070907 (1, 5, and 10µM) for up to 9 days, with media, IL-2 and/or T0070907 being refreshed every 3 days. Shown are HIV-p24 levels (B) and IL-17A     Figure 6A-C), ii) inflammation/immune response to type I interferon (Supplemental Figure 5D), and iii) cytokines, chemokines and adhesion molecules (Supplemental Figure 5E-H). Differentially expressed genes linked to the GO term lipid/phospholipid metabolism, include the upregulation of the transcription factors PPARy, PPARα, KLF4, and NR4A3; the pattern recognition receptor NOD2; the tetraspanin CD81; the signaling molecules PTK2, PLA2G6, FGF2, and FLT1; the guanine nucleotide exchange factor VAV3; the hormone ADIPOQ/adiponectin; the cytokines TNF and IFNG; the downregulation of the ATP transporter ABCG1; the G protein RAC1; and the cell cycle regulator CDC42 (Supplemental Figure 5A-B). Differentially expressed genes linked to the GO term glucose metabolism include the upregulation of the glycosylphosphatidylinositol (GPI) degrading enzyme GPLD1, the insulin-like growth factors IGF1 and IGF2, and the phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1); and the downregulation of the enzymes tyrosine-protein phosphatase non-receptor type 2 (PTPN2) and diglyceride acyltransferase (DGAT2) (Supplemental Figure  5C). Differentially expressed genes linked to the GO term inflammation/immune response to type I interferon were mainly downregulated by T0070907 and included genes documented to play a positive/negative regulatory role in HIV replication such as ADAR, MX2, MX1, OAS1, RNASEL, SAMHD1, ISG15, ISG20, IFITM2, IFITM3, and TRIM56; of note, transcripts coding for the restriction factor BST2 were upregulated (Supplemental Figure 5D). Finally, Differentially  These results reveal a previously unrecognized complex network of cellular processes that are positively/negatively controlled by PPARy in Th17-polarized CCR6 + T cells, with relevance for understanding the dichotomous effects of T0070907 on the various steps or HIV replication.
A Tfh-specific transcriptional signature induced upon PPARy inhibition Ingenuity Pathway Analysis revealed the upregulation and downregulation of transcripts previously linked to the negative (eg, IL-21, CAV1, BST2) and positive (eg, furin) regulation of HIV replication, respectively ( Figure 5D). Considering the well-documented role of IL-21 in modulating Th17/Tfh survival [14,15], as well as its antiviral properties [44, 45, 56], we pursued the validation of IL-21 at the protein level. Results generated with memory CCR6 + T cells from 5 individuals confirmed the significant upregulation of IL-21 protein production by T0070907 ( Figure 5E)

HIV-dependency factors modulated by PPARy inhibition
A meta-analysis using the NCBI HIV-1 interactions database allowed the identification of human genes previously involved in HIV-1 infection that are modulated by T0070907 in CCR6 + T cells. Specifically, TRIM5, TNF, TRIM22, BST2, IL-2, IL-3, LIF, IL-10, CXCR4, SERP1, and CD4 were Together, these RNA-Seq results reveal that T0070907-mediated transcriptional reprogramming is associated with the negative regulation of multiple steps of the viral replication cycle such as CCR5-mediated entry, the uncoating (eg, TRIM5), reverse transcription (eg, SAMHD1), Nef-mediated functions (eg, IL-21, miR29), viral particle production (eg, TRIM22), release (eg, BST2), . Unexpectedly, the PPARy antagonism prevented de novo production/release of virions from reservoir cells by negatively interfering with multiple steps of the HIV replication cycle, from virion maturation (eg, furin) and viral particle release (eg, BST2), to viral entry into new target cells (eg, CCR5), as well as the IL-21/miR-29 antiviral axis. Thus, the PPARy antagonism may represent a new strategy to eradicate HIV reservoirs in Th17 cells. Table 1: Clinical parameters of ART-treated PLWH study participants.

DISCUSSION
In this study, we reveal the unique features combined by the PPARy antagonist T0070907 including the positive regulation of HIV transcription/translation and Th17/Tfh-specific effector functions in memory CD4 + T cells of ART-treated PLWH, together with its capacity to reduce de novo virion production and/or spread from HIV reservoir cells. By using a genome-wide transcriptional profiling in Th17-polarized CCR6 + CD4 + T cells, we revealed a complex transcriptional reprogramming underlying the observed immunological/virological features of T0070907, with antiviral mechanisms located at multiple steps of the HIV replication cycle downstream translation, including the BST-2-mediated restriction of HIV release ( Figure 8).
In addition to the knowledge that PPARy acts as a repressor of HIV [31] and RORyt transcription [32, 34], we demonstrate that the pharmacological inhibition of PPARy using the antagonist T0070907 [53] boosted HIV transcription and RORyt-mediated transcription of Th17-specific genes. Conversely, we observed an unexpected block in the viral production and release and/or spread in culture observed during viral outgrowth ex vivo and HIV infection in vitro. Of particular importance, T0070907 acted preferentially on CCR6 + Th17-polarized T cells, a subset known to be enriched in HIV reservoirs in ART-treated PLWH [16,63], to increase IL-17A production and reduce CCR5 expression and viral replication in vitro. Similar to T0070907, the literature documents that PKC-q activators such as prostratin, a non-tumor-promoting phorbol ester, also acts as an LRA while blocking de novo HIV production to mediate the elimination of HIV reservoirs by a kick and kill strategy [64][65][66][67]. Whether the effects of prostratin and its derivatives [68] also involve PPARy-modulated processes remains to be determined. However, one major difference is that PKC-q activators downregulate CD4, while T0070907 does not.
The PPARy/RXR heterodimer is known to target genes involved in lipid metabolism such as cholesterol and fatty acids that influence multiple aspects of antiviral immunity [26, 28, 69]. Among oxysterols presenting antiviral properties, 25HC, metabolized from cholesterol by the enzyme CH25H, blocks the replication of HIV by acting on the viral entry but not transcription [70], with the effects on the post-transcriptional steps of the replication cycle remaining unexplored.
In addition, 25HC has been identified as a natural ligand for RORyt [71,72]. The fact that PPA-Ry deficiency was linked to CH25H overexpression [73], prompted our initial hypothesis that T0070907 blocks HIV outgrowth ex vivo and infection in vitro and boosts Th17 effector functions via CH25H/25HC-dependent mechanisms. In agreement, T0070907 upregulated the expression of CH25H mRNA in TCR-activated CCR6non-Th17 cells (data not shown), further explaining their relative resistance to HIV infection [14,15]. However, CH25H mRNA was undetectable in CCR6 + Th17 cells (data not shown), indicating that T0070907 exerts its antiviral effects in Th17 cells via CH25H/25HC-independent mechanisms.
To investigate mechanisms by which T0070907 disconnects HIV transcription from downstream viral replication steps, we performed a genome-wide transcriptional profiling using the RNA-Seq Illumina technology. GSVA identified activation of pathways linked to lipid/phospholipid and glucose metabolism. Metabolic reprogramming during TCR triggering trains T cells to integrate immunological and metabolic information required for the subsequent acquisition of specific effector functions [74]. Glucose metabolism has been identified to play a central role in HIV replication, with the glucose transporter GLUT1 being a marker for HIV permissive T cells [75]. Metabolism disruption is associated with HIV disease progression, with higher glucose uptake being observed in CD4 + T cells of PLWH compared to non-infected individuals [76]. Recent studies linked the susceptibility to HIV infection to the metabolic status of specific CD4 + T-cell subsets [77]. Changes in the CD4 + T-cell metabolic program are controlled by the mTORC1/PPARy axis [74,78]. In line with this, T0070907 upregulated genes associated with PI3K/Akt signaling, a pathway known to promote mTOR activation [15]. Indeed, several groups including ours, identified mTOR as a positive regulator of HIV replication [20], acting at the level of viral entry [79] and transcription [80,81]. Indeed, in preliminary studies, we demonstrated that TCR triggering in the presence of T0070907 leads to increased mTOR phosphorylation. Therefore, the activation of the PI3K/Akt pathway in the presence of T0070907 might be in part responsible of the increase in HIV transcription, likely via mTOR-dependent mechanisms.
GSVA identified pathways modulated by T0070907 in CCR6 + T cells revealed that PPARy antagonism produces profound transcriptional modifications linked to the metabolism of cellular membrane components, including glycosaminoglycan, glycosphingolipid, and sphingolipid. These components of the cellular membrane play a key role in membrane organization and membrane raft formations [29]. Membrane receptors such as the HIV co-receptors CCR5/ CXCR4 are recruited to the membrane raft, and the clustering of these receptors promotes HIV entry into target cells [82]. In addition, membrane rafts play a crucial role in HIV-1 assembly and release [83,84]. Therefore, modification of the cellular composition and membrane raft formation by T0070907 may contribute to the decreased HIV entry/release; additional investigations are needed to clarify this. The formation of biofilms rich in collagen and cell-host molecules such as tetherin/BST2 has been reported for human T-cell leukemia virus type 1 (HTLV-1) [85]. The possibility that other viruses such as HIV form biofilms remains to be determined [86]. Of note, the main upregulated gene by T0070907 is fibromodulin (FMOD), a component of the extracellular matrix which participates in the assembly of collagen fibers. In line with this, the collagen triple helix repeat containing 1 (CTHRC1) and the tetherin/BST2 transcripts were upregulated by T0070907. These findings indicate that T0070907 facilitates the establishment of biofilms able to trap newly produced virions thus preventing their spreading.
The GSVA of GO pathways also revealed the downregulation of pathways/transcripts linked to interferon responses. Multiple interferon-stimulated genes (ISG), documented to restrict HIV replication, were downregulated by T0070907 in CCR6 + T cells. Among these transcripts, we noted a decreased expression of SAMHD1, which limits HIV reverse transcription and promotes HIV-RNA degradation [87]; MX2, which limits viral decapsidation, pre-integration complex formation and nuclear import [88,89]; IFITM2 and IFITM3, known to interact with HIV-1 Env in infected cells and impair Env processing and incorporation into virions [90]; and ISG15, known to induce ISGylation of viral Gag proteins and impeded HIV release [91]. These results point to a previously unrecognized implication of PPARy in the positive transcriptional regulation of specific HIV-restriction factors, including SAMHD1, MX2, IFITM2, IFITM3, BST2, and ISG15, in line with the antiviral program promoted by PPARy activation [17].
Our RNA-Seq results also revealed a T0070907-mediated increase in the expression of the classical Tfh markers CXCR5, ICOS, BCL6, PD- . Therefore, the IL-21/ miR-29 axis is highly likely to contribute to the antiviral effects of PPARy antagonism.
The meta-analysis performed using the NCBI HIV-1 interaction database pointed to additional T0070907-mediated antiviral mechanisms. Specifically, T0070907 upregulated expression of CAV1, reported to inhibit HIV particle production in macrophages [92]; SERINC5, which is incorporated into virions and prevents the fusion of the virion with the cellular membrane of a new target cell [93]; TRIM22, which blocks Gag migration to the plasma membrane and inhibits HIV particle production [94]; and BST2, which limits viral particle release [87]. A T0070907-mediated upregulation of the HIV restriction factor TRIM5α, which interacts with the HIV capsid and induces its proteasomal degradation leading to premature decapsidation [95], was also observed. Finally, T0070907 downregulated furin, a protease preferentially expressed in Th17 cells [17,18] and involved in HIV protein Env maturation and virion infectivity [96]. Thus, the antiviral features of T0070907 involve mechanisms dependent on CAV1, SERINC5, TRIM22, and BST2 over-expression, as well as furin downregulation, thus explaining a post-transcriptional block in HIV virion production and/or release.
Finally, the counterintuitive capacity of PPARy antagonism to decrease viral release/outgrowth while increasing viral transcription prompted us to focus on Tetherin/BST-2, an HIV restriction factor counteracted by Vpu and documented to mediate HIV tethering on the surface of infected cells [59-61]. Of note, T0070907 increased BST-2 mRNA expression in uninfected CCR6 + CD4 + T cells. In a model of single round VSV-G/HIV infection in vitro, as expected, BST-2 protein expression was downregulated on infected T cells in the absence of T0070907. In contrast, the BST-2 expression was significantly higher on the surface of infected cells exposed to T0070907. An in silico search using the ENCODE database revealed that BST-2 encodes PPREs in its promoter and represents a putative direct PPARy target in CD4 + T cells. Thus, PPARy inhibition boosts HIV reactivation, while preventing progeny virion release from infected cells via BST-2-dependent mechanisms. The recognition of such reactivated viral reservoirs by antibodies and immune cells for subsequent clearance will be key for HIV cure. Future studies in vitro and in preclinical models are needed to determine whether PPARy antagonism promotes HIV reservoir purging in shock and kill strategies.
In conclusion, our results reveal complex previously unrecognized PPARy-dependent host-cell molecular circuits involved in the positive, as well as the negative regulation of various steps of the HIV replication cycle and demonstrate the possibility of disconnecting HIV transcription and translation from viral particle production/release (Figure 8). The efficacy of the PPARy antagonism in boosting IL-21 production is of major importance, considering IL-21 paucity during HIV infection [14,15] and its documented antiviral/immune-regulatory features [44-46, 56]. Therefore, the pharmacological inhibition of PPARy may represent a new promising therapeutic strategy to boost Th17-effector functions that are key for mucosal immunity restoration and to promote HIV-reservoir purging in ART-treated PLWH.

Supplementary Figure 3. RGZ limits HIV outgrowth in memory CD4+ T cells of ART-treated PLWH.
A VOA was performed with memory CD4+ T cells of ART treated PLWH (Table 1;