Galectin-9 Mediates HIV Transcription by Inducing TCR-Dependent ERK Signaling

Endogenous plasma levels of the immunomodulatory carbohydrate-binding protein galectin-9 (Gal-9) are elevated during HIV infection and remain elevated after antiretroviral therapy (ART) suppression. We recently reported that Gal-9 regulates HIV transcription and potently reactivates latent HIV. However, the signaling mechanisms underlying Gal-9-mediated viral transcription remain unclear. Given that galectins are known to modulate T cell receptor (TCR)-signaling, we hypothesized that Gal-9 modulates HIV transcriptional activity, at least in part, through inducing TCR signaling pathways. Gal-9 induced T cell receptor ζ chain (CD3ζ) phosphorylation (11.2 to 32.1%; P = 0.008) in the J-Lat HIV latency model. Lck inhibition reduced Gal-9-mediated viral reactivation in the J-Lat HIV latency model (16.8–0.9%; P < 0.0001) and reduced both Gal-9-mediated CD4+ T cell activation (10.3 to 1.65% CD69 and CD25 co-expression; P = 0.0006), and IL-2/TNFα secretion (P < 0.004) in primary CD4+ T cells from HIV-infected individuals on suppressive ART. Using phospho-kinase antibody arrays, we found that Gal-9 increased the phosphorylation of the TCR-downstream signaling molecules ERK1/2 (26.7-fold) and CREB (6.6-fold). ERK and CREB inhibitors significantly reduced Gal-9-mediated viral reactivation (16.8 to 2.6 or 12.6%, respectively; P < 0.0007). Given that the immunosuppressive rapamycin uncouples HIV latency reversal from cytokine-associated toxicity, we also investigated whether rapamycin could uncouple Gal-9-mediated latency reactivation from its concurrent pro-inflammatory cytokine production. Rapamycin reduced Gal-9-mediated secretion of IL-2 (4.4-fold, P = 0.001) and TNF (4-fold, P = 0.02) without impacting viral reactivation (16.8% compared to 16.1%; P = 0.2). In conclusion, Gal-9 modulates HIV transcription by activating the TCR-downstream ERK and CREB signaling pathways in an Lck-dependent manner. Our findings could have implications for understanding the role of endogenous galectin interactions in modulating TCR signaling and maintaining chronic immune activation during ART-suppressed HIV infection. In addition, uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of recombinant Gal-9 within the reversal of HIV latency eradication framework.


INTRODUCTION
Antiretroviral therapy (ART) effectively suppresses HIV replication but does not achieve viral eradication due to the persistence of latently-infected, long-lived CD4 + T cells (1,2). This persistent infection leads to continued immune activation, chronic inflammation, and ongoing damage to multiple organ systems (3,4). Many studies indicate that HIV persistence is regulated, at least in part, by the immune system (5)(6)(7)(8). Thus, understanding the host immune factors driving and maintaining HIV persistence is needed to develop new strategies to cure HIV and/or prevent the development of HIV-associated co-morbidities, which remain prevalent despite suppressive therapy.
One key regulator of immunological functions, and several cellular processes, is interactions between cell-surface glycans and glycan-binding proteins (lectins) (9)(10)(11)(12). One class of lectins that play critical roles in T cell function are galectins, a family of β-galactoside-binding, soluble lectins (13)(14)(15)(16)(17). Among galectins, galectin-9 (Gal-9) has recently been recognized to play an essential role in regulating both adaptive and innate defense mechanisms and thus may be involved in HIV pathogenesis (18)(19)(20). Our prior work showed that endogenous secretion of Gal-9 is rapidly increased after HIV infection, and that elevated levels of Gal-9 do not return to normal after suppressive ART (20). More recently, we reported that the endogenous levels of Gal-9 are associated with HIV transcription in vivo, in plasma of HIV-infected, ART-suppressed individuals (8). We also demonstrated that treating CD4 + T cells with recombinant Gal-9 (rGal-9) induces HIV transcription and reverses HIV latency in vitro and ex vivo (8). However, the signaling pathways by which Gal-9 modulates HIV transcriptional activity remain unclear.
The goal of this work was to identify the signaling mechanisms underlying Gal-9-mediated HIV transcription. We hypothesized that Gal-9 modulates HIV transcriptional activity through T cell receptor (TCR) signaling transduction, based on results in non-HIV contexts showing that galectins, including Gal-9, modulate TCR-signaling (21)(22)(23), and that Gal-9 interacts with various cell-surface proteins known to induce TCR signaling, including CD44 (24) and 41-BB (25)(26)(27). We show that Gal-9 modulates HIV transcription through activating the TCR-downstream ERK and CREB signaling pathways in a lymphocyte-specific protein tyrosine kinase (Lck)-dependent manner. This signaling pathway also induces an undesirable, pro-inflammatory response, namely secretion of IL-2 and TNF-α, and activating CD4 + T cells. This pro-inflammatory response can be inhibited using the mTOR pathway inhibitor, rapamycin, without impacting Gal-9mediated viral reactivation. Our results could have implications for understanding the role of endogenous galectin-9 in modulating TCR signaling in vivo and maintaining chronic immune activation during ART-suppressed HIV infection. In addition, uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of rGal-9 within the reversal of HIV latency eradication framework.

Cell Lines
As a model of HIV latency, we used "J-Lat" cells, which harbor latent, transcriptionally competent HIV provirus that encodes green fluorescent protein (GFP) as an indicator of viral reactivation (28,29). We have shown in our previous work (8) that this latency model mimics the impact of Gal-9 on HIV transcription ex vivo using CD4 + T cells from HIVinfected ART-suppressed individuals. Therefore, it can be used to investigate the signaling mechanisms underlying Gal-9-mediated viral transcription. J-Lat 5A8 clone was kindly provided by Dr. Warner Greene (The Gladstone Institute of Virology and Immunology). J-Lat 15.4 (catalog number 9850), 10.6 (catalog number 9849), and Jurkat E6-1 (catalog number 177) clones were provided by the NIH AIDS Reagent Program (Germantown, MD). J.CaM1.6 clone, a derivative mutant of Jurkat cells, which is deficient in Lck activity (30) Isolation and Treatment of Primary CD4 + T Cells CD4+ T cells were enriched from the cryopreserved PBMCs by negative selection using the EasySep Human CD4 + T Cell Enrichment Kit (Stemcell Technologies), according to the manufacturer's instructions. Primary CD4 + T cells were maintained in RPMI with L-glutamine supplemented with 20% FBS. Primary CD4 + T cells were pre-incubated with 1 µM for Lck inhibitor or 5 µM for rapamycin. Cells were then treated with 500 nM of rGal-9, ImmunoCult human CD3/CD28 T Cell Activator, or an equivalent volume of PBS, for 24 h. Cells were centrifuged for 5 min at 250 g, and cells and supernatants were collected separately.

Measurement of TNF-α and IL-2 Levels Using ELISA
Supernatants from J-Lat 5A8 and primary CD4 + T cells cultures treated or not with rGal-9, αCD3/αCD28, and inhibitors were collected. Levels of TNFα and IL-2 cytokines were quantified using human TNF and IL-2 DuoSet Elisa kits (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. Optical density was measured at 450 and 540 nm using Versa Max microplate reader. Data were analyzed using GraphPad Prism.

Statistical Analysis
Two-tailed paired and unpaired t-tests were used for comparisons. All statistical analyses were conducted using GraphPad Prism release 7.0 (GraphPad Software, San Diego, CA, United States) and statistical significance was set at a p-value of 0.05.
Confirming dose-dependent relationship, we found that rGal-9 is able to phosphorylate CD3ζ starting from 25 nM in the J-Lat 5A8 HIV latency model (Supplementary Figure 2). These data demonstrate that Gal-9 induces TCR-signaling in HIV latentlyinfected cells through Lck-mediated CD3ζ phosphorylation.

Gal-9 Induces Phosphorylation of ERK and CREB Signaling Molecules
To identify components of the TCR signaling cascade that are induced by Gal-9 downstream of CD3ζ phosphorylation, we used Proteome Profiler Human Phospho-Kinase arrays to assess the phosphorylation levels of 43 kinases and related transcription factors. rGal-9 treatment of J-Lat 5A8 cells induced the phosphorylation of several downstream effectors of TCR signaling, including ERK1/2 (26.7-fold) and CREB (6.6-fold) (Figures 1B,C). These data demonstrate that Gal-9-mediated induction of CD3ζ phosphorylation results in activation of ERK1/2 and CREB signaling pathways.

Gal-9-Mediated CD4 + T Cell Activation Is Lck-Dependent
Next, to determine whether Gal-9-mediated T cell activation is dependent on the same mechanism as Gal-9-mediated viral activation, we examined the impact of Lck inhibition of Gal-9-mediated CD4 + T cell activation in primary cells from HIV-infected ART-suppressed individuals. Cell viability was determined using Zombie Aqua Fixable Viability and apoptosis was determined using Propidium iodide and Annexin V staining (Supplementary Figure 6). T cell activation was measured by co-expression of the T cell-surface activation markers CD69 and CD25. rGal-9 induced CD4 + T cell activation [from 0.14 ± 0.03% to 10.34 ± 1.1% (mean ± SEM)]. This effect was significantly reduced by Lck inhibition (from 10.34 ± 1.1% to 1.65 ± 0.5%; P = 0.0006) (Figure 3). Lck inhibition did not fully inhibit Gal-9-mediated T cell activation, suggesting that while TCR signaling plays a major role in Gal-9-mediated T cell activation, other signaling pathways may contribute to this effect.

Gal-9 Induces IL2 and TNF-α Secretion by Inducing Lck-Dependent ERK and CREB Signaling
Given that Gal-9 induces TCR signaling and activates T cells, we posited that the Gal-9-mediated reactivation of HIV latency would be accompanied by secretion of pro-inflammatory cytokines. Indeed, rGal-9 treatment of J-Lat 5A8 cells was associated with a significant induction of IL2 [from < 15.6 pg/ml to 375.6 ± 28.1 pg/ml (mean ± SD)] and TNFα (from <15.6 pg/ml to 208.8 ± 9.7 pg/ml). This effect was significantly reduced by inhibiting Lck, ERK, or CREB activity (P < 0.001; Figures 4A,B). Similar results were obtained using primary CD4 + T cells from HIV-infected, ART-suppressed individuals: rGal-9 significantly induced the production of IL2 (from < 15.6 pg/ml to 700.2 ± 93.1 pg/ml (mean ± SEM)] and TNFα (from <15.6 pg/ml to 1372.68 ± 240.75 pg/ml); Lck inhibition significantly reduced IL2 (from 700.2 ± 93.1 pg/ml to 47.9 ± 47.9 FIGURE 3 | Gal-9-mediated CD4 + T cell activation is Lck-dependent. CD4 + T cells isolated from 5 HIV-infected ART-suppressed individuals were treated for 24 h with Gal-9 (500 nM) or the equivalent volume of PBS (Control) in the presence of 1 µM of Lck inhibitor or the equivalent volume of DMSO. Cells were analyzed by flow cytometry for expression of CD69 and CD25 activation markers. (A) % of CD4 + T cells expressing CD69, (B) % of CD4+ T cells expressing CD25, (C) % of CD4 + T cells co-expressing CD69 and CD25. (D) A representative flow cytometry plot of one individual. αCD3/αCD28 antibodies were used as a positive control. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed paired t-tests. **p < 0.01, and ***p < 0.001. pg/ml; P = 0.0006) and TNFα secretion (from 1372.68 ± 240.75 pg/ml to 37.8 ± 37.8 pg/ml; P = 0.0035) (Figures 4C,D). Further, we examined the impact of rGal-9 on the secretion of a panel of pro and anti-inflammatory cytokines. Supplementary Figure 7 shows that rGal-9 induces the secretion of several pro-and anti-inflammatory cytokines. Together the data in Figures 3,  4 demonstrate that Gal-9-mediated T cell activation and proinflammatory cytokine secretion are dependent on the same mechanism that induces HIV transcriptional activity, namely Lck-mediated induction of the TCR downstream ERK/CREB signaling. These undesirable, pro-inflammatory effects of Gal-9 limit its potential to be used as an HIV latency reversal agent. Thus, we asked whether it would be possible to separate the desirable (HIV latency reversal) from the undesirable (T cell activation and cytokine secretion) effects of Gal-9.

DISCUSSION
Gal-9 promotes HIV transcription by a previously unidentified mechanism (8). Given that Gal-9 is known to cross-link several surface proteins, some of which are involved in TCR signaling and that Gal-9 can induce TCR signaling (22), we hypothesized that Gal-9 promotes HIV transcriptional activity through TCR signaling transduction. In this study, we demonstrate that Gal-9 modulates HIV transcription through activating the TCR-downstream ERK and CREB signaling pathways, in an Lck-dependent manner. We also show that this same signaling pathway that induces HIV transcription also induces an undesirable pro-inflammatory response, namely secretion of IL-2 and TNFα as a consequence of T cell activation. These undesirable, Gal-9-mediated pro-inflammatory responses can be inhibited using the mTOR pathway inhibitor, rapamycin, without blunting Gal-9-mediated viral reactivation.
Gal-9 has several effects on T cells during HIV infection. It renders CD4 + T cells less susceptible to HIV infection via induction of the host restriction factor cyclin-dependent kinase inhibitor 1 (p 21) (42). It can also increase HIV entry by inducing the CD4 + T cell-surface concentration of protein disulfide isomerase (PDI) (43). We have previously shown that the endogenous levels of Gal-9 are induced after HIV infection and that these levels do not return to normal levels after ART suppression (20). We also found a positive correlation between endogenous levels of Gal-9 and levels of HIV transcription in CD4 + T cells during ART suppression (8). The impact of this chronically elevated levels of Gal-9 on immune functions during ART-suppressed HIV infection is not FIGURE 5 | Rapamycin reduces Gal-9-mediated cytokine-secretion without impacting the ability of Gal-9 to reactivate HIV. (A) Viral reactivation in J-Lat 5A8 cells by Gal-9 (200 nM) in the presence or absence of 5 µM of rapamycin. Concentration of (B) IL2, and (C) TNF-α quantified by ELISA in cell culture supernatants. Mean ± SD is displayed, and statistical comparisons were performed using two-tailed unpaired t-tests. ***p < 0.001. Concentration of IL2 (D) and TNF-α (E) secretion quantified by ELISA in culture supernatants of CD4 + T cells isolated from five HIV+ ART-suppressed individuals after 24 h of Gal-9 stimulation (500 mM) in the presence of absence of 5 µM rapamycin. Mean ± SEM are displayed, and statistical comparisons were performed using two-tailed paired t-tests. **p < 0.01, and ***p < 0.001.
clear. Our current study demonstrates that Gal-9 induces Lckdependent ERK/CREB signaling in HIV-infected latently infected cells, which may explain the correlation between Gal-9 and HIV transcriptional activity in vivo. However, this induction of TCR signaling by Gal-9 raises an important question of whether elevated endogenous Gal-9 levels contribute to the state of chronic inflammation and chronic immune activation during suppressive ART. Elevated T cell activation persists during suppressive ART (44) and is associated with the development of HIV-associated co-morbidities and premature mortality (44)(45)(46)(47); it may also contribute to HIV persistence (48)(49)(50)(51). This chronic immune activation involves multifactorial mechanisms (52)(53)(54)(55)(56)(57)(58)(59), and the persistent induction of TCR signaling by the elevated levels of endogenous Gal-9 may be playing an important, unrecognized, role in sustaining it. Interventions that target Gal-9 may prove useful in inhibiting chronic immune activation, which might ultimately reduce the development of HIV-associated co-morbidities and levels of HIV persistence during suppressive ART.
Gal-9 is a multifaceted lectin, with opposing roles in modulating innate and adaptive immune responses. Gal-9 was mainly described to exhibit immunosuppressive activities (24,(60)(61)(62)(63)(64)(65)(66)(67). Gal-9 can also increase the function of regulatory T cells (T-regs) through interaction with CD44 (24) and may impair Natural Killer cells (NK) cytotoxicity and cytokine production through a Tim-3 independent mechanism (68). Along the same line, two recent studies demonstrated that Gal-9 suppresses B cell receptor signaling and B cell activation through interaction with CD45 and IgM-BCR complex (69,70). However, other studies showed that Gal-9 exhibits immunopotentiating activity in the setting of immunosuppression (71) and induces TCR signaling (22), similar to our current study. Also, the endogenous levels of Gal-9 are induced during many inflammatory conditions (66,(72)(73)(74). Together, it is likely that Gal-9 effects are contextdependent and cell-type-dependent. The effect of Gal-9 on cellmediated immunity in different subsets of T cells and other immune cells, during ART-suppressed HIV infection, warrants a broader investigation. Clarifying the signaling pathways induced or inhibited by Gal-9 in different cell-population, during HIV infection, may provide insights that may lead to the development of novel therapies to improve immune functionality, and reduce inflammation-associated co-morbidities, in the setting of viral suppression by ART.
The ability of Gal-9 to potently induce latent HIV transcription suggested that it could be considered within the "shock and kill" HIV eradication framework (8). However, the adverse Gal-9-mediated induction of pro-inflammatory cytokines, by the same pathway, limits the potential use of Gal-9 as a shock and kill agent. The recent study by Martin et al. (40) raised the possibility of using rapamycin, an immunosuppressive agent that does not affect TCR signaling, to prevent the adverse effects of T cell activation without impacting HIV transcription. Unlike several immunosuppressive agents that impact TCR signaling, rapamycin suppresses IL-2 downstream signaling (75,76). Our data show that rapamycin is able to inhibit the pro-inflammatory impact of Gal-9 without affecting its ability to reactivate latent infection. That is in agreement with a previous study that used rapamycin to inhibit the proinflammatory effects of galectin-9 on dendritic cells and promote allograft tolerance in mice (41). Uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of recombinant Gal-9 within the "reversal of HIV latency" eradication framework.
In summary, we identified TCR/Lck-dependent ERK1/2-CREB as the signaling pathways underlying Gal-9 modulation of HIV transcriptional activity. We also found that rapamycin can uncouple the Gal-9 impact on HIV transcription from the undesirable, pro-inflammatory secretions associated with inducing TCR signaling. Our data highlight the further investigations needed to comprehensively understand the immunologic consequences of Gal-9 in vivo, during ARTsuppressed HIV infection. Our findings could have implications for understanding the role of endogenous galectin interactions in modulating TCR signaling and maintaining chronic immune activation, that persists during ART-suppressed HIV infection. Finally, uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of recombinant Gal-9 within the reversal of HIV latency eradication framework.

ACKNOWLEDGMENTS
We would like to thank Rachel E. Locke, Ph.D. for providing comments and editing.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00267/full#supplementary-material Supplementary Figure 1 | Gal-9-mediated CD3ζ phosphorylation is Lck-dependent. Jurkat E6-1 and J.CaM1.6 (a derivative mutant of Jurkat cells, which are deficient in Lck activity) cells were treated with Gal-9 (200 nM), or an equivalent volume of PBS, for 15 min and stained with PE-conjugated anti-phospho-CD3ζ antibody. Cell staining/phosphorylation was quantified by flow cytometry. Mean ± SD is displayed, and statistical comparisons were performed using two-tailed unpaired t-tests. * * * p < 0.001.
Supplementary Figure 2 | Low concentrations of Gal-9 induce CD3ζ phosphorylation. J-Lat 5A8 cells were treated with escalating doses of Gal-9 (0-200 nM) for 15 min and stained with PE-conjugated anti-phospho-CD3ζ antibody. Cell staining/phosphorylation was quantified by flow cytometry. Mean ± SD is displayed, and statistical comparisons were performed using two-tailed unpaired t-tests comparing each concentration to the 0 nM control. * p < 0.05, and * * p < 0.01.
Supplementary Figure 3 | Gal-9-mediated HIV latency reactivation is Lck-dependent. J-Lat 5A8 were transfected with Lck siRNA or non-target siRNA control using Amaxa Nucleofector4D. After 48 h, cells were treated with rGal-9 (200 nM), or an equivalent volume of PBS, for 24 h. HIV-encoded GFP expression was detected by flow cytometry. Mean ± SD is displayed, and statistical comparisons were performed using two-tailed unpaired t-tests. * * * p < 0.001, and * * * * p < 0.0001. Figure 4 | Low concentrations of Gal-9 reactivate latent HIV in the J-Lat HIV latency model. J-Lat 5A8 cells were treated with escalating doses of Gal-9 (0-200 nM) for 24 h. HIV-encoded GFP expression was detected by flow cytometry. Mean ± SD is displayed, and statistical comparisons were performed using two-tailed unpaired t-tests comparing each concentration to the 0 nM control. * p < 0.05, * * * p < 0.001, and * * * * p < 0.0001. Mean ± SD is displayed, and statistical comparisons were performed using two-tailed unpaired t-tests. * * * * p < 0.0001. Figure 6 | Impact of Gal-9 on CD4+ T cell viability and apoptosis. (A) CD4 + T cells isolated from 5 HIV-infected ART-suppressed individuals were treated for 24 h with Gal-9 (500 nM) or DMSO Control in the presence of 1 µM of Lck inhibitor or the equivalent volume of DMSO. Cell viability was determined using Zombie Aqua Fixable Viability staining. (B) A representative flow cytometry plot from one individual. (C) CD4 + T cells isolated from one HIV-infected ART-suppressed individual were treated for 24 h with Gal-9 (500 nM) or DMSO Control. Apoptosis was determined using Propidium iodide and Annexin V Pacific blue (Biolegend). anti-CD95 (1 ug/ml) stimulation for 6 h was used as positive control. Experiment was performed in duplicates. Mean ± SD is displayed (D) A representative flow cytometry plot of one replicate. Figure 7 | Gal-9 induces the secretion of several pro-and anti-inflammatory cytokines. CD4 + T cells isolated from 3 HIV-infected ART-suppressed individuals were treated for 24 h with Gal-9 (200 nM), rGal-9 (500 nM), or DMSO Control for 4 h, 24 h, or 3 days. Culture supernatants were collected on day 3 and levels the 13 indicated pro-and anti-inflammatory cytokines were determined using Luminex assay.