Table_1_Galectin-9 Mediates HIV Transcription by Inducing TCR-Dependent ERK Signaling.docx

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.

内源性血浆免疫调节碳水化合物结合蛋白半乳糖凝集素-9（galectin-9，Gal-9）的水平在HIV感染期间升高，且在抗逆转录病毒治疗（antiretroviral therapy，ART）实现病毒抑制后仍维持在较高水平。我们此前的研究报道称，Gal-9可调控HIV转录，并能高效激活潜伏状态的HIV。然而，Gal-9介导的病毒转录调控信号机制仍未明确。
鉴于半乳糖凝集素家族可调控T细胞受体（T cell receptor，TCR）信号通路，我们提出假说：Gal-9至少部分通过诱导TCR信号通路来调控HIV转录活性。在J-Lat HIV潜伏模型中，Gal-9可诱导T细胞受体ζ链（CD3ζ）发生磷酸化（磷酸化水平从11.2%提升至32.1%；P = 0.008）。
Lck抑制剂可降低J-Lat HIV潜伏模型中Gal-9介导的病毒激活效率（激活率从16.8%降至0.9%；P < 0.0001），同时可抑制接受ART病毒抑制治疗的HIV感染者原代CD4+ T细胞中Gal-9介导的CD4+ T细胞活化（CD69与CD25共表达阳性率从10.3%降至1.65%；P = 0.0006）以及IL-2、TNF-α的分泌（P < 0.004）。
通过磷酸化激酶抗体芯片，我们发现Gal-9可提升TCR下游信号分子ERK1/2（26.7倍）与CREB（6.6倍）的磷酸化水平。ERK与CREB抑制剂可显著降低Gal-9介导的病毒激活效率（分别从16.8%降至2.6%和12.6%；P < 0.0007）。
鉴于免疫抑制剂雷帕霉素（rapamycin）可将HIV潜伏逆转与细胞因子相关毒性解偶联，我们同时探究了雷帕霉素能否将Gal-9介导的潜伏病毒激活与其伴随的促炎细胞因子产生解偶联。雷帕霉素可降低Gal-9介导的IL-2（4.4倍，P = 0.001）与TNF（4倍，P = 0.02）分泌，但不影响病毒激活效率（激活率从16.8%变为16.1%；P = 0.2）。
综上，Gal-9通过以Lck依赖的方式激活TCR下游ERK与CREB信号通路，从而调控HIV转录。我们的研究结果有助于理解内源性半乳糖凝集素相互作用在调控TCR信号通路以及维持ART抑制状态下HIV感染慢性免疫激活中的作用。此外，通过雷帕霉素将Gal-9介导的病毒激活与不良促炎效应解偶联，可提升重组Gal-9在HIV潜伏清除策略中的应用潜力。