Galectin-3 facilitates cell-to-cell HIV-1 transmission by altering the composition of membrane lipid rafts in CD4 T cells.

Galectin-3 (GAL3) is a ß-galactoside-binding lectin expressed in CD4 T cells infected with human immunodeficiency virus-1 (HIV-1). GAL3 promotes HIV-1 budding by associating with ALIX and Gag p6. GAL3 has been shown to localize in membrane lipid rafts in dendritic cells and positively regulate cell migration. HIV-1 spreads between T cells by forming supramolecular structures (virological synapses [VSs]), whose integrity depends on lipid rafts. Here, we addressed the potential role of GAL3 in cell-to-cell transmission of HIV-1 in CD4 T cells. GAL3 expressed in donor cells was more important for facilitating HIV-1 cell-to-cell transfer than GAL3 expressed in target cells. GAL3 was found to be co-transferred with Gag from HIV-1-positive donor to HIV-1-negative target T cells. HIV-1 infection induced translocation of GAL3 together with Gag to the cell-cell interfaces and colocalize with GM1, where GAL3 facilitated VS formation. GAL3 regulated the coordinated transfer of Gag and flotillin-1 into plasma membrane fractions. Finally, depletion of GAL3 reduced the cholesterol levels in membrane lipid rafts in CD4 T cells. These findings provide evidence that endogenous GAL3 stimulates lipid raft components and facilitates intercellular HIV-1 transfer among CD4 T cells, offering another pathway by which GAL3 regulates HIV-1 infection. These findings may inform the treatment of HIV-1 infection based on targeting GAL3 to modulate lipid rafts.

Galectin-3 promotes HIV-1 budding via association with Alix and Gag p6.

Galectin-3 has been reported to regulate the functions of a number of immune cell types. We previously reported that galectin-3 is translocated to immunological synapses in T cells upon T-cell receptor engagement, where it associates with ALG-2-interacting protein X (Alix). Alix is known to coordinate with the endosomal sorting complex required for transport (ESCRT) to promote human immunodeficiency virus (HIV)-1 virion release. We hypothesized that galectin-3 plays a role in HIV-1 viral budding. Cotransfection of cells of the Jurkat T line with galectin-3 and HIV-1 plasmids resulted in increased HIV-1 budding, and suppression of galectin-3 expression by RNAi in Hut78 and primary CD4+ T cells led to reduced HIV-1 budding. We used immunofluorescence microscopy to observe the partial colocalization of galectin-3, Alix and Gag in HIV-1-infected cells. Results from co-immunoprecipitation experiments indicate that galectin-3 expression promotes Alix-Gag p6 association, whereas the results of Alix knockdown suggest that galectin-3 promotes HIV-1 budding through Alix. HIV-1 particles released from galectin-3-expressing cells acquire the galectin-3 protein in an Alix-dependent manner, with proteins primarily residing inside the virions. We also found that the galectin-3 N-terminal domain interacts with the proline-rich region of Alix. Collectively, these results suggest that endogenous galectin-3 facilitates HIV-1 budding by promoting the Alix-Gag p6 association.

A salicylate-based small molecule HS-Cm exhibits immunomodulatory effects and inhibits dipeptidyl peptidase-IV activity in human T cells.

Activated T cells are key players in chronic inflammatory diseases, including atherosclerosis. Salicylates, like aspirin, display not only anti-inflammatory, anti-thrombotic, anti-atherosclerotic activities, but also immunomodulatory effects in T cells at high dosages. Here, we aimed to identify potent immunomodulators for T cells through cell-based screening from a mini-library of 300 salicylate-based small molecules, and elucidate the mechanisms. Human peripheral blood T cells were isolated from buffy coat. Phorbol 12-myristate 13-acetate plus ionomycin (P/I) was used to stimulate T cells. Cytokine production was measured by enzyme-linked immunosorbent assays. T cell activation markers were determined by flow cytometry. The activation of transcription factors and kinases was analyzed by western blotting, electrophoretic mobility shift assay, or kinase assay. Through library screening, we identified a small molecule named HS-Cm [C13H9ClFNO2; N-(4-chloro-2-fluorophenyl)-2-hydroxybenzamide] that exhibited potent immunomodulatory effects on T cells with low cytotoxicity. In P/I-stimulated T cells, HS-Cm inhibited the production of interleukin-2, tumor necrosis factor-alpha, and interferon-gamma and suppressed the expression of surface activation markers CD25, CD69, and CD71, but not CD45RO. HS-Cm down-regulated DNA-binding activities of activator protein-1 and nuclear factor-kappa B, but not nuclear factor of activated T-cells, through inhibiting c-Jun N-terminal kinase/p38 and inhibitor of kappaB alpha (IκBα) kinase (IKK)/IκBα pathways, respectively. On the basis of structure-activity relationship, HS-Cm exerted considerable inhibition of dipeptidyl-peptidase IV/CD26 activity in T cells. Our results suggested that the small molecule HS-Cm exhibiting immunomodulatory effects on T cells may be useful for therapeutics in chronic inflammatory diseases, like atherosclerosis, diabetes and autoimmune arthritis.