Reactivation of latent HIV-1 in vitro using an ethanolic extract from Euphorbia umbellata (Euphorbiaceae) latex

Abstract

Euphorbia umbellata (E. umbellata) belongs to Euphorbiaceae family, popularly known as Janauba, and its latex contains a combination of phorbol esters with biological activities described to different cellular protein kinase C (PKC) isoforms. Here, we identified deoxi-phorbol esters present in E. umbellata latex alcoholic extract that are able to increase HIV transcription and reactivate virus from latency models. This activity is probably mediated by NF-kB activation followed by nuclear translocation and binding to the HIV LTR promoter. In addition, E. umbellata latex extract induced the production of pro inflammatory cytokines in vitro in human PBMC cultures. This latex extract also activates latent virus in human PBMCs isolated from HIV positive patients as well as latent SIV in non-human primate primary CD4+ T lymphocytes. Together, these results indicate that the phorbol esters present in E. umbellata latex are promising candidate compounds for future clinical trials for shock and kill therapies to promote HIV cure and eradication.

Fig 1. Isolation of JALEx.

Gas chromatograms of (A) whole extract JALEx and (B) Phorbol ester-enriched JALEx fractions. T1 and T2 = major triterpenes constituents; Ph1 and Ph2 = major phorbol esters constituents. The chemical classes were indicated by the characteristic mass fragmentation in the GC-MS analysis and data from literature.

Fig 2. JALEx reactivates virus transcription in HIV-1 latent models.

J-Lat 8.4 (A) and 10.6 (C) cells were exposed to increasing concentrations of JALEx and HIV-1 activation was assessed 24 hours post-treatment by GFP positive cells screening with flow cytometry assay. Mock stands for DMSO treated cells. PMA (1 μM) and INGB (0.32 μM) were used as positive induction controls. The cytotoxicity of JALEx was evaluated using CellTiter Blue vital stain 5 days after treatment in J-Lat 8.4 (B) and 10.6 cells (D). GFP positivity and cytotoxicity were expressed as a percentage of cells relative to mock (DMSO-treated) cells (n = 3). Dashed lines in Fig 2B and 2D represents 100% of viability measures in MOCK. The bars in the Fig represent the SD around the mean.

Fig 3. JALEx induces cellular relocation of PKC isoforms.

Cellular location analysis of different PKC (alpha, delta and gamma) isoforms after treatment of HeLa cells with two concentrations of JALEx: 1 μg / mL and 0.1 μg / mL. All cells were submitted to nuclear staining with DAPI (blue). HeLa cells were transfected with 3 different PKC isoforms for 24h and treated with JALEx for 10 min and 24 hours. PMA (1 μM) was used as positive control (n = 3) and Mock images were taken with the results from 24 hours.

Fig 4. JALEx-mediated HIV-1 transcriptional activation by NF-κB nuclear translocation.

HeLa cells were treated with two concentrations of the JALEx (1 and 0.1 μg/mL) for 6 hours (A) and 24 hours (B). After, cells were subjected to immunofluorescence labeling of NF-κB (red) and cell nucleus (blue). As a positive control, PMA (1 μM) was used. (C) Quantification of NF-κB nuclear translocation of 100 HeLa cells for each condition (n = 3; Two-way ANOVA and (***) indicate p<0,0001). (D) Jurkat cells were transfected with pBlue30LTR-Luc (NF-κB WT) and the mutant construct, pBlue30LTR NF-κB MUT-Luc (NF-κB MUT), which is absent for the NF-κB binding site. After transfections, cells were treated with 1μg / mL of the JALEx for 24 hours and luciferase expression was measured after lysis. The fold-change of the luciferase expression between the wild-type plasmid and the mutant is shown in the graph (n = 4, Mann-Whitney test and (*) indicate p<0,05). The bars in Fig 4C and 4D represents the SD around the mean.

Fig 5. JALEx modulates the expression of cell surface receptors, activation markers and cytokine secretion in primary CD4 + T cells from HIV-1 infected individuals.

CD4+ T cells were isolated from PBMCs from 4 healthy donors and treated for 24 hours with JALEx. Then, the expression of a set of surface molecules was evaluated. (A) Expression of the CD4 receptor and CCR5 and CXCR4 co-receptors in CD4 + T cells isolated after treatment with JALEx (n = 4). (B) Expression of CD25, CD38 and HLA-DR activation markers in CD4 + T cells isolated after treatment with JALEx (n = 4). (C) Increasing of the CD69 activation marker expression on CD4+ T cells isolated after treatment with JALEx extract. The black bar represents the positive control (PMA 1 μM) and the gray bar represents the treatment with 10 μg / mL of JALEx (n = 4). Data is expressed as an increasing percentage relative to controls (DMSO treated cells). (D) CD4+ T cells were treated with 1 μM PMA, 10 μg/mL (-3) or 1 μg/mL (-4) of JALEx for 24 hours. After, supernatants from the CD4+ T cells were collected and cytokine analysis were performed by Bio-plex Pro Human Cytokines 17-plex platform (n = 5, Kruskal-Wallis test and (*) indicate p<0.05). The bars in the Fig represents the SD around the mean.

Fig 6. Effect of JALEx on T cell viability, HIV-1 replication in PBMC cultures and cytokine secretion in T cells from AIDS patients.

Different concentrations of JALEx (0.01, 0.1 and 1 μg/mL) were added to PBMC cultures (1 x 10⁶/mL) obtained from 15 AIDS patients. 24h after, (A) the HIV-1 viral load into supernatants was determined through RT-qPCR and (B) CD4⁺ and CD8⁺ T cells viability were analyzed by FACS with 7-AAD staining. AIDS-derived PBMC cultures (1 x 10⁶/mL, n = 15) were maintained for 24h in the presence of medium alone or with different JALEx concentrations (0.01 and 0.1 μg/mL). As a positive control, specific wells were treated with PMA plus ionomycine (PMA/IO). The percentage of CD4+ and CD8+ T cells producers of IFN-γ, IL-17, IL-21 and IL-10 was determined by cytometry (C). The mean values were compared and the p-values are shown. In (D), the proportion of different (CD4+ and CD8+) T cell subsets of secreting different combinations of IL-17, IFN-γ and IL-21 in response to JALEx (0.1 μg/mL) was also evaluated by FACS. In the Fig, (*), (**), and (***) indicate p<0.05, p<0.001 and p<0.0001, respectively. The bars in the Fig represent the SD around the mean.