Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

Abstract

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.

Fig 1. EBOV induces cell death of T lymphocytes.

A. Percentages of annexin-V⁺ cells in preparations of CD4⁺ T lymphocytes cultured alone, activated overnight with CD3/CD28 beads, or co-cultured with imDCs or mDCs in the absence (mock) and presence of EBOV (MOI 0.3 PFU/cell) for 7 days. B. Percentages of annexin-V⁺ CD4⁺ T cells following incubation of with EBOV at MOI of 0.3, 1.0 or 10.0 PFU/cell for 4 days. Staurosporine (1 μM final concentration) treatment was used as a positive control for cell death. C. Percentages of CFSE_low (divided) CD4⁺ T lymphocytes treated as indicated in panel A. D. Percentages of CFSE_low (divided) CD4⁺ and CD8⁺ T lymphocytes cultured in the presence of EBOV for 4 days at MOI 0.3 PFU/cell. E. Flow cytometry analysis of CD4⁺ T lymphocytes cultured with EBOV for 4 days at MOI of 0.3 or 1.0 PFU/cell: dead (Live/Dead⁺) cells, cells positive for activated caspase-8 and activated caspase-9, and CFSE_low (divided) cells. F, G. Percentages of dead (Live/Dead⁺) cells and proliferation of isolated CD4⁺ T cells exposed to EBOV for 4 days in the presence of the apoptosis inhibitor zVAD-FMK. Data are representative of triplicate samples from one of 7 independent donors. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, n.s., non-significant (Student T-test).

Fig 2. Cell death of T lymphocytes is triggered by direct binding of GP.

A. Percentages of annexin V⁺ CD3⁺ T cells in PBMCs incubated with HPIV3/ΔF-HN/EboGP or HPIV3 for 1, 4 and 7 days determined by flow cytometry. Mean values ±SE based on 3 donors. B. Percentages of annexin-V⁺ isolated primary CD3⁺ T cells incubated with HPIV3/ΔF-HN/EboGP or HPIV3 for 1, 4 or 7 days at 37°C determined by flow cytometry. Mean values ±SE based on 3 donors. A, B, Two-Way ANOVA followed by a Tukey’s multiple comparisons test, P values are indicated in Fig 1C, 1D and 1E. Activation of caspases in Jurkat cells incubated with HPIV3 or HPIV3/ΔF-HN/EboGP for 7 days or stimulated with staurosporine for 6 h determined by Western blotting: non-activated and activated caspases 3 (C), 8 (D) and 9 (E). Amounts of active caspases as percentages of total (active and inactive) caspases. Panels C, D and E are one set of representative data from two independent experiments. F, G. Flow cytometry analysis of activated caspase 8 in Jurkat cells incubated with HPIV3/ΔF-HN/EboGP or HPIV3 for 7 days or stimulated with staurosporine (control) for 6 h: representative primary data with percentages of positive populations indicated for each treatment (F) and mean percentages of caspase-8⁺ cells ±SE based on triplicate samples from one of three independent experiments. P values * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (Student T-test) (G).

Fig 3. EBOV GP mediates binding to T lymphocytes through TLR4.

A, B. Western blotting analysis of immunoprecipitation of TLR4 or TLR-4 FLAG with EBOV GP (A) or EBOV VP40 (B). Representative data from one of two independent experiments. C. Confocal microscopy of HPIV3/ΔF-HN/EboGP bound to primary CD4⁺ T lymphocytes, Jurkat cells and 293T-TLR4 cells. Insets show the formation of plasma membrane associated GP-positive puncti. D. Flow cytometry analysis demonstrating the binding of EBOV and HPIV3/ΔF-HN/EboGP to SupT1 T cells. E. Flow cytometry analysis of TLR4 expression by isolated CD4⁺ T cells. T cells were activated with CD3/CD28 beads and then cultured with EBOV. Results are representative of 3 donors. F. Inhibition of EBOV binding to SupT1 cells by anti-TLR4 serum: % of no serum control. Mean values ±SE based on triplicate samples of one of two independent experiments, *** P<0.001 (Student T-test).

Fig 4. EBOV GP activates the TLR4 pathway that leads to T cell death.

A, B. Western blot analysis of proteins involved in TLR4 signaling pathway TRAM1, p-TRAM1, MyD88, IRAK4, p-IRAK4, Pyk2, p-Pyk2, p38, p-p38 in SupT1 cells and monocytes (A) or THP-1, THP-1 MyD88-/- and SupT1 cells (B) following stimulations with LPS, poly I:C, HPIV3/EboGP, HPIV3/ΔF-HN/EboGP, HPIV3 or EBOV (A) or empty beads or EBOV GP beads (B) in the presence or absence of the TLR4 inhibitor CLI-095. C, D, E. Western blot analysis of p65 phosphorylation in SupT1 cells (C), monocytes (D) or THP-1, THP-1 MyD88-/- and SupT1 cells (E) following stimulation with CD3/CD28 beads, LPS (+, 100 ng/ml, ++; 500 ng/ml), VLP (+, 100 μl; ++, 250 μl) and HPIV3/ΔF-HN/EboGP (+, MOI 0.1 PFU/cell; ++, MOI 1 PFU/cell), empty beads or EBOV GP beads, as indicated, with or without CLI-095. Western blots in panels A-E are representative of two independent experiments. F. Percentages of dead (Live/Dead⁺) cells, cell positive for caspase 8 and 9 and proliferated CD4⁺ T lymphocytes following a 4 day-long incubation with EBOV or LPS with or without CLI-095. Mean values ±SE based on triplicates from one of two independent experiments with P values * P<0.05, ** P<0.01, n.s., non-significant (Student T-test).

Fig 5. Mechanisms of cell death caused by EBOV.

A. Concentrations of TNFα in medium of purified CD4⁺ T cells cultured with EBOV or SEB for 4 days. Mean values ±SE based on 4 donors analyzed in duplicates. P values * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (Student T-test). B, C. Flow cytometry analysis of the percentages of TNFα⁺ SupT1 cells cultured with medium (mock), TPA/ionomycin or EBOV, mean values ±SE based on triplicate samples from one of two independent experiments shown (C). D. Percentages of Live/Dead⁺ SupT1 cells following daily additions of TNFα at 80 pg/ml for 4 days: mean values ±SE based triplicate samples from one of two independent experiments. E. Effects of TNFα antagonist III on CD4⁺ T cell death induced by EBOV at 4 days post infection: percentages of Live/Dead⁺ cells (left panel) and proliferated cells (right panel). Mean values ±SE based on triplicate samples from one of 3 independent experiments. F. Percentages of dead (Live/Dead⁺) cells (left panel) and proliferation (right panel) in the presence of necrosis inhibitors NecroX5 (X5), geldanamycin (GA) or N-(3-aminomethyl)benzylacetamindine (1400W) added immediately prior to the addition of EBOV at 4 days post infection. Mean values ±SE based on triplicate samples from one of 3 independent experiments. P values for panels E, F: ** P<0.01, *** P<0.001, n.s., non-significant (Student T-test).

Fig 6. EBOV promotes differentiation and infection of THP-1 cells through TLR4 activation.

A-C, Effects of EBOV or GP on differentiation of THP-1 cells. Cells were mock-treated or treated with CLI-095, cultured with medium (mock), LPS, EBOV (no GFP) or HPIV3/ΔF-HN/EboGP for 24 or 96 h and analyzed for activation markers CD14 and CD11b by flow cytometry. Representative primary data (A) and mean values ±SE for 24 h (B) and 96 h based on triplicate samples from one of two independent experiments. (C). D. The experimental design to evaluate the role of GP in EBOV infection of THP-1 cells. E, F. Infection of THP-1 cells following stimulation with LPS, EBOV-no GFP or HPIV3/ΔF-HN/EboGP for 24 or 96 h and infection with EBOV-GFP analyzed by flow cytometry: representative primary flow cytometry data showing expression of GFP (E) and mean values ±SE based on triplicate samples from one of two independent experiments with P values (Two-Way ANOVA followed by a Tukey’s multiple comparison test and multiple T-tests), * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, n.s., non-significant, black asterisks, difference to CLI-095-treated cells, red asterisks, difference to mock-stimulated cells (F).