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Abstract

Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a "cytokine storm." Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1^-/- mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1^-/- mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine-Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4^Hi CD3^Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4⁺ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4⁺ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD.IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is consistently linked with fatal disease outcome. Previous findings have demonstrated that specific T-cell subsets are key contributors to the onset of a cytokine storm. In this study, we investigated the role of Tim-1, a T-cell-receptor-independent trigger of T-cell activation. We first demonstrated that Tim-1-knockout (KO) mice survive lethal Ebola virus challenge. We then used a series of in vitro assays to demonstrate that Ebola virus directly binds primary T cells in a Tim-1-phosphatidylserine-dependent manner. We noted that binding induces a cytokine storm-like phenomenon and that blocking Tim-1-phosphatidylserine interactions reduces viral binding, T-cell activation, and cytokine production. These findings highlight a previously unknown role of Tim-1 in the development of a cytokine storm and "immune paralysis."

Keywords: Ebola virus; T lymphocytes; cytokine storm; cytokines; transcriptome; viral pathogenesis.

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Figures

👁 FIG 1

FIG 1

Role of Tim-1 in pathogenesis of EBOV disease. Wild-type (WT) mice and Tim-1^−/− mice were infected with mouse-adapted EBOV at 5 animals per group. (A) Survival curves, P < 0.0001 (Mantel-Cox test). (B) Clinical scores assigned as described in Materials and Methods. (C) Changes of mouse body weight in percent.

👁 FIG 2

FIG 2

Tim-1^−/− mice exhibit reduced Th1/2 responses. Serum cytokines/chemokines, CD4⁺ T-cell functional responses, and plasma viremia levels were analyzed 6 days following EBOV infection. (A) Heat map representing global variations in cytokine/chemokine responses in wild-type C57BL/6J mice and Tim-1^−/− as determined by multiplex analysis. G-CSF, granulocyte colony-stimulating factor; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; VEGF, vascular endothelial growth factor. (B and C) Graphical representation of Th1-associated (B) and Th2-associated (C) cytokines. Gray circles, mock-infected wild-type mice; red squares, EBOV-infected wild-type mice; black triangles, Tim-1^−/− EBOV-infected mice. (D) Intracellular cytokine staining for IL-2, IFN-γ, and TNF-α in gated CD4⁺ CD3⁺ T cells. Black line, isotype control, an EBOV-infected wild-type mouse; red line, a mock-infected wild-type mouse; gray line, an EBOV-infected wild-type mouse; blue line, an EBOV-infected Tim-1^−/− mouse. (E) Plasma viremia levels were determined by quantitative PCR. Histograms of cells are representative of individual mice within each group. Plasma viremia is shown by the mean number ± SE of NP copies from 5 wild-type C57BL/6J mice and 4 Tim-1^−/− mice infected with mouse-adapted EBOV. Asterisks denote statistical significances between the mean averages for wild-type mice and Tim-1^−/− mice, where P is <0.05 (Student’s t test).

👁 FIG 3

FIG 3

EBOV binds to CD4⁺ T cells and induces rapid TCR internalization in a PS-dependent manner. (A) EBOV binding to T cells. Isolated CD4⁺ T lymphocytes and Jurkat cells were incubated with EBOV for 2 h at 4°C, stained for GP, and analyzed by flow cytometry. FSC, forward scatter. (B) EBOV binding to CD4⁺ T lymphocytes (upper panel) and Jurkat cells (lower panel) demonstrating the GP^Hi population and the shift of the bulk population with mean fluorescence intensity indicated. (C) Confocal microscopy of EBOV bound to primary CD4⁺ T lymphocytes, Jurkat cells, and 293T cells. Insets show the formation of plasma membrane-associated GP-positive puncta. (D and E) Rapid induction of a CD4^Hi CD3^Low population by EBOV following a 2-h incubation at 4°C. Primary naive (upper panel) and activated (lower panel) CD4⁺ T lymphocytes (D) and Jurkat cells (E). The EBOV-GP⁺ populations were back-gated onto the CD4-versus-CD3 plots. (F) CD4 and CD3 expression levels 24 h after stimulation of CD4⁺ T cells with SEB or EBOV. (G) Activation of PS signaling, including significant upregulation of Tim-1 (HAVCR1), in isolated primary CD4⁺ T cells incubated with EBOV. Deep-sequencing-based transcriptional profiling in which solid lines represent direct interactions and dashed lines represent indirect interactions from IPA’s Knowledge Base. Red and blue indicate increased and decreased transcriptional activity, respectively. (H) Preincubation of CD4⁺ T cells with PS-containing liposomes or preincubation of EBOV with annexin V reduces viral binding to cells. ****, P < 0.0001 (Student t-test). (I) Addition of PS-containing liposomes reduces EBOV-induced development of CD4^Hi CD3^Low population. (J) Following EBOV binding, CD4⁺ T cells were either immediately treated with trypsin or incubated for 1 h at 37°C followed by trypsin treatment. EBOV binding and/or internalization was assessed by Western blotting for NP. In addition, blotting for CD3ε was used to determine the relative internalization of TCR. Binding assays are representative of over a dozen independent experiments; histograms are representative of one donor. Inhibition assays are representative of one of four individual experiments conducted in triplicate.

👁 FIG 4

FIG 4

EBOV activates CD4⁺ T cells and induces the release of inflammatory mediators. (A to C) Expression levels of the activation markers CD25 (A), CD69 (B), and intracellular Ki-67 (C) as assessed by flow cytometry at 48 h after addition of EBOV to CD4⁺ T cells. (D to F) Intracellular cytokine staining for IL-2 (D), IFN-γ (E), and TNF-α (F) performed on primary CD4⁺ T cells at 48 h after the addition of EBOV at an MOI of 0.3; histograms of cells from representative donors and mean percentages of cells from 4 donors ± SE. Statistical significances between the mean percentages are shown as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test). (G and H) Heat maps and levels of cytokines (picograms per milliliter) associated with Th1, Th2, and Th17 responses in supernatants of EBOV-stimulated CD4⁺ T cells at 48 h after addition of EBOV. Heat maps in panel G are shaded as in Fig. 2A. Grey circles, red squares, and black triangles represent mock- (medium only), EBOV-, and SEB-stimulated cells, respectively. (I) Transcriptional profiling shows the coordinated upregulation in inflammation pathways indicative of a cytokine storm in CD4⁺ T cells on both day 1 and day 4 after addition of EBOV. Solid lines represent direct interactions, and dashed lines represent indirect interactions from IPA’s Knowledge Base. Expression data from EBOV-infected samples relative to mock samples at day 1 and day 4 are overlaid onto each gene, where red represents significant relative upregulation and blue represents significant relative downregulation.

👁 FIG 5

FIG 5

Role of Tim-1 in EBOV-induced activation of CD4⁺ T cells. (A and B) Disabling of Tim-1 reduces activation of EBOV-exposed T cells. Jurkat cells were transfected with control siRNA or siRNA targeting Tim-1, incubated for 48 h, stimulated with EBOV for an additional 48 h, stained, and analyzed for CD25 (A) and CD69 (B). (C) The EBOV-mediated activation of T cells depends on Lck kinase. Primary CD4⁺ T cells were incubated with Src inhibitor Ly294 for 1 h, stimulated with EBOV for 48 h, and analyzed for CD25 and CD69. (D) The EBOV-mediated activation of T cells depends on the PI3K pathway. Primary CD4⁺ T cells were incubated with PI3K inhibitor PP2 and EBOV and analyzed as described for panel F. (E) Binding of EBOV GP and EBOV-mediated downregulation of CD3 depend on Lck kinase and PI3K. Primary CD4⁺ T cells were incubated with Ly294 or PP2, and the percentages of binding and the CD4^Hi CD3^Low population were determined. (F) EBOV-induced phosphorylation of Akt depends on Src kinase and PI3K. CD4⁺ T cells were treated with Ly294 and PP2. Akt and its phosphorylated form were analyzed by Western blotting. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student t-test).

👁 FIG 6

FIG 6

EBOV preferentially binds to T_CM cells. (A) Activation of isolated CD4⁺ T cells by EBOV. Expression of HLA-DR versus CD38 (left panel). The EBOV GP⁺ population (middle panel) was back-gated on HLA-DR⁺ CD38⁺ flow cytometry plots (right panel) to determine correlation of GP binding with cell activation. (B) HLA-DR versus CD45RO staining used to determine if activated cells are derived from the naive (CD45RO⁻) or memory (CD45RO⁺) subsets. GP⁺ population (middle panel) was back-gated to HLA-DR⁺ CD45RO⁺ plots (right panel). (C) EBOV binding to T_EM or T_CM determined following staining for CCR7 versus CD45RO. GP binding for each of the 3 quadrants corresponding to T_EM, T_CM, and naive cells is shown. GP⁺ cells were back-gated to CCR7⁺ CD45RO⁺ plots for T_CM quadrant (right panel). (D) Tim-1 expression profile on T-cell subsets. (E) Analysis of Th subsets in primary CD4⁺ T cells following 1- and 4-day-long stimulation with EBOV. Percentages of naive (CD45RO⁻), Th2/17 (CD45RO⁺ CXCR3⁻), and Th1 (CD45RO⁺ CXCR3⁺) cell populations. (F) Analysis of CD4^Hi CD38⁺ T cells after 4-day-long stimulation with EBOV, CD3/CD28 beads, or both EBOV and CD3/CD28 beads. (G) Analysis of HLA-DR⁺ CD38⁺ T cells at 4 days following stimulation with EBOV, CD3/CD28 beads, or both EBOV and CD3/CD28 beads. Data in all panels are representative of 1 of 2 independent donors and experiments performed in triplicate wells.

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URL: https://pubmed.ncbi.nlm.nih.gov/28951472/

⇱ Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm - PubMed

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