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. 2008 Oct;69(10):634-46.
doi: 10.1016/j.humimm.2008.07.012. Epub 2008 Aug 22.

Influenza-induced expression of functional tumor necrosis factor-related apoptosis-inducing ligand on human peripheral blood mononuclear cells

Erik L Brincks  1 , Tamara A Kucaba, Kevin L Legge, Thomas S Griffith
Affiliations

Influenza-induced expression of functional tumor necrosis factor-related apoptosis-inducing ligand on human peripheral blood mononuclear cells

Erik L Brincks et al. Hum Immunol. 2008 Oct.

Abstract

The immunologic response to influenza virus infection, like many other viruses, is characterized by robust production of proinflammatory cytokines, including type I and II interferon (IFN), which induce a number of antiviral effects and are essential for priming the innate and adaptive cellular components of the immune response. Here, we demonstrate that influenza virus infection induces the expression of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on human peripheral blood mononuclear cell (PBMC) populations. Consistent with previous studies examining TRAIL upregulation, increased TRAIL expression correlated with increased type I and II IFN levels in PBMC cultures. Interestingly, dilution of these cytokines resulted in decreased expression of TRAIL. TRAIL upregulation was not dependent on active viral infection, and TRAIL was observed on NS-1-negative cells. Furthermore, influenza virus infection of lung adenocarcinoma cells (A549) resulted in increased sensitization to TRAIL-induced apoptosis compared with uninfected A549. Infected PBMC expressing TRAIL preferentially killed infected A549, but did not affect uninfected cells, and the addition of soluble TRAIL-R2:Fc blocked the lysis of infected cells, demonstrating TRAIL-dependent killing of infected cells. Collectively, these data demonstrate that TRAIL expression is induced on primary human innate and adaptive immune cells in response to cytokines produced during influenza infection and that TRAIL sensitivity is increased in influenza virus-infected cells. These data also suggest that TRAIL is a primary mechanism used by influenza-stimulated human PBMC to kill influenza-infected target cells and reinforce the importance of cytokines produced in response to TLR agonists in enhancing cellular immune effector functions.

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Figures

Figure 1
Figure 1
TRAIL expression on human PBMC after influenza virus infection or TLR agonist stimulation. PBMC were infected with (A) viable or (B) UV-inactivated influenza (as described in the Materials and Methods) or stimulated with the TLR agonists (C) poly I:C, (D) ssRNA, or (E) CpG ODN. Surface TRAIL expression was analyzed 24 h later on CD3+, CD14+, CD19+, and CD56+ cells using two-color flow cytometry. Representative results are shown in histograms based on 104 gated cells in all conditions, and cell viability was >95%, as assessed by propidium iodide exclusion. Similar results were observed using at least 4 different PBMC donors.
Figure 2
Figure 2
TRAIL-mediated cytotoxicity by human PBMC occurs after influenza virus infection or stimulation with TLR agonists. PBMC were infected with (A) viable or (B) UV-inactivated influenza (as described in the Materials and Methods) or stimulated with the TLR agonists (C) poly I:C, (D) ssRNA, or (E) CpG ODN. After 24 h, the PBMC were harvested and cultured for 14 h with 51Cr-labeled WM 793 target cells at the indicated effector-target cell ratios. For each condition, TRAIL-R2:Fc (20 μg/ml) inhibited target cell killing, while Fas:Fc (20 μg/ml) did not. Data points represent the mean of triplicate wells, and experiments were repeated at least three times using different donor PBMC with similar results. For clarity, SD bars were omitted from the graphs, but were <10% of the value of all points.
Figure 3
Figure 3
Influenza RNA stimulates TRAIL expression on human T cells. (A) RNA was isolated from UV-inactivated influenza particles (as described in the Materials and Methods), and used to stimulate PBMC at the indicated concentrations. To facilitate uptake, RNA was mixed with DOTAP. As controls, PBMC were incubated with DOTAP alone or influenza RNA without DOTAP. After 24 h, cells were collected, and processed to examine TRAIL expression on CD3+ T cells. Results are representative of 3 independent experiments using different donor PBMC. (B) PBMC were infected with influenza and cultured for 24 hours. Surface TRAIL expression and intracellular NS1 expression were analyzed on CD3+, CD14+, and CD56+ cells using three-color flow cytometry. Representative results are shown in histograms based on 104 gated cells in all conditions, and cell viability was >95%, as assessed by propidium iodide exclusion. Similar results were observed using 2 different PBMC donors.
Figure 4
Figure 4
Influenza stimulates IFN-α and –γ production from PBMC. (A) PBMC were infected with viable or UV-inactivated influenza (UV-flu) or stimulated with the TLR agonists poly I:C, ssRNA, or CpG ODN. After 24 h, IFN-α and –γ levels were quantitated in the culture supernatant by ELISA. Cytokine levels represent the average amount measured from at least four independent experiments using different donors. (B) IFN-α is made by pDC within PBMC after influenza virus infection. PBMC or PBMC depleted of pDC were infected with influenza. After 24 h culture, IFN-α and –γ levels in the culture supernatants were then determined by ELISA. Results represent the average amount measured from 3 independent experiments using different donors. (C) IFN-γ expression by PBMC after influenza virus infection or TLR agonist stimulation. PBMC were infected with viable influenza or stimulated with the TLR agonists poly I:C or ssRNA. Intracellular IFN-γ levels were analyzed 24 h later in CD3+, CD14+, and CD56+ cells using two-color flow cytometry. Representative results are shown based on 104 gated cells in all conditions, and cell viability was >95%, as assessed by propidium iodide exclusion. Similar results were observed using at least 3 different PBMC donors.
Figure 5
Figure 5
TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (106 – 1.25 × 105 cells) were aliquoted into 2 ml of media. After 24 h culture, IFN-α levels in the culture supernatants were determined by ELISA. Results represent the average amount measured from 2 independent experiments using different donors. * p < 0.05 compared to uninfected level. (B) After infection and 24 h culture (as described in 5A), PBMC from cell dilution cultures were analyzed for surface TRAIL expression on CD3+ cells using two-color flow cytometry. Representative results are shown in histograms based on at least 5 × 103 gated cells. Similar results were observed using 2 different PBMC donors. (C) After infection and 24 h culture (as described in 5A), supernatants from 106-cell PBMC cultures were transferred to uninfected cells from the same donor. After 24 h incubation, TRAIL expression on uninfected cells (uninfected), uninfected cells with supernatants from influenza-infected cells (Flu supe), or receiving media plus IFN-α (IFN-α) was determined. Representative results for CD3+ cells are shown in histograms based on at least 104 gated cells. Similar results were observed using 2 different PBMC donors. (D) PBMC were depleted of pDC, infected with influenza virus, and cultured for 24 h (as described in 5A). Supernatants from 106-cell cultures were incubated with an anti-IFN-γ neutralizing mAb, and then transferred to uninfected cells from the same donor. TRAIL expression on uninfected cells cultured with isotype or anti-IFN-γ mAb only, or in supernatants from influenza-infected cells treated with anti-IFN or isotype antibody. Representative results for CD3+ cells are shown in histograms based on at least 104 gated cells. Similar results were observed using 2 different PBMC donors.
Figure 6
Figure 6
Influenza virus infection alters cell sensitivity to TRAIL. (A) The lung adenocarcinoma cell line, A549, was infected with influenza (1 MOI) for 24 h. The cells were then added to 96-well microtiter plates (4 × 105 cells/well) and cultured with increasing concentrations of recombinant TRAIL (rTRAIL) at the indicated concentrations. Cell death was measured 24 h later. Uninfected A549 cells were tested at the same time. Results are representative of 3 independent experiments, where each data point is the average of 3 wells. (B) Influenza-infected PBMC readily kill influenza-infected A549 cells, but not uninfected A549 cells. PBMC were infected with influenza. After 24 h, the PBMC were harvested and cultured for 14 h with 51Cr-labeled uninfected or influenza-infected (1 MOI for 24 h) A549 target cells at the indicated effector-target cell ratios. (C) Inhibition of influenza-infected PBMC killing of influenza-infected A5499 target cells is blocked by TRAIL-R2:Fc (20 μg/ml), while Fas:Fc (20 μg/ml) did not significantly inhibit killing. The effector:target cells ratio was 50:1. Data points represent the mean of triplicate wells, and experiments were repeated at least three times using different donor PBMC with similar results. * p < 0.05.
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