Activation of lymphocytes induced by bronchial epithelial cells with prolonged RSV infection

Abstract

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells,which might be responsible for susceptibility to asthma; however, the underlying mechanism is not clear. This study determined the activation of lymphocytes and drift of helper T (Th) subsets induced by RSV-infected human bronchial epithelial cells (HBECs) in vitro. HBECs had prolonged infection with RSV, and lymphocytes isolated from human peripheral blood were co-cultured with RSV-infected HBECs. Four groups were established, as follows: lymphocytes (group L); lymphocytes infected with RSV (group RL); co-culture of lymphocytes with non-infected HBECs (group HL); and co-culture of lymphocytes with infected HBECs (group HRL). After co-culture with HBECs for 24 hours, lymphocytes were collected and the following were determined in the 4 groups: cell cycle status; apoptosis rate; and concentrations of IL-4, IFN-γ, and IL-17 in the supernatants. Cell cycle analysis for lymphocytes showed a significant increase in S phase cells, a decrease in G1 phase cells, and a higher apoptosis rate in group HRL compared with the other three groups. In group HRL, the levels of IL-4, IFN-γ, and IL-17 in supernatants were also higher than the other three groups. For further study, lymphocytes were individually treated with supernatants from non-infected and RSV-infected HBECs for 24 h. We showed that supernatants from RSV-infected HBECs induced the differentiation of Th2 and Th17 subsets, and suppressed the differentiation of Treg subsets. Our results showed that HBECs with prolonged RSV infection can induce lymphocyte proliferation and apoptosis, and enhance the release of cytokines by lymphocytes. Moreover, subset drift might be caused by RSV-infected HBECs.

Figure 1. The cytopathic effects in HBECs after infection with RSV were observed under phase contrast microscopy.

A. normal HBECs; B. The classical pathologic changes of RSV-infected cells: enlarged fusion cell (×100). In this vision, variant shape, shrinkage, and enhanced refraction were present in cells, as well as enlarged fusion cells and intracytoplasmic eosinophilic inclusion, which were considered to be proof of RSV infection. A fusion cell is indicated by arrows.

Figure 2. The model of RSV-infected HBECs was examined by immunofluorescence.

A. Infected cells can be seen under phase contrast microscopy (×40); B. Infected cells conjugated with FITC can be seen under fluorescence microscopy (×40). Bright yellow green particles of various sizes were distributed within cytoplasm, which suggested the cells were positive for RSV infection and RSV infected model was constructed successfully.

Figure 3. Subcellular structure of infected cells and subcellular localization of virus under electron microscopy.

A is normal HBECs (×10000); B is RSV-prolonged infected HBECs in early infection stage, fissure around nucleus is seen (×10000); C is also in early infection stage, expansion of endoplasmic reticulum and a large number of lysosomes in cytoplasm were present (×15000). Both cells in B and C had double nucleoli, which indicated the cell was proliferating rapidly; D is in late infection stage, enlarged fusion cells were present (×10000). Intracellular virus particles indicated by arrows were observed both in nucleus and endoplasmic reticulum.

Figure 4. Effect of RSV- infected HBECs on cell cycle of lymphocyte.

Flow cytometry analysis showed significant more cells in S phase and less cells in G1 phase in group HRL compared with the other three groups. And even cells in S phase were more in group HL than the rest two groups.

Figure 5. Effect of RSV-infected HBECs on concentrations of IL-4, IFN-γ and IL-17 secreted by lymphocytes (pg/mL, n = 10/group, mean±SEM; *P<0.05 vs. L ^▵P<0.05 vs. RL ^▴P<0.05 vs. HL.

The levels of IL-4, IL-17 and IFN-γ in group HRL were highest in four groups. The level of IFN-γ in group HL was higher than those of groups L and RL.

Figure 6. The change of Th subsets in lymphocytes after treatment with supernatants from RSV-infected HBECs.

a. The change of Th2 cells in lymphocytes after treatment with supernatants from RSV-infected HBECs. Th2 cells mainly secreted IL-4. Monensin inhibited the secretion of newly produced cytokines in Golgi body. The positive cells in M2 which were conjugated with anti IL-4 were Th2 cells. In Fig 2.6a, the percentage of Th2 cells in lymphocytes co-cultured with RSV-infected HBECs was highest among three groups. The percentage of Th2 cells in lymphocytes co-cultured with normal HBECs was still higher than control. b: The change of Th17 cells in lymphocytes after treatment with supernatants from RSV-infected HBECs. Th17 cells mainly secreted IL-17. The positive cells in M2 which were conjugated with anti IL-17 were Th17 cells. In Fig 2.6b, the percentage of Th17 cells in lymphocytes co-cultured with RSV-infected HBECs was highest among three groups. The percentage of Th2 cells in lymphocytes co-cultured with normal HBECs was still higher than control. c: The change of Treg cells in lymphocytes after treatment with supernatants from RSV-infected HBECs. CD25 was surface marker and Foxp3 was intracellular activating factor of regulatory T cells (Treg). The positive cells in right upper region which were conjugated with anti-CD25 and anti-Foxp3 antibodies were Treg cells. In Fig 2.6c, the percentage of Treg cells in lymphocytes co-cultured with RSV-infected HBECs was lower than the other two groups. There was no difference between control group and normal HBECs group.

Figure 7. The distribution of Th subsets after treatment with supernatants from RSV-infected HBECs (%, n = 6/group, mean±SEM; *P<0.01 vs. control ^▵P<0.05 vs. normal HBECs).

The percentages of Th2 and Th17 cells in RSV-infected group were higher than the other two groups, while the percentage of Treg was less than the other groups. The percentages of Th2 and Th17 cells in normal HBECs group were higher than control group.