POU Class 2 Associating Factor 1 Exerts a Protective Effect on the Respiratory Syncytial Virus-Induced Acute Bronchiolitis by the NF- κ B Pathway

Abstract

Background: Respiratory syncytial virus (RSV) is the main pathogen causing acute bronchiolitis, which is common in infants and young children. A previous study revealed the possible involvement of POU class 2 associating factor 1 (POU2AF1) in RSV-triggered acute bronchiolitis. We attempted to clarify the specific action mechanism of POU2AF1 underlying RSV-triggered inflammation.

Methods: RT-qPCR measured POU2AF1 levels in RSV-infected children, mice, and airway epithelial cell lines (HBECs). HE staining showed histopathological features in the lung tissue of RSV-infected mice. ELISA examined the contents of proinflammatory cytokines in RSV-infected mice. Western blotting evaluated the protein abundance of proinflammatory cytokines in RSV-infected HBECs and assessed NF-κB pathway-associated protein expression in RSV-infected mice and RSV-treated HBECs.

Results: POU2AF1 presented depletion in RSV-infected children, mice, and HBECs. RSV-infected triggered lung injury and inflammatory cell infiltration in the mouse lung tissue, while POU2AF1 overexpression rescued these changes. RSV-infected induced inflammatory impairment in HBECs, whereas POU2AF1 reversed this effect. POU2AF1 suppressed the upregulated NF-κB pathway-associated protein expression in mice and HBECs under RSV infection.

Conclusion: POU2AF1 exerts a protective impact on RSV-induced acute bronchiolitis in vitro and in vivo through the NF-κB pathway. Our research may provide a novel direction for better therapy of RSV-triggered acute bronchiolitis.

Figure 1

POU2AF1 presented downregulation in NPA of RSV-infected children. (a) Flow chart for grouping of RSV-infected children (n = 22) and controls (n = 15). (b) RT-qPCR detected POU2AF1 level in NPA of RSV-infected children and controls. (c) RT-qPCR detected IL-6 level in NPA of RSV-infected children and controls. (d) Pearson correlation analysis assessed relationship of POU2AF1 level and clinical symptom scores in NPA of RSV-infected children. (e) Pearson correlation analysis assessed relationship of POU2AF1 level and IL-6 level in NPA of RSV-infected children. ^∗∗∗P < 0.001, RSV vs. Control group.

Figure 2

RSV-induced lung injury and inflammation and downregulated POU2AF1 in mice. (a) HE staining detected histopathological features in the lung tissue of RSV mice (n = 6) and controls (n = 6). (b, c) Cell counts for neutrophils and monocytes in the lung tissue of RSV mice and controls. (d–g) ELISA assessed INF-γ, TNF-α, IL-1β, and IL-6 concentrations in the lung tissue of RSV mice and controls. (h) RT-qPCR detected POU2AF1 level in RSV mice and controls. ^∗∗∗P < 0.001, RSV vs. Control group.

Figure 3

POU2AF1 attenuated lung injury and inflammation in RSV-infected mice. (a) RT-qPCR measured POU2AF1 level in RSV mice after injection of adv-POU2AF1 (n = 6) and adv-NC (n = 6). (b) HE staining detected histopathological features in the lung tissue of RSV mice under POU2AF1 overexpression. (c, d) Cell counts for neutrophils and monocytes in the lung tissue of RSV mice under POU2AF1 overexpression. (e–h) ELISA assessed INF-γ, TNF-α, IL-1β, and IL-6 concentrations in the lung tissue of RSV mice under POU2AF1 overexpression. ^∗∗P < 0.01, ^∗∗∗P < 0.001, RSV + Adv-POU2FA1 vs. RSV + Adv-NC group.

Figure 4

POU2AF1 inactivated the NF-κB pathway in RSV-infected mice. (a–d) Western blotting evaluated p-Iκ-Ba, p-p65, p-p50, Iκ-Ba, p65, and p50 protein abundance. ^∗∗∗P < 0.001, RSV vs. Control group; ^###P < 0.001, RSV + Adv-POU2FA1 vs. RSV + Adv-NC group.

Figure 5

POU2AF1 ameliorated inflammation in RSV-infected airway epithelial cells via the NF-κB pathway. (a) RT-qPCR detected POU2AF1 level in RSV-treated HBECs and controls. (b) RT-qPCR detected POU2AF1 overexpression efficacy in RSV-treated HBECs. (c–e) Western blotting detected TNF-α, IL-1β, and IL-6 protein expressions under indicated treatment. (f, g) Western blotting detected p-Iκ-Ba, p-p65, p-p50, Iκ-Ba, p65, and p50 protein expressions under indicated treatment. ^∗∗∗P < 0.001, RSV vs. Control group; ^###P < 0.001, RSV + pcDNA POU2FA1 vs. RSV + pcDNA NC group.