Close Relationship between cIAP2 and Human ARDS Induced by Severe H7N9 Infection

Abstract

Background: cIAP2 is involved in necroptosis as a key upstream regulation factor. We aimed to investigate the role of cIAP2 in ARDS/ALI induced by H7N9 virus through regulating the RIPK1/3 necroptosis pathway.

Methods: Lung tissues of 11 patients who died from ARDS-complicated H7N9 infection between 2013 and 2016 were obtained as the H7N9-ARDS group. Lung tissues near benign lung nodules were acquired as the control group. Histological changes were evaluated by H&E staining. Protein levels of cIAP2, RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL in the lung tissues were detected by Western Blot. The mRNA levels of cIAP2, RIPK1, and RIPK3 were detected by real-time PCR.

Results: H7N9 virus infection had a high mortality, with ARDS being the leading cause of death. The protein level of cIAP2 in the experimental group was lower than that in the control group (P<0.05). However, the experimental group showed higher RIPK1, RIPK3, and p-RIPK3 protein levels than the control group (P<0.05), as well as the expression level of MLKL and p-MLKL protein, which is a key downstream protein in necroptosis (P<0.05).

Conclusion: In tissues from patients with fatal H7N9, downregulation of cIAP2 and induction of necroptosis was observed. We could speculate that necroptosis of the pulmonary epithelium is associated with severe H7N9 infection leading to ARDS. Thus, necroptosis inhibition may be a novel therapy for H7N9 influenza virus.

Figure 1

Pathological changes in the lung tissues (H&E staining, 200x). The lung tissues in control group had few abnormal pathological changes (a). The experimental group showed severe alveolar congestion (b), exudation of fibrin (c), broadened alveolar septum (d), disordered arrangement of bronchial epithelium (e), and inflammatory cells infiltration (f).

Figure 2

Lung injury scores. Each pathological feature was scored by a 5-scale point: 0=no injury or very mild injury, 1=mild injury, 2= moderate injury, 3=severe injury, and 4=very severe injury. The total lung injury score of each section was the sum of each score. The lung injury score of the experimental group was significantly higher than the control group (P<0.05).

Figure 3

cIAP2 expression level. cIAP2 protein had a lower expression level, in the lung tissues of the patients dying from human infection with H7N9 virus than the control group. ((a) and (b) P<0.05). The experimental group exhibited a lower cIAP2 mRNA level than the control group. ((c) P<0.05).

Figure 4

The mRNA level of RIPK1 and RIPK3. The mRNA level of RIPK1 and RIPK3 was enhanced in the lung tissues of severe patients dying from human infection with H7N9 virus (P<0.05).

Figure 5

The expression of RIPK1 and RIPK3. The protein level of RIPK1 and RIPK3 was increased in the lung tissues of severe patients dying from human infection with H7N9 virus (P<0.05). The protein level of p-RIPK3 and p-MLKL was increased in the lung tissues of severe patients dying from human infection with H7N9 virus (P<0.05).

Figure 6

The protein level of MLKL. The protein level of MLKL was increased in the lung tissues of severe patients dying from human infection with H7N9 virus (P<0.05).

Figure 7

Necroptosis was observed in SM-164 treated alveolar epithelium cells during LPS-induced ARDS. The mouse alveolar epithelium cell line MLE12 was exposed to SM-164 for 24h with or without LPS for 24h. RIPK3 and MLKL were downregulated along with the inhibition of cIAP2 by SM-164 during LPS-induced ARDS (a). The loss of viability is correlated with cell death as detected by LDH release in the cell supernatant ((b) and (c)). ^∗p<0.05.