XB130 deficiency enhances lipopolysaccharide-induced septic response and acute lung injury

Abstract

XB130 is a novel oncoprotein that promotes cancer cell survival, proliferation and migration. Its physiological function in vivo is largely unknown. The objective of this study was to determine the role of XB130 in lipopolysaccharide (LPS)-induced septic responses and acute lung injury. LPS was intraperitoneally administrated to Xb130 knockout (KO) and wild type (WT) mice. There was a significant weight loss in KO mice at Day 2 and significantly higher disease scores during the 7 days of observation. The levels of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, interleukin-6 and interleukin-10 in the serum were significantly higher in KO mice at Day 2. In KO mice there were a significantly higher lung injury score, higher wet/dry lung weight ratio, more apoptotic cells and less proliferative cells in the lung. Macrophage infiltration was significantly elevated in the lung of KO mice. There was significantly increased number of p-GSK-3β positive cells in KO mice, which were mainly neutrophils and macrophages. XB130 is expressed in alveolar type I and type II cells in the lung. The expression in these cells was significantly reduced after LPS challenge. XB130 deficiency delayed the recovery from systemic septic responses, and the presence of XB130 in the alveolar epithelial cells may provide protective mechanisms by reducing cell death and promoting cell proliferation, and reducing pulmonary permeability.

Keywords: cell death; cell proliferation; inflammatory response; septic response; transgenic mouse.

Figure 1. XB130 deficiency increased LPS-induced septic response in mice

(A) Kaplan-Meier survival curves of WT mice and Xb130 KO mice after LPS treatment (25 mg/kg). Difference in mortality was evaluated according to log-rank test (p = 0.114). Weight loss (B) and composite disease score (C) were recorded on different days. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout.

Figure 2. XB130 deficiency enhanced LPS-induced cytokine production

(A and B) Cytokine/chemokine levels in serum were measured at 2, 6 and 48 hours after LPS treatment. *p < 0.05 and **p < 0.01 (WT vs. KO), ^#p < 0.05 and ^##p < 0.01 (PBS groups vs. LPS groups), WT = Wild type, KO = Xb130 knockout.

Figure 3. XB130 deficiency enhanced LPS-induced lung injury

Weight loss (A) and composite disease score (B) were recorded for 2 days after LPS treatment. (C) Hematoxylin and Eosin staining showed severe lung injury in Xb130 KO mice at Day 2 after LPS challenge. Scale Bars = 50 μm. Lung injury score (D) and W/D weight ratio (E) were significantly higher in Xb130 KO mice. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout, W/D = wet/dry.

Figure 4. XB130 deficiency resulted in more apoptosis and less cell proliferation in LPS-induced lung injury

(A) The number of apoptotic cells (red) was significantly higher (A–C), and the number of Ki-67+ cells (brown) was significantly lower (B and D) in Xb130 KO mice compared to WT mice at Day 2 after LPS administration. Scale Bars = 50 μm. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout.

Figure 5. The number of neutrophils infiltrated into the alveoli at Day 2 after LPS treatment was not significantly different between WT mice and Xb130 KO mice (A–C)

On the other hand, there was significantly higher number of macrophages infiltrated into the alveoli in Xb130 KO mice (B) (D). The neutrophils and macrophages were immunohistochemically stained with antibodies for Ly-6B2 and F4/80 antibodies, respectively. Scale Bars = 50 μm. *p < 0.05, WT = Wild type, KO = Xb130 knockout.

Figure 6

(A, B) Immunostaining for p-GSK-3β showed the number of p-GSK-3β+ cells (brown) were significantly higher at Day 2 after LPS treatment in Xb130 KO mice compared to WT mice. Scale Bars = 50 μm. p-GSK-3β was mainly stained in neutrophils and macrophages (C, D) but not in alveolar type II cells (E). *p < 0.05, WT = Wild type, KO = Xb130 knockout.

Figure 7. Immunofluorescence staining shows XB130 expression (XB130+: red) in alveolar type II cells (Sftpc+: green) (A) and type I cells (PDPN+: green) (B)

PDPN = podoplanin, SFTPC = surfactant protein C.

Figure 8

(A) In WT mice, Xb130 mRNA levels were significantly reduced in the lung tissue 2 days after LPS treatment. (B) Immunofluorescence studies shows that the intensity of XB130 (red) expression in alveolar epithelial cells was extremely weak after LPS administration. WT = Wild type.