Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

Abstract

Background: Axl is a cell surface receptor tyrosine kinase, and activation of the Axl attenuates inflammation induced by various stimuli. Growth arrest-specific 6 (Gas6) has high affinity for Axl receptor. The role of Gas6/Axl signaling in ischemia-reperfusion-induced acute lung injury (IR-ALI) has not been explored previously. We hypothesized that Gas6/Axl signaling regulates IR-induced alveolar inflammation via a pathway mediated by suppressor of cytokine signaling 3 (SOCS3).

Methods: IR-ALI was induced by producing 30 min of ischemia followed by 90 min of reperfusion in situ in an isolated and perfused rat lung model. The rats were randomly allotted to a control group and IR groups, which were treated with three different doses of Gas6. Mouse alveolar epithelium MLE-12 cells were cultured in control and hypoxia-reoxygenation (HR) conditions with or without Gas6 and Axl inhibitor R428 pretreatment.

Results: We found that Gas6 attenuated IR-induced lung edema, the production of proinflammatory cytokines in perfusates, and the severity of ALI ex vivo. IR down-regulated SOCS3 expression and up-regulated NF-κB, and Gas6 restored this process. In the model of MLE-12 cells with HR, Gas6 suppressed the activation of TRAF6 and NF-κB by up-regulating SOCS3. Axl expression of alveolar epithelium was suppressed in IR-ALI but Gas6 restored phosphorylation of Axl. The anti-inflammatory effect of Gas6 was antagonized by R428, which highlighted that phosphorylation of Axl mediated the protective role of Gas6 in IR-ALI.

Conclusions: Gas6 up-regulates phosphorylation of Axl on alveolar epithelium in IR-ALI. The Gas6/Axl signaling activates the SOCS3-mediated pathway and attenuates IR-related inflammation and injury.

Fig 1. Effects of Gas6 on lung edema.

(A) Lung weight gain, (B) pulmonary microvascular permeability (Kf), (C) ratios of lung wet/dry (W/D) weight, (D) lung weight/body weight (LW/BW), and (E) protein concentration in bronchoalveolar lavage fluid (BALF) in IR-ALI. The increase of these parameters in the IR group was significantly attenuated by pretreatment with Gas6. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 6 per group). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ## P < 0.01 compared with the IR group; ### P < 0.001 compared with the IR group.

Fig 2. Effects of Gas6 on proinflammatory cytokines.

(A) CINC-1, (B) IL-1β, (C) IL-6, and (D) TNF-α in IR-ALI. Gas6 attenuated the production of proinflammatory cytokines induced by IR. IR: ischemia–reperfusion. Data are expressed as the means ± SD (n = 6 per group). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ### P < 0.001 compared with the IR group.

Fig 3. Effects of Gas6 on lung tissues.

(A) Hematoxylin and eosin staining for lung tissue (200× magnification), (B) neutrophil count, and (C) lung injury score in IR-ALI. Gas6 attenuates the severity of IR-ALI. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 6 per group). ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; ### P < 0.001 compared with the IR group.

Fig 4. Effects of Gas6 on NF-κB of lung tissues.

(A) Bronchoalveolar lavage fluid (BALF) TNF-α, (B) BALF IL-6, (C) NF-κB p65 levels of lung tissues, and (D) cytoplasmic IκB-α levels of lung tissues in IR-ALI. Gas6 modulates TNF-α and IL-6 levels in BALF and NF-κB expressions of lung tissues in IR-ALI. IR: ischemia–reperfusion. Data are expressed as the means ± SD (n = 6 per group for BALF and N = 4 per group for lung tissue). ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ## P < 0.01 compared with the IR group; ### P < 0.001 compared with the IR group.

Fig 5. SOCS3 expression of lung tissues and alveolar epithelium.

(A) SOCS3 protein levels in lung tissues determined by western blot analysis. (B) SOCS3 mRNA expressions in lung tissues. (C) Representative images of SOCS3 immunofluorescence staining (FITC-labeled green; original magnification ×400) of rat lung. Nuclei were counterstained with DAPI (blue). SOCS3 expressions of alveolar epithelium are significantly decreased after IR and restored by Gas6 treatment. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 4 per group). ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; ## P < 0.01 compared with the IR group; ### P < 0.001 compared with the IR group.

Fig 6. The SOCS3-mediated pathway in MLE-12 cells with HR.

(A-B) SOCS3, TRAF6, cytoplasmic IκB-α, and nuclear NF-κB p65 levels determined by western blot analysis. TATA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. Gas6 up-regulates SOCS3 and down-regulates TRAF6 and NF-κB in HR model. HR: hypoxia-reoxygenation. Data are expressed as the means ± SD (n = 4 per group). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; # P < 0.05 compared with the HR group; ## P < 0.01 compared with the HR group; ### P < 0.001 compared with the HR group.

Fig 7. Modulation of Axl in MLE-12 cells with HR.

(A) T-Axl protein levels determined by western blot analysis. (B) Axl mRNA expressions. (C) p-Axl, (D) SOCS3 and (E) nuclear NF-κB p65 levels determined by western blot analysis. HR resulted in lower p-Axl and SOCS3 levels and a higher NF-κB p65 level. Pretreatment with the Axl inhibitor R428 reversed the effects of Gas6 on p-Axl, SOCS3 and NF-κB p65. Data are expressed as the means ± SD (n = 4 per group). T-Axl: total Axl. p-Axl: phosphorylated Axl. HR: hypoxia-reoxygenation. Gas6: recombinant Gas6 20 ng/ml. R428: R428 100 nM. All values are expressed as the means ± SD (n = 4 per group). * P < 0.05 compared with the control group; *** P < 0.001 compared with the control group; ## P < 0.01 compared with the HR group.

Fig 8. Gas6 and p-Axl of lung tissues.

(A) Gas6 mRNA expressions in lung tissues. (B) Endogenous Gas6 levels in perfusate determined by ELISA analysis. (C) p-Axl levels determined by western blot analysis. Recombinant Gas6, but not endogenous Gas6, up-regulated phosphorylation of Axl in IR-ALI. p-Axl: phosphorylated Axl. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 4 per group for tissue mRNA and n = 6 per group for perfusate). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ### P < 0.001 compared with the IR group.