Adenovirus-mediated overexpression of soluble ST2 provides a protective effect on lipopolysaccharide-induced acute lung injury in mice

Abstract

Acute lung injury is characterized by a diffuse inflammatory parenchymal process, implicated in the context of significant morbidity and mortality. Previously, we have reported that soluble ST2 (sST2), a member of the Toll-interleukin (IL)-1 receptor (TIR) superfamily, represses proinflammatory cytokine production of macrophage exposed to lipopolysaccharide (LPS). In this study, we examined the possibility of modulating LPS-induced murine inflammatory pulmonary damage by recombinant adenovirus-mediated sST2-Fc (Ad-sST2-Fc) gene transfer. Single intranasal administration of Ad-sST2-Fc led to a profound decrease in LPS-induced bronchoalveolar lavage leucocyte exudation and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Histological examination revealed alveolitis with inflammatory cell infiltration and alveolar haemorrhage in the alveolar airspace was less severe in Ad-sST2-Fc-treated mice when compared with control groups. In addition, high levels of sST2-Fc in vivo reduced the transcription of tumour necrosis factor-α, IL-6 and Toll-like receptor-4 gene remarkably, and suppressed the nuclear translocation of nuclear factor-κB in lung tissues in response to LPS challenge. Taken together, these results suggested that administration of Ad-sST2-Fc gene transfer may have therapeutic potential for the immunomodulatory treatment of LPS-mediated inflammatory lung injury.

Fig. 1

Expression of recombinant adenovirus-mediated soluble ST2 (Ad-sST2-Fc) in vivo. (a) Ad-sST2-Fc was administered intranasally to BALB/c mice at a dose of 5 × 10⁸ plaque-forming units (pfu)/mice. Time–course of sST2-Fc expression in bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). (b) Animals were treated with different virus doses (high: 1 × 10⁹ pfu/mice; medium: 5 × 10⁸ pfu/mice; low: 1 × 10⁸ pfu/mice). Bronchoalveolar lavage was performed 2 days later after viral administration and sST2-Fc expression in BALF was determined by ELISA. Data are presented as the mean ± standard error of the mean (n = 4–6 in each group); n.s.: not significant.

Fig. 2

Pretreatment with adenovirus-mediated soluble ST2 (Ad-sST2-Fc) attenuates lung tissue damage in mice challenged with lipopolysaccharide (LPS). (a) Lungs from each experimental group were processed for histological examination after haematoxylin and eosin staining (400×). Compared with control mice, mice exposed to intranasal LPS alone or LPS + adenovirus expressing green fluorescence protein (Ad-EGFP) for 24 h led to profound neutrophil infiltration and alveolar haemorrhage. These features were decreased dramatically in mice pretreated with Ad-sST2-Fc prior to LPS challenge. (b) Bronchoalveolar lavage fluid (BALF) was collected and cell differentiation was determined at 24 h after LPS challenge. Exposure to intranasal LPS markedly enhanced the total cells and percentage of neutrophils that was attenuated in mice pretreated with Ad-sST2-Fc. (c) Myeloperoxidase (MPO) content was also assessed in lung tissue homogenates, as described in Materials and methods. Exposure to LPS resulted in a significant increase in lung MPO activity assessed at 24 h after intranasal LPS administration. The level of tissue MPO activity was reduced markedly in mice pretreated with Ad-sST2-Fc. Data are presented as the mean ± standard error of the mean (n = 4–6 in each group). *P < 0·05 versus the LPS group.

Fig. 3

Overexpression of soluble ST2 (sST2-Fc) decreases the production of proinflammatory cytokines. (a) Lung tumour necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were determined in mice pretreated with adenovirus-mediated sST2-Fc (Ad-sST2-Fc) following lipopolysaccharide (LPS) challenge at 3 h. Results were obtained using real-time reverse transcription–polymerase chain reaction and expressed as relative increase of mRNA expression compared with control animals. (b) The levels of TNF-α and IL-6 protein in bronchoalveolar lavage fluid (BALF) were also evaluated by enzyme-linked immunosorbent assay in mice after LPS stimulation at 6 h. Data are presented as the mean ± standard error of the mean (n = 4–6 in each group). *P < 0·05 versus the LPS group.

Fig. 4

Overexpression of soluble ST2 (sST2-Fc) suppresses the transcription of Toll-like receptor (TLR)-4. Lung TLR-4 mRNA expression was measured in mice pretreated with adenovirus-mediated sST2-Fc (Ad-sST2-Fc) followed by lipopolysaccharide (LPS) exposure at 3 h. Results were obtained using real-time reverse transcription–polymerase chain reaction and expressed as relative increase of mRNA expression compared with control animals. Data are presented as the mean ± standard error of the mean (n = 4–6 in each group). *P < 0·05 versus the LPS group.

Fig. 5

High levels of soluble ST2 (sST2-Fc) in vivo inhibit nuclear factor-κB activation. Mice werepretreated with adenovirus-mediated sST2-Fc (Ad-sST2-Fc) following lipopolysaccharide (LPS) challenge. Nuclear extracts were prepared at 3 h from the lung tissues and subjected to electrophoretic mobility shift assay. Assay shown is representative of three experiments with similar results.