Shock/Sepsis/Trauma/Critical Care
TLR4 Mediates Lung Injury and Inflammation in Intestinal Ischemia-Reperfusion

https://doi.org/10.1016/j.jss.2010.12.005Get rights and content

Background

Splanchnic ischemia is common in critically ill patients, and it can result in injury not only of the intestine but also in distant organs, particularly in the lung. Local inflammatory changes play a pivotal role in the development of acute lung injury after intestinal ischemia, but the underlying molecular mechanisms are not fully understood. We sought to examine the role of Toll-like receptor 4 (TLR4) in the mouse model of intestinal ischemia-reperfusion (I/R)-induced lung injury and inflammation.

Materials and Methods

Adult male TLR4 mutant (C3H/HeJ) mice and TLR4 wild-type (WT) (C3H/HeOuJ) mice were subjected to 40 min of intestinal ischemia by clamping the superior mesenteric artery followed by 6 h of reperfusion. Lung histology was assessed and parameters of pulmonary microvascular permeability, inflammatory cytokine expression, and neutrophil infiltration were measured. Activation of mitogen-activated protein kinases (MAPKs) and the transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) in the lungs were also detected.

Results

After intestinal I/R, lungs from TLR4 mutant mice demonstrated a significantly lower histological injury, a marked reduction of epithelial apoptosis associated with the decreased level of cleaved caspase-3 and the increased ratio of Bcl-xL to Bax proteins, and a large reduction in pulmonary vascular permeability and myeloperoxidase (MPO) activity in comparison with WT mice. TLR4 mutant mice also displayed marked decreases in tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) expression. Following intestinal I/R, phosporylation of p38 MAPK and activation of NF-κB and AP-1 were significantly inhibited in lung tissue from TLR4 mutant mice compared with WT controls.

Conclusions

These data suggest that TLR4 plays an important role in the pathogenesis of intestinal I/R-induced acute lung injury and inflammation and that p38 kinase and NF-κB may be involved in TLR4 signaling-mediated lung inflammatory processes during intestinal I/R.

Introduction

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, develop frequently and remain a considerable clinical challenge in the intensive care unit, which affect about 200,000 patients each year in the United States with an average mortality rate of approximately 40% [1]. A better understanding of the molecular pathophysiology of ALI may lead to more effective management of critically ill patients with this life-threatening disorder.

Acute lung injury can occur from a direct insult to the lung and, more usually, indirectly as a result of the systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS). The gut is increasingly recognized to have a pivotal role in the development of SIRS/MODS and ALI in the critical care setting 2, 3. Mesenteric ischemia due to circulatory stress is an early and common phenomenon in critical illness or during major surgical procedures [4]. Mesenteric ischemia leads to increased mucosal permeability, bacterial translocation, and further mucosal hypoperfusion. Mucosal injury in mesenteric ischemia may be substantially exacerbated by targeted improvement in organ tissue perfusion with resuscitation. A solid body of evidence has demonstrated that intestinal ischemia/reperfusion (I/R) rapidly activates local, remote, and systemic inflammatory responses, which contribute substantially to injury of the intestine and in distant organs including the lung, heart, kidney and liver, leading finally to multiple organ failure and death [5]. Intestinal I/R is thus a common feature of critical care and represents a useful and clinically relevant model for studying the pathophysiologic aspects of indirect ALI. Despite having established a key inflammatory mechanism in the pathogenesis of ALI in gut I/R, however, the understanding is lacking.

Toll-like receptors (TLRs) are a family of signaling receptors that play a critical role in the regulation of inflammatory and innate immune responses [6]. Among these receptors, TLR4 specifically recognizes bacterial lipopolysaccharide (LPS) and also conjugates with endogenous ligands such as heat shock proteins, heparan sulfate, and hyaluronan. Ligand-specific binding to TLR4 can activate multiple intracellular signaling systems including the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 mitogen activated protein kinase (MAPK) as well as the nuclear factor κB (NF-κB) pathways, all of which are pivotal regulators of inflammatory immune responses and cell survival and death [6]. Recent studies have demonstrated a critical role for TLR4 in the pathophysiology of ALI in response to a variety of stimuli including LPS challenge [7]. However, the functional relevance of TLR4 signaling in the setting of gut I/R-induced ALI remains unclear. In this study, we examined whether deficiency of TLR4 ameliorated lung damage and inflammation in a mouse model of intestinal I/R. To further understand the function of TLR4 in lung inflammatory processes, we also investigated whether TLR4 deficiency affected the activation of lung MAPKs or NF-κB following intestinal I-R.

Section snippets

Animal

TLR4 mutant mice (C3H/HeJ) and wild-type (WT) mice (C3H/HeOuJ) were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in a specific pathogen-free environment and the male TLR4 mutant mice and WT controls (8–10-wk-old; 22–30 g) were used for experiments. All protocols were approved by the Laboratory Animal Care and Use Committee of Second Military Medical University.

Intestinal Ischemia/Reperfusion

Animals were starved for 24 h before the experimentation but allowed free access to water. Animals were

TLR4 Deficiency Ameliorates Lung Injury after Intestinal I/R

After a 6-h period of intestinal I/R, a severe lung injury was observed in TLR4 WT C3H/HeOuJ mice, as indicated by extensive interstitial edema, alveolar congestion, and infiltration of leukocytes. These changes were significantly ameliorated in TLR4 mutant C3H/HeJ mice (Fig. 1). No evidence of marked lung injury was found in the sham animals.

TLR4 Deficiency Inhibits Lung Epithelial Cell Apoptosis after Intestinal I/R

TUNEL staining indicated that intestinal I/R resulted in significant apoptosis of alveolar and bronchiolar epithelial cells in WT mice, while TLR4 mutant

Discussion

In the present study, we demonstrated that multiple parameters for lung inflammation, including histologic changes, epithelial apoptosis, microvascular leak, and neutrophil accumulation were substantially ameliorated by TLR4 deficiency in intestinal I/R. Moreover, deficiency of TLR4 abrogated the local inflammatory cytokine expression in the lungs. Furthermore, we demonstrated that TLR4 deficiency inhibited the lung activation of proinflammatory signaling pathways, including p38 kinase, NF-κB,

Acknowledgments

This study was supported by the National Nature Science Foundation of China Emphasis Grant (No. C03730091), the National Nature Science Foundation of China (No. 30973117, 30901428), and the Shanghai Youth Science and Technology Venus Program (No. 10QA1408500).

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