Elsevier

Gene

Volume 344, 3 January 2005, Pages 193-202
Gene

Gene expression of Toll-like receptor-2, Toll-like receptor-4, and MD2 is differentially regulated in rabbits with Escherichia coli pneumonia

https://doi.org/10.1016/j.gene.2004.09.032Get rights and content

Abstract

Sepsis, a common sequela to Gram-negative pneumonia, results in considerable morbidity and mortality in hospitalized patients. The goal of this study was to determine whether Gram-negative pneumonia alters the expression TLR2, TLR4, and MD2 in lungs or in organs distant to the site of the primary infection. The cDNA sequence coding open reading frames for rabbit TLR2, TLR4, and MD2 were cloned and expressed in Escherichia coli, and specific polyclonal antibodies and polymerase chain reaction (PCR) probes were produced to identify changes in these receptors in rabbits with Gram-negative pneumonia. Using tissues from lungs and distant organs, we show that TLR2, TLR4, and MD2 gene expression is differentially regulated in rabbits with E. coli pneumonia. The increased expression of TLR2 and TLR4 could play an important role in the innate immune response to bacterial infection in the lungs, and improve pathogen recognition and bacterial clearance. In contrast, the increased gene expression of TLR2, TLR4, and MD2 in organs distant to the primary site of infection may contribute to the deleterious systemic inflammatory response observed in patients with sepsis.

Introduction

Sepsis, a leading cause of mortality in hospitalized patients, results in over 200,000 attributable deaths annually (Angus et al., 2001). Despite preventative measures, broad-spectrum antibiotics, and complex supportive care, bacterial pneumonia remains a leading source of infection that results in sepsis (Alberti et al., 2003). The innate immune system is responsible for the successful clearance of bacteria from the lungs, and an effective innate immune response clears bacterial pathogens from the lungs with minimal tissue injury or systemic inflammatory response. In contrast, an ineffective innate immune response results in bacterial proliferation, increased lung injury, and the development of systemic manifestations of tissue infection, which include sepsis and septic shock (Fox-Dewhurst et al., 1997).

Recent advances have increased our understanding of the mechanisms responsible for the recognition and clearance of bacteria by the innate immune system (Ulevitch, 2003). This began with the discovery that CD14 and lipopolysaccharide (LPS)-binding protein were responsible for the recognition of LPS (Wright et al., 1990). Subsequent work showed that Toll-like receptors (TLRs) were responsible for bacterial recognition and signaling, and are a mechanism that provides specificity to the innate immune system (reviewed in Beutler et al., 2003). TLR2 recognizes peptidoglycans and lipoteichoic acid on Gram-positive bacteria as well as lipoproteins on Gram-negative bacteria, whereas TLR4 recognizes LPS on Gram-negative bacteria (Poltorak et al., 1998, Schwandner et al., 1999, Takeuchi et al., 1999, Lee et al., 2002). MD2 confers responsiveness on TLR4, suggesting that LPS recognition by the innate immune system requires the complex of CD14/TLR4/MD2 (da silva Correia et al., 2001).

A number of studies provide insight into the gene regulation of CD14 during tissue infection (Matsuura et al., 1994, Maus et al., 2001). Studies have been performed on the expression of TLR2, TLR4, and MD2 using macrophages, epithelial cells, and neutrophils stimulated with LPS and cytokines in vitro (Droemann et al., 2003, Oshikawa and Sugiyama, 2003). However, little is known about the changes that occur in the expression of TLR2, TLR4, and MD2 during Gram-negative pneumonia.

The goal of this study was to determine whether Gram-negative pneumonia alters the expression TLR2, TLR4, and MD2 in lungs or in organs distant to the site of the primary infection. To accomplish this goal, rabbit TLR2, TLR4, and MD2 cDNA were cloned and expressed in Escherichia coli, and specific polyclonal antibodies and polymerase chain reaction (PCR) probes were produced. Using tissues from lungs and distant organs obtained from rabbits with E. coli pneumonia, we show that TLR2, TLR4, and MD2 gene expression is differentially regulated in lungs and distant organs of rabbits with E. coli pneumonia. Whereas the increased expression of these genes in the lungs is probably important for maintaining pulmonary host defenses, increased expression of TLR2, TLR4, and MD2 at distant sites may play a role in the development of systemic manifestations of tissue infection such as sepsis and septic shock.

Section snippets

cDNA cloning

A partial sequence of rabbit TLR2 cDNA was cloned from a rabbit spleen cDNA library (Stratagene, La Jolla, CA) using a 32P-labeled PCR-amplified cDNA (245 bp) probe. The probe had been prepared using the same spleen cDNA library as a template and a PCR primer pair designed on the consensus cDNA sequence of human and mouse TLR2 (GenBank accession nos. AF051152 and AK005043). The complete sequence of rabbit TLR2 cDNA was obtained using the partial clone of rabbit TLR2 cDNA and extension of the

Cloning of cDNA of rabbit TLRs and MD2

The cloned rabbit TLR2 cDNA was 3271 bp and coded 784 amino acid residues (Fig. 1; GenBank accession no. AY101393); the cloned rabbit TLR4 cDNA was 2729 bp and coded 839 amino acid residues (Fig. 2; GenBank accession no. AY101394), and the cloned cDNA of rabbit MD2 was 547 bp and coded 160 amino acid residues (Fig. 3; GenBank accession no. AY101395). The open reading frames for rabbit TLR2, TLR4, and MD2 start at the methionine codon, exactly as reported for human TLR2, TLR4, and MD2 (Medzhitov

Conclusions

These studies show a considerable amount of shared amino acid sequence identity between rabbit and human TLR2, TLR4, and MD2. This is especially true for regions of TLR2 and TLR4 that have functional significance, such as the extracellular domain that plays a role in pathogen recognition and the cytoplasmic domain that plays a role in intracellular signaling pathways. The considerable amount of shared identity observed between rabbits and humans suggests that the inflammatory response to

Acknowledgements

This work was supported, in part, by NIH grant GM37696, VA Research.

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