Response of Human Pulmonary Epithelial Cells to Lipopolysaccharide Involves Toll-like Receptor 4 (TLR4)-dependent Signaling Pathways

Abstract

Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.

Airway epithelial cells modify immune responses by inducing an anti‐inflammatory microenvironment

“…However, only limited information is available for the situation in the upper airways. It has been reported that the sensitivity and the activation threshold of TLR in bronchial epithelial cells is indeed regulated [6,[14][15][16], supporting the concept that airway epithelial cells actively restrict pattern recognition principles.…”

“…This has two important implications: First, epithelial cells at non-sterile surfaces have to regulate their TLR sensitivity to avoid continuous threat of inflammation and, second, organ-specific mechanisms of regulating immunity have to exist. The first fact has now been intensively studied and multiple mechanisms including threshold regulation of TLR stimulation [6,10], anatomical sequestration [14,26], inhibitory signaling molecules [27,28], coreceptor modulation [6] and specific signaling pathways [29] have been described. The second hypothesis was the subject of this study.…”

Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells

“…Other epithelial cell types (e.g. bronchial epithelial cells [18], nasopharynx epithelial cells [48], etc.) have also been shown to respond to LPS.…”

Airway epithelial cell signaling in response to bacterial pathogens

“…9,10 However, not all of these TLRs and the associated adaptors are equally available on the surface of the airway epithelium where they can respond to luminal contaminants. Whereas TLR2 is apically expressed and TLR5 is readily mobilized in response to bacteria, 5,6 TLR4 is intracellular 11 and is not mobilized to the cell surface in response to the major lung pathogens P. aeruginosa or S. aureus. 5 The expression of MD2, a co-stimulatory molecule that it is required for TLR4 signaling, is limited on airway epithelial cells 11 which explains the minimal response to LPS.…”