Chemokine production by human alveolar epithelial cells in response to Mycobacterium tuberculosis infection.

摘要

Mycobacterium tuberculosis (M. tuberculosis ), the etiologic agent of tuberculosis, is the leading cause of death in the adult population worldwide and infects more than one-third of the global population. Upon inhalation of M. tuberculosis, four potential infection outcomes can be observed: (1) immediate clearance of the organism and no disease development; (2) primary disease; (3) latent infection that will never reactivate or (4) latent infection and disease reactivation months or years later.;Most of the studies on the host response to M. tuberculosis infection focus on the effector functions of macrophages to eliminate the pathogen. In fact, it is well established that M. tuberculosis-activated macrophages produce inflammatory mediators required for the recruitment and activation of monocytes and lymphocytes to the site of infection, resulting in the formation of a protective granuloma and the establishment of acquired immunity. However, due to the high number of alveolar epithelial cells that constitute the human alveolus and their potential involvement in innate immunity to M. tuberculosis, we have hypothesized that the aerosolized M. tuberculosis bind to and invade epithelial cells upon arrival into the alveolar space, even prior to infecting alveolar macrophages, triggering a cascade of innate immune responses that ultimately results in granuloma formation.;Our laboratory has previously shown that M. tuberculosis disrupted in the mce1 operon is hypervirulent, fails to stimulate protective granuloma formation and causes rapidly progressive disease in the murine model of infection, leading to faster death of the animals. Recent findings show that the mce1 mutant strain has an altered lipid profile, with an increased abundance of mycolic acids localized on the surface of the organism.;The focus of the second chapter of this dissertation is the role of alveolar epithelial cells in the initiation of innate immune response to M. tuberculosis infection. Our results demonstrate that the human alveolar epithelial cell line A549 produces a series of chemokines involved in the recruitment of immune cells to the site of infection responsible for the formation of a protective granuloma. On the other hand, the mce1 operon mutant strain demonstrated reduced expression of chemokines by infected A549 cells, suggesting that the presence of the Mce1 proteins is required for an efficient initiation of innate immune response in human lungs.;M. tuberculosis has been shown to mainly activate the host pro-inflammatory response through toll-like receptors (TLRs) 2, in particular TLR-2, by secreting a number of specific agonists. The third chapter of this dissertation focuses on understanding the involvement of TLR-2 as potential pattern recognition receptor to M. tuberculosis pathogen-associated molecular patterns, in human alveolar epithelial cells. Anti-TLR-2 antibodies were used as blocking elements of TLR-2-induced intracellular signaling pathway in A549 cells. We observed that the difference in chemokine upregulation in response to infection by wild-type (WT) M. tuberculosis versus mce1 mutant strains no longer occurred. These data suggest the role of TLR-2 as the receptor involved in initiation of innate immunity in A549 cells.;Recent work in our laboratory has demonstrated by thin-layer chromatography and mass spectrometry that the mce1 mutant strain has an altered lipid profile, with an increased accumulation of free mycolic acids in the outermost region of the bacteria cell wall, when compared to the WT strain. This observation led us to hypothesize, in the fourth chapter of this dissertation, that the abundance of mycolic acids observed in the mce1 operon mutant strain might be related to bacterial hypervirulence. Mycolic acids may block the immune response through TLR-2. In order to determine the role of excess free mycolic acids in M. tuberculosis pathogenesis, A549 cells and RAW macrophages were challenged with different concentrations of free M. tuberculosis mycolic acids, followed by the addition of a TLR-2 agonist. The amount of chemokines produced in response to the TLR-2 agonist was diminished in a dose-dependent manner. These observations led us to conclude that the excess free mycolic acids observed in the cell wall of mce1 mutant strain can block TLR-2-dependent intracellular signaling in A549 cells.;These data show for the first time that a M. tuberculosis cell wall component can modulate the innate immune response in human alveolar epithelial cells through TLR-2. Furthermore, they show that alveolar epithelial cells are likely to play an important role in the early phase of infection and affect the granuloma formation in an infected host. Clarifications on the interaction of M. tuberculosis cell wall components with host receptors can shed light into the development of effective drug targets or therapeutic vaccines against tuberculosis.