Differential Roles of Toll-Like Receptors 2 and 4 in In Vitro Responses of Macrophages toLegionella pneumophila

Abstract

The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2 ؊/؊ ), TLR4 ؊/؊ , and wild-type (WT) littermate (C57BL/6 ؋ 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2 ؊/؊ macrophages compared to WT and TLR4؊/؊ macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2 ؊/؊ macrophages compared to WT and TLR4 ؊/؊ macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2 ؊/؊ macrophages was significantly lower than those of WT and TLR4 ؊/؊ macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.Legionella pneumophila is the major etiologic agent of Legionnaires' disease, a potentially fatal type of pneumonia affecting immunocompromised and immunocompetent subjects (28,30). This gram-negative bacterium can multiply within the mononuclear cells in vivo and in vitro (6, 14) and evades phagosome-lysosome fusion within these cells (3). Several L. pneumophila virulence factors that facilitate intracellular growth have been identified in screenings with macrophages or in an in vivo screening system with signature-tagged mutagenesis (12). One important set of virulence factors is the dot/icm system, which is a type IV secretion system and is required for evasion of phagosome-lysosome fusion (5, 37, 38) and for the establishment of phagosomes permissive for the growth of L. pneumophila within them (8). However, the effector molecule(s) of this system remains undetermined. The innate immunity to L. pneumophila has been extensively studied. The A/J mouse strain is permissive for the intracellular growth of L. pneumophila (48), whereas other inbred strains of mice, such as BALB/c (49), 57BL/6, and 129X1 (10), are not. In terms of permissiveness of A/J macrophages, the genetic determinant of this permissiveness has been confirmed to be within the Lgn locus, and specifically the Bircle/Naip5 gene (4, 9, 10, 40, 52, 53). However, the role of this gene in regulation of intracellular growth of the bacterium is still unknown.Toll-like receptors (TLRs) have been identified as receptors of pathogen-as...

Toll-like receptor 4 polymorphisms are associated with resistance to Legionnaires' disease

“…Legionella is an intracellular Gram-negative bacterium with an unusual LPS structure that is primarily recognized by TLR2 rather than TLR4 (12). We and others (13,15) have shown that TLR4 does not mediate recognition of Legionella in mouse bone marrow macrophages stimulated in vitro, a result that contrasts with many other Gram-negative bacteria that are recognized by TLR4. We also found that TLR4 does not affect bacterial growth during in vivo Legionella infections in mice inoculated via an intranasal route (36).…”

“…Legionella pneumophila, described in 1976 as the agent of Legionnaires' disease (LD), is a flagellated Gram-negative bacterium that causes from 1% to 30% of cases of community-acquired pneumonia (7)(8)(9)(10)(11). In vitro studies indicate that Legionella is recognized by several TLRs, including TLR2, TLR4, and TLR5 (12)(13)(14)(15). We recently demonstrated (13,16,17) that TLR5 recognizes bacterial flagellin and that a common dominant TLR5 stop codon variant is associated with susceptibility to LD.…”

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…Previous studies used knockout mice and BMD macrophages from these knockout mice to demonstrate that during intracellular infection, L. pneumophila stimulates TLRs, including both cell surface TLR2 (via the recognition of lipoprotein and LPS) and TLR5 (flagellin) and intracellular TLR9 (CpG oligodeoxynucleotide) (64)(65)(66)(67)(68)(69). In order to test, for the first time, the importance of these pathways in human macrophages, we started out by generating a stable knockdown of MyD88 with a 76% decrease in protein expression (Fig.…”

Airway epithelial cell signaling in response to bacterial pathogens

“…22 The protective role of TLR2-mediated signaling in the clearance of L. pneumophila in the lungs was demonstrated using a murine model of Legionnaires' disease. 23 In vivo growth of wild-type L. pneumophila was enhanced in the lungs of TLR2-deficient mice, resulting in a delay in bacterial clearance whereas L. pneumophila dotA mutants that cannot replicate intracellularly were efficiently cleared.…”