Expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is associated with lower innate immune responses to infection. Here we show that ESAT-6 inhibited activation of transcription factor NF-kappaB and interferon-regulatory factors (IRFs) after Toll-like receptor (TLR) signaling; inhibition of TLR signaling by ESAT-6 required the kinase Akt. Direct binding of ESAT-6 to TLR2 activated Akt and prevented interaction between the adaptor MyD88 and 'downstream' kinase IRAK4, thus abrogating NF-kappaB activation. The six carboxy-terminal amino acid residues of ESAT-6 were required and sufficient for the TLR2-mediated inhibitory effect. A critical function for the carboxy-terminal peptide of ESAT-6 in restricting MyD88-dependent TLR signaling emphasizes the possibility that mimetic inhibitory peptides could be used to restrict innate immune responses in situations in which prolonged TLR signaling has deleterious effects.
Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1 (IL-1) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1 release and dissected the signaling pathways leading to LPS-induced IL-1 secretion. We demonstrate that both the NF-B and the C/EBP-binding elements of the IL-1 promoter drive LPS-induced IL-1 gene expression. NF-B activation requires the classical TLR4-initiated signaling cascade leading to I B phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBP activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBP, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBP. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser 376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1 gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1 gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.
Apoptosis contributes to the pathology of gastric epithelial cell damage that characterizes Helicobacter pylori infection. The secreted peptidyl prolyl cis, trans-isomerase of H. pylori, HP0175 executed apoptosis of the gastric epithelial cell line AGS in a dose- and time-dependent manner. The effect of HP0175 was confirmed by generating an isogenic mutant of H. pylori disrupted in the HP0175 gene. The apoptosis-inducing ability of this mutant was impaired compared with that of the wild type. The effect of HP0175 was mediated through TLR4. Preincubation of the gastric epithelial cell line AGS with anti-TLR4 mAb inhibited apoptosis induced by HP0175. Downstream of TLR4, apoptosis signal-regulating kinase 1 activated MAPK p38, leading to the caspase 8-dependent cleavage of Bid, its translocation to the mitochondria, mitochondrial pore formation, cytochrome c release, and activation of caspases 9 and 3. We show for the first time that a secreted bacterial Ag with peptidyl prolyl cis,trans-isomerase activity signals through TLR4, and that this Ag executes gastric epithelial cell apoptosis through a signaling pathway in which TLR4 and apoptosis signal-regulating kinase 1 are central players.
Mycobacterum tuberculosis is the causative agent of tuberculosis, which leads to an estimated 2 to 3 million deaths worldwide each year. The organism evades the immune response of the host by manipulating host cell signaling pathways (1). One of the important mechanisms is the suppression of interleukin-12 (IL-12) 3 production. IL-12 is a heterodimeric cytokine composed of two disulfide-linked subunits of 35 (p35) and 40 (p40) kDa encoded by two separate genes (2-4). It is produced by macrophages and dendritic cells in response to infection with bacteria or exposure to bacterial constituents such as lipopolysaccharide (LPS) (2, 4). It is required for cell-mediated immunity and host defense against intracellular microbes (5). IL-12 is critical for the generation of Th1 responses against M. tuberculosis infection. In support of this view are the observations that individuals with mutations affecting the genes of IL-12 (6) or its receptor (7,8) have increased susceptibility to mycobacterial infections. IL-12-deficient mice are susceptible to Mycobacterium bovis bacillus Calmette-Guerin infection (9). This underscores the need to understand how M. tuberculosis dampens IL-12 production. Intracellular bacteria and bacterial products regulate IL-12 production at the transcriptional level (10). The best studied transcriptional regulation of the IL-12 p40 promoter is mediated by binding of Rel family members effecting induction of a Th1 immune response (11-13). We therefore considered the possibility of M. tuberculosis modulins dampening this pathway.Lipoarabinomannan (LAM) is a cell wall lipoglycan. LAMs have been considered modulins because of their capacity to manipulate the host immune system. To date, three families of LAMs have been described (14). A glycosylphosphatidylinositol anchor, a D-mannan core, and a large terminal D-arabinan represents the core structure. The arabinan domain is capped by either mannosyl (15-18) or phosphoinositol residues (19,20). The mannose-capped LAMs (Man-LAMs) are found in pathogenic species including M. tuberculosis (14). Phosphoinositol-capped LAMs have been isolated from non-pathogenic species such as Mycobacterium smegmatis. Phosphoinositol-capped LAMs activate macrophages in a Toll-like receptor (TLR) 2-dependent manner by activating NF-B-dependent pathways (21). Mycobacterial Man-LAMs on the other hand, modulate the immune response by dampening IL-12 production in macrophages and dendritic cells (22,23) and attenuating apoptotic signaling (24,25). The emerging consensus is that Man-LAM contributes to the capacity of M. tuberculosis to survive inside macrophages by mediating immunosuppressive effects. Recent studies have demonstrated that lipomannans induce IL-12 production (26) in a TLR-dependent manner and may also inhibit IL-12 production in a TLR-independent manner (27), suggesting that the ratio of lipomannans to Man-LAM may govern the capacity of the organism to circumvent IL-12 production. The anti-inflammatory effects of Man-LAM have been attributed to their binding to t...
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