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.

Understanding how pathogenic mycobacteria subvert the protective immune response is crucial to the development of strategies aimed at controlling mycobacterial infections. Prostaglandin E 2 exerts an immunosuppressive function in the context of mycobacterial infection. Because cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in prostaglandin biosynthesis, there is a need to delineate the mechanisms through which pathogenic mycobacteria regulate COX-2 expression in macrophages. Our studies demonstrate that the NF-B and CRE elements of the COX-2 promoter are critical to Mycobacterium avium-induced COX-2 gene expression. M. avium-triggered signaling originates at the Toll-like receptor 2 (TLR2). Ras associates with TLR2 and activates the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), whereas tumor necrosis factor receptor-associated factor 6 (TRAF6)/transforming growth factor ␤-activated kinase 1 (TAK1)-dependent signaling activates p38 MAPK. Both ERK and p38 MAPK activation converge to regulate the activation of mitogen-and stress-activated kinase 1 (MSK1). MSK1 mediates the phosphorylation of the transcription factor CREB accounting for its stimulatory effect on CRE-dependent gene expression. M. aviumtriggered cytoplasmic NF-B activation following IB phosphorylation is necessary but not sufficient for COX-2 promoter-driven gene expression. MSK1 activation is also essential for M. avium-triggered NF-Bdependent gene expression, presumably mediating nucleosomal modifications. These studies demonstrate that the nuclear kinase MSK1 is necessary in regulating the pathogen-driven expression of a gene by controlling two transcription factors. The attenuation of MSK1 may therefore have potential benefit in restricting survival of pathogenic mycobacteria in macrophages.To design novel therapeutics and to rationalize vaccination strategies against mycobacterial diseases, it is of foremost importance to understand the mechanisms by which mycobacteria subvert the protective immune response. The response of macrophages to invasion by intracellular pathogens such as mycobacteria depends on the signals triggered during the initial contact of the bacterium with the macrophages. Prostaglandin E 2 (PGE 2 ) 1 is believed to exert an immunosuppressive function in the context of mycobacterial infections. PGE 2 interferes with T lymphocyte responses by inhibiting the production of interleukin-2 (1). PGE 2 also inhibits the secretion of interferon-␥, which is important in activating T cells and macrophages (2). Cooper and colleagues (3) reported that the ability of the virulent Mycobacterium avium to attenuate macrophage activation was dependent on its ability to trigger p38 mitogenactivated protein kinase (MAPK)-dependent production of PGE 2 . These observations were supported by a case report in which monocytes from a child with disseminated M. avium infection demonstrated a defect in bactericidal activity which could be offset by treatment with indomethacin (4). The critical role of PGE 2 is al...

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