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.

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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...

Helicobacter pylori infection is associated with the local production of chemokines and cytokines, of which IL-6 is overexpressed at the margin of gastric ulcer in H. pylori-positive gastritis. Cells of the monocytic lineage are the major sources of IL-6, and mononuclear cell infiltration in the lamina propria is characteristic of H. pylori-induced chronic infection. Our study shows for the first time that a secreted peptidyl prolyl cis-, trans-isomerase, HP0175 elicits IL-6 gene expression and IL-6 release from macrophages. An isogenic strain inactivated in the HP0175 gene (knockout) was attenuated in its IL-6-inducing ability, which was restored after complementation with the HP0175 gene. The specificity of the HP0175-induced effect was confirmed by the fact that rHP0175 purified from HEK293 cells could also induce IL-6 release, ruling out the possibility that the observed effect was due to bacterial contaminants. HP0175 was capable of interacting directly with the extracellular domain of TLR4. HP0175-induced IL-6 gene expression was critically dependent on TLR4-dependent NF-κB and MAPK activation. TLR4/PI3K-dependent ERK1/2 and p38 MAPK signaling converged upon activation of mitogen- and stress-activated protein kinase 1 (MSK1). The central role of MSK1 was borne out by the fact that silencing of MSK1 expression abrogated HP0175-mediated NF-κB-dependent IL-6 gene transcription. MSK1 regulated the recruitment of p65 and phopho-Ser10-histone H3 to the IL-6 promoter. HP0175 therefore regulated IL-6 gene transcription through chromatin modification at the IL-6 promoter.

The pathophysiology of Helicobacter pylori-associated gastroduodenal diseases, ulcerogenesis, and carcinogenesis is intimately linked to activation of epidermal growth factor receptor (EGFR) and production of vascular endothelial growth factor (VEGF). Extracellular virulence factors, such as CagA and VacA, have been proposed to regulate EGFR activation and VEGF production in gastric epithelial cells. We demonstrate that the H. pylori secretory protein, HP0175, by virtue of its ability to bind TLR4, transactivates EGFR and stimulates EGFR-dependent VEGF production in the gastric cancer cell line AGS. Knock-out of the hp0175 gene attenuates the ability of the resultant H. pylori strain to activate EGFR or to induce VEGF production. HP0175-induced activation of EGFR is preceded by translocation of TLR4 into lipid rafts. In lipid rafts, the Src kinase family member Lyn interacts with TLR4, leading to tyrosine phosphorylation of TLR4. Knockdown of Lyn prevents HP0175-induced activation of EGFR and VEGF production. Tyrosine-phosphorylated TLR4 interacts with EGFR. This interaction is necessary for the activation of EGFR. Disruption of lipid rafts with methyl ␤-cyclodextrin prevents HP0175-induced tyrosine phosphorylation of TLR4 and activation of EGFR. This mechanism of transactivation of EGFR is novel and distinct from that of metalloprotease-dependent shedding of EGF-like ligands, leading to autocrine activation of EGFR. It provides new insight into our understanding of the receptor cross-talk network.Receptor-tyrosine kinases are among key cell surface receptors that transduce external signals through the membrane to regulate biological processes, such as cell proliferation, differentiation, and survival. Within this group, the epidermal growth factor receptor (EGFR) 3 family is closely associated with the pathophysiology of cancer. EGFR is mutated or overexpressed in a wide variety of epithelial tumors (1-3). The classical pathway of activation of the EGFR occurs through binding of a ligand to its extracellular domain and subsequent autophosphorylation of two receptor molecules (4). Ligands such as epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF) are synthesized as membrane-spanning molecules that are proteolytically cleaved to become active. Ligand binding initiates the formation of activated dimers/oligomers that undergo autophosphorylation of tyrosine residues in the cytoplasmic tail. This serves as the trigger for the recruitment of adaptor proteins and the initiation of signaling cascades within the cell. Mutations of EGFR, such as deletion of 801 bp encoded by exons 2-7 (5), result in a truncated receptor lacking 267 amino acid residues in the extracellular domain. This receptor shows ligand-independent, constitutive activity associated with increased tumorigenicity of tumors in vivo (6, 7). The paradigm for transactivation of EGFR has been the G protein-coupled receptor (GPCR)-mediated pathway (8). GPCR-dependent activation of matrix metalloproteases leads to cleavage of pro-HB-EGF and release...