Sushil Kumar Pathak

Execution of macrophage apoptosis by PE_PGRS33 of Mycobacterium tuberculosis is mediated by toll-like receptor 2-dependent release of tumor necrosis factor-α

Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-α (TNF-α) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-α release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-α and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-α release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-α plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-α-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.

Mycobacterium tuberculosis lipoarabinomannan-mediated IRAK-M induction negatively regulates Toll-like receptor-dependent interleukin-12 p40 production in macrophages.

Mannose-capped lipoarabinomannans (Man-LAMs) are members of the repertoire of Mycobacterium tuberculosis modulins that the bacillus uses to subvert the host innate immune response. Interleukin-12 (IL-12) production is critical for mounting an effective immune response by the host against M. tuberculosis. We demonstrate that Man-LAM inhibits IL-12 p40 production mediated by subsequent challenge with lipopolysaccharide (LPS). Man-LAM inhibits LPS-induced IL-12 p40 expression in an IL-10-independent manner. It attenuates LPS-induced NF-κB-driven luciferase gene expression, suggesting that its effects are likely directly related to inhibition of NF-κB. This is probably because of dampening of the Toll-like receptor signaling. Man-LAM inhibits IL-1 receptor-associated kinase (IRAK)-TRAF6 interaction as well as IκB-α phosphorylation. It directly attenuates nuclear translocation and DNA binding of c-Rel and p50. Man-LAM exerts these effects by inducing the expression of Irak-M, a negative regulator of TLR signaling. Knockdown of Irak-M expression by RNA interference reinstates LPS-induced IL-12 production in Man-LAM-pretreated cells. The fact that Irak-M expression could be elicited by yeast mannan suggested that ligation of the mannose receptor by the mannooligosaccharide caps of LAM was the probable trigger for IRAK-M induction.

TLR4-Dependent NF-κB Activation and Mitogen- and Stress-Activated Protein Kinase 1-Triggered Phosphorylation Events Are Central to Helicobacter pylori Peptidyl Prolyl cis-, trans-Isomerase (HP0175)-Mediated Induction of IL-6 Release from Macrophages

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 Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175 of Helicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4- and Apoptosis Signal-Regulating Kinase 1-Dependent Manner

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.

Toll-like Receptor 2 and Mitogen- and Stress-activated Kinase 1 Are Effectors of Mycobacterium avium-induced Cyclooxygenase-2 Expression in Macrophages

Understanding how pathogenic mycobacteria subvert the protective immune response is crucial to the development of strategies aimed at controlling mycobacterial infections. Prostaglandin E2 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-κB 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 β-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. avium-triggered cytoplasmic NF-κB activation following IκB phosphorylation is necessary but not sufficient for COX-2 promoter-driven gene expression. MSK1 activation is also essential for M. avium-triggered NF-κB-dependent 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.

A TNF- and c-Cbl-dependent FLIP S -degradation pathway and its function in Mycobacterium tuberculosis –induced macrophage apoptosis

Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIPS, which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIPS, which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis–triggered macrophage cell death.

Helicobacter pylori Protein HP0175 Transactivates Epidermal Growth Factor Receptor through TLR4 in Gastric Epithelial Cells

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 β-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.

Exogenous Nef Is an Inhibitor of Mycobacterium tuberculosis-induced Tumor Necrosis Factor-α Production and Macrophage Apoptosis

Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-α (TNF-α)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-α production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-α production in macrophages. However, TNF-α production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-α promoter. Mtb-induced TNF-α production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-α promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-α production probably by inhibiting binding of ATF2 to the TNF-α promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-α production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-α production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-α promoter activation, Nef also reduces Mtb-dependent TNF-α mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-α production.

IFN-α Induces APOBEC3G, F, and A in Immature Dendritic Cells and Limits HIV-1 Spread to CD4+ T Cells

Cytokines and IFNs, such as TNF-α and IFN-α, upregulate costimulatory molecules in monocyte-derived dendritic cells (MDDCs), enabling effective Ag presentation to T cells. This activation of MDDCs is often accompanied by upregulation of apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) (A3) family proteins that are able to restrict HIV-1 replication in MDDCs by inducing hypermutations in the viral genome. In this study, we show that TNF-α upregulates costimulatory molecules and are able to restrict HIV-1BaL replication in MDDCs without significant induction of A3G, A3A, or A3F. Conversely, low quantities of IFN-α failed to upregulate costimulatory molecules, did not induce IL-12p40 or migration, but significantly induced A3G, A3A, and A3F mRNA expression and restricted viral replication in MDDCs. We also showed that transmission of HIV-1 from MDDCs to autologous T cells was significantly reduced in the presence of IFN-α. Sequence analyses detected the induction of high frequency of G-to-A hypermutations in the env genes from HIV-1BaL–infected MDDCs treated with low quantities of IFN-α2b. These findings show that low quantities of IFN-α can induce functional A3 family proteins and restrict HIV-1 replication in MDDCs while keeping an immature nonmigratory phenotype, supporting further investigations of modalities that enhance retroviral restriction factors. In addition, the findings highlight the role of IFN-α as a double-edged sword in HIV-1 infection, and we show that IFN-α can be powerful in reducing HIV-1 infection both in MDDCs and T cells.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Background: Activated apoptotic lymphocytes provide activation/maturation signals to human monocyte-derived dendritic cells (DCs). Results: Cell-cell contact-dependent signaling involved β2 integrins, DC-SIGN, and TLR4, which resulted in activation of multiple signaling pathways. Conclusion: These studies provide mechanistic insight into DC responses during encounter with cells undergoing immunogenic cell death. Significance: Learning how DCs respond to certain cell death has implications for vaccine design. Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4−/− mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.