MAPK-directed phosphatases preferentially regulate pro- and anti-inflammatory cytokines in experimental visceral leishmaniasis: involvement of distinct protein kinase C isoforms

Kar, Susanta Kumar; Ukil, Anindita; Sharma, Gunjan; Das, Pritha

doi:10.1189/jlb.0909644

Cited by 58 publications

(79 citation statements)

References 31 publications

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“…BMDM were isolated from femur and tibia by sacrificing 6-8 week old BALB/c mice as described previously. 40 The murine macrophage cell line RAW264.7 was cultured at 37°C with 5% CO 2 in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS, 100 μg/ml streptomycin and 100 U/ml penicillin. Reagents, antibodies and constructs.…”

Section: Methodsmentioning

confidence: 99%

Leishmania donovani inhibits macrophage apoptosis and pro-inflammatory response through AKT-mediated regulation of β-catenin and FOXO-1

et al. 2016

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In order to establish infection, intra-macrophage parasite Leishmania donovani needs to inhibit host defense parameters like inflammatory cytokine production and apoptosis. In the present study, we demonstrate that the parasite achieves both by exploiting a single host regulator AKT for modulating its downstream transcription factors, β-catenin and FOXO-1. L. donovaniinfected RAW264.7 and bone marrow-derived macrophages (BMDM) treated with AKT inhibitor or dominant negative AKT constructs showed decreased anti-inflammatory cytokine production and increased host cell apoptosis resulting in reduced parasite survival. Infection-induced activated AKT triggered phosphorylation-mediated deactivation of its downstream target, GSK-3β. Inactivated GSK-3β, in turn, could no longer sequester cytosolic β-catenin, an anti-apoptotic transcriptional regulator, as evidenced from its nuclear translocation during infection. Constitutively active GSK-3β-transfected L. donovani-infected cells mimicked the effects of AKT inhibition and siRNA-mediated silencing of β-catenin led to disruption of mitochondrial potential along with increased caspase-3 activity and IL-12 production leading to decreased parasite survival. In addition to activating antiapoptotic β-catenin, phospho-AKT inhibits activation of FOXO-1, a pro-apoptotic transcriptional regulator. Nuclear retention of FOXO-1, inhibited during infection, was reversed when infected cells were transfected with dominant negative AKT constructs. Overexpression of FOXO-1 in infected macrophages not only documented increased apoptosis but promoted enhanced TLR4 expression and NF-κB activity along with an increase in IL-1β and decrease in IL-10 secretion. In vivo administration of AKT inhibitor significantly decreased liver and spleen parasite burden and switched cytokine balance in favor of host. In contrast, GSK-3β inhibitor did not result in any significant change in infectivity parameters. Collectively our findings revealed that L. donovani triggered AKT activation to regulate GSK-3β/β-catenin/FOXO-1 axis, thus ensuring inhibition of both host cell apoptosis and immune response essential for its intra-macrophage survival.

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Section: Methodsmentioning

confidence: 99%

Leishmania donovani inhibits macrophage apoptosis and pro-inflammatory response through AKT-mediated regulation of β-catenin and FOXO-1

et al. 2016

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“…BMDM were isolated from the femurs and tibiae of euthanized BALB/c mice (6 to 8 weeks old) as described earlier (27). Splenic macrophages from BALB/c mice were isolated as described earlier (28).…”

Section: Methodsmentioning

confidence: 99%

“…The adherent murine macrophage cell line RAW 264.7 was cultured in 5% CO 2 atmosphere at 37°C in presence of RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FBS, 100 g/ml of streptomycin, and 100 units/ml of penicillin. In vitro infection of macrophages was carried out with L. donovani promastigotes at a parasite:cell ratio of 10:1 (27) for specific periods of incubation.…”

Section: Methodsmentioning

confidence: 99%

Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis

Giri

Srivastav

Basu

et al. 2016

Journal of Biological Chemistry

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Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.Leishmania donovani, an obligate intracellular parasite, is the causative agent of the fatal visceral leishmaniasis (1). After entry into the macrophages, it survives and replicates inside the acidified phagolysosomes of macrophages (2). Its persistence to resist the host immune system determines the extent of pathogenicity and requires extensive manipulation of macrophage defense (3). One of the vital mechanisms by which host cells defend themselves against intracellular pathogens is the induction of apoptosis (4, 5). On the contrary, pathogens relentlessly try to defeat the host defense systems and evolved a number of ways to inhibit host cell apoptosis, which allows them more time to replicate (6). Various studies have documented the molecular mechanisms in virus (7), bacteria (8), and protozoan parasites (9) that tamper with the host defensive apoptotic machinery. Leishmania, being an obligate intracellular parasite, makes host cells resistant to a variety of pro-apoptotic signals in murine and human cell lines. L. donovani infection protects bone marrow-derived macrophages (BMDM) 2 from growth factor withdrawal-induced apoptosis (10). Infection with Leishmania infantum reduces actinomycin D-induced cell dea...

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“…Data are represented as Leishman-Donovan units (26). For in vivo cytokine production, splenocytes were isolated from mice and cultured as described earlier (27). All the animal care and experimental procedures were carried out in strict accordance with the recommendations in the Guide for the Care …”

Section: Mice and Infectionsmentioning

confidence: 99%

Prostaglandin E2 Negatively Regulates the Production of Inflammatory Cytokines/Chemokines and IL-17 in Visceral Leishmaniasis

Saha

Biswas

Srivastav

et al. 2014

The Journal of Immunology

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Persistence of intracellular infection depends on the exploitation of factors that negatively regulate the host immune response. In this study, we elucidated the role of macrophage PGE2, an immunoregulatory lipid, in successful survival of Leishmania donovani, causative agent of the fatal visceral leishmaniasis. PGE2 production was induced during infection and resulted in increased cAMP level in peritoneal macrophages through G protein–coupled E-series prostanoid (EP) receptors. Among four different EPs (EP1–4), infection upregulated the expression of only EP2, and individual administration of either EP2-specific agonist, butaprost, or 8-Br–cAMP, a cell-permeable cAMP analog, promoted parasite survival. Inhibition of cAMP also induced generation of reactive oxygen species, an antileishmanial effector molecule. Negative modulation of PGE2 signaling reduced infection-induced anti-inflammatory cytokine polarization and enhanced inflammatory chemokines, CCL3 and CCL5. Effect of PGE2 on cytokine and chemokine production was found to be differentially modulated by cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). PGE2-induced decreases in TNF-α and CCL5 were mediated specifically by PKA, whereas administration of brefeldin A, an EPAC inhibitor, could reverse decreased production of CCL3. Apart from modulating inflammatory/anti-inflammatory balance, PGE2 inhibited antileishmanial IL-17 cytokine production in splenocyte culture. Augmented PGE2 production was also found in splenocytes of infected mice, and administration of EP2 antagonist in mice resulted in reduced liver and spleen parasite burden along with host-favorable T cell response. These results suggest that Leishmania facilitates an immunosuppressive environment in macrophages by PGE2-driven, EP2-mediated cAMP signaling that is differentially regulated by PKA and EPAC.

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MAPK-directed phosphatases preferentially regulate pro- and anti-inflammatory cytokines in experimental visceral leishmaniasis: involvement of distinct protein kinase C isoforms

Cited by 58 publications

References 31 publications

Leishmania donovani inhibits macrophage apoptosis and pro-inflammatory response through AKT-mediated regulation of β-catenin and FOXO-1

Leishmania donovani inhibits macrophage apoptosis and pro-inflammatory response through AKT-mediated regulation of β-catenin and FOXO-1

Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis

Prostaglandin E2 Negatively Regulates the Production of Inflammatory Cytokines/Chemokines and IL-17 in Visceral Leishmaniasis

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