Anindita Ukil

Successful Therapy of Visceral Leishmaniasis With Curdlan Involves T-Helper 17 Cytokines

The aim of this study was to evaluate and characterize the therapeutic potential of curdlan, a naturally occurring β-glucan immunomodulator, against visceral leishmaniasis, a fatal parasitic disease. Curdlan eliminated the liver and spleen parasite burden in a 45-day BALB/c mouse model of visceral leishmaniasis at a dosage of 10 mg/kg/day as determined by Giemsa-stained organ impression smears. Curdlan was associated with production of the disease-resolving T-helper (Th) 1 and Th17-inducing cytokines interleukin (IL)-6, IL-1β, and IL-23, as well as with production of Th17 cytokines IL-17 and IL-22, as determined by enzyme-linked immunosorbent assay (ELISA) and real time polymerase chain reaction (RT-PCR). Reversal of curdlan-mediated protection by anti-IL-17 and anti-IL-23 monoclonal antibodies showed the importance of Th17 cytokines. Significantly decreased production of both IL-17 and IL-22 by mice that received anti-IL-23 antibody suggested the essential role of IL-23 in Th17 differentiation. Although administration of recombinant IL-17 or IL-23 caused significant suppression of the organ parasite burden, with marked generation of interferon γ and nitric oxide (NO), effects were much faster for IL-17. These results documented that although both IL-23 and IL-17 play major roles in the antileishmanial effect of curdlan, the effect of IL-23 may occur indirectly, through the induction of IL-17 production.

Cystatin cures visceral leishmaniasis by NF-κB-mediated proinflammatory response through co-ordination of TLR/MyD88 signaling with p105-Tpl2-ERK pathway.

Cystatin could completely cure experimental visceral leishmaniasis by switching the differentiation of Th2 cells to Th1 type, as well as upregulating NO, and activation of NF‐κB played a major role in these processes. Analysis of upstream signaling events revealed that TLR 2/4‐mediated MyD88‐dependent participation of IL‐1R‐activated kinase (IRAK)1, TNF receptor‐associated factor (TRAF)6 and TGFβ‐activated kinase (TAK)1 is essential to induce cystatin‐mediated IκB kinase (IKK)/NF‐κB activation in macrophages. Cystatin plus IFN‐γ activated the IKK complex to induce phosphorylation‐mediated degradation of p105, the physiological partner and inhibitor of the MEK kinase, tumor progression locus 2 (Tpl‐2). Consequently, Tpl‐2 was liberated from p105, thereby stimulating activation of the MEK/ERK MAPK cascade. Cystatin plus IFN‐γ‐induced IKK‐β post‐transcriptionally modified p65/RelA subunit of NF‐κB by dual phosphorylation in infected phagocytic cells. IKK induced the phosphorylation of p65 directly on Ser‐536 residue whereas phosphorylation on Ser 276 residue was by sequential activation of Tpl‐2/MEK/ERK/MSK1. Collectively, the present study indicates that cystatin plus IFN‐γ‐induced MyD88 signaling may bifurcate at the level of IKK, leading to a divergent pathway regulating NF‐κB activation by IκBα phosphorylation and by p65 transactivation through Tpl‐2/MEK/ERK/MSK1.

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

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.

Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease

ABSTRACT Cystatin, a natural cysteine protease inhibitor, has strong antileishmanial activity, which is due to its potential to induce nitric oxide (NO) generation from macrophages. Cysteine protease-inhibitory activity and NO-up-regulatory activity correspond to different regions, as revealed by the dissection of cystatin cDNA into nonoverlapping fragments. By using synthetic overlapping peptides, the NO-up-regulatory activity was found to be confined to a 10-mer sequence. In addition to having reasonable inhibitory effects on amastigote multiplication within macrophages (50% inhibitory concentration, 5.2 μg/ml), 97 and 93% suppression, respectively, of liver and spleen parasite burdens was achieved with the 10-mer peptide at a dose of 0.5 mg/kg of body weight/day, given consecutively for 4 days along with a suboptimal dose of gamma interferon in a 45-day mouse model of visceral leishmaniasis. Peptide treatment modulated the levels of cytokine secretion by infected splenocytes, with increased levels of interleukin-12 and tumor necrosis factor alpha and increased inducible NO synthase production, and also resulted in resistance to reinfection. The generation of a natural peptide from cystatin with robust immunomodulatory potential may therefore provide a promising therapeutic agent for macrophage-associated diseases.

Leishmania donovani Prevents Oxidative Burst-mediated Apoptosis of Host Macrophages through Selective Induction of Suppressors of Cytokine Signaling (SOCS) Proteins

Background: Leishmania inhibits oxidative burst-mediated apoptosis of macrophages during phagocytosis. Results: L. donovani induces (SOCS) 1 and 3, which suppress macrophage apoptosis through thioredoxin-mediated stabilization of protein-tyrosine phosphatases. Conclusion: Leishmania exploits macrophage SOCS proteins for inhibition of apoptosis, thus protecting its niche for survival and replication. Significance: This study demonstrates a novel anti-apoptotic mediator for parasite infection. One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.

Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response

Intramacrophage pathogen Leishmania donovani escapes host immune response by subverting Toll‐like receptor (TLR) signaling, which is critically regulated by protein ubiquitination. In the present study, we identified tumor necrosis factor receptor‐associated factor (TRAF) 3, degradative ubiquitination of which is essential for TLR4 activation, as a target for Leishmania to deactivate LPS‐mediated TLR4 signaling. We used LPS‐treated RAW 264.7 cells and compared the TLR4‐mediated immune response in these cells with L. donovani and L. donovani + LPS costimulated macrophages. TRAF3, which was ubiquitinated (2.1‐fold over control) at lys 48 position and subsequently degraded following LPS treatment, persisted in L. donovani and L. donovani + LPS costimulated cells due to defective lys 48 ubiquitination. Lys 63‐linked ubiquitination of upstream proteins in the cascade (cIAP1/2 and TRAF6), mandatory for TRAF3 degradation, was also reduced postinfection. This may be attributed to reduced association between ubiquitin‐conjugating enzyme Ubc13 and TRAF6 during infection. Inhibition of TRAF3 before infection by shRNA in Balb/c mice showed enhanced IL‐12 and TNF‐α (10.8‐ and 8.1‐fold over infected control) and decreased spleen parasite burden (61.3% suppression, P<0.001), thereby marking reduction in disease progression. Our findings identified TRAF3 as a novel molecular regulator exploited by Leishmania for successful infection.—Gupta, P., Giri, J., Srivastav, S., Chande, A. G., Mukhopadhyaya, R., Das, P. K., Ukil, A. Leishmania donovani targets tumor necrosis factor receptor‐associated factor (TRAF) 3 for impairing TLR4‐mediated host response. FASEB J. 28, 28–1756 (1768). www.fasebj.org

Curative effect of 18β-glycyrrhetinic acid in experimental visceral leishmaniasis depends on phosphatase-dependent modulation of cellular MAP kinases.

We earlier showed that 18β-glycyrrhetinic acid (GRA), a pentacyclic triterpenoid from licorice root, could completely cure visceral leishmaniasis in BALB/c mouse model. This was associated with induction of nitric oxide and proinflammatory cytokine production through the up regulation of NF-κB. In the present study we tried to decipher the underlying cellular mechanisms of the curative effect of GRA. Analysis of MAP kinase pathways revealed that GRA caused strong activation of p38 and to a lesser extent, ERK in bone marrow-derived macrophages (BMDM). Almost complete abrogation of GRA-induced cytokine production in presence of specific inhibitors of p38 and ERK1/2 confirmed the involvement of these MAP kinases in GRA-mediated responses. GRA induced mitogen- and stress-activated protein kinase (MSK1) activity in a time-dependent manner suggested that GRA-mediated NF-κB transactivation is mediated by p38, ERK and MSK1 pathway. As kinase/phosphatase balance plays an important role in modulating infection, the effect of GRA on MAPK directed phosphatases (MKP) was studied. GRA markedly reduced the expression and activities of three phosphatases, MKP1, MKP3 and protein phosphatase 2A (PP2A) along with a substantial reduction of p38 and ERK dephosphorylation in infected BMDM. Similarly in the in vivo situation, GRA treatment of L. donovani-infected BALB/c mice caused marked reduction of spleen parasite burden associated with concomitant decrease of individual phosphatase levels. However, activation of kinases also played an important role as the protective effect of GRA was significantly abrogated by pharmacological inhibition of p38 and ERK pathway. Curative effect of GRA may, therefore, be associated with restoration of proper cellular kinase/phosphatase balance, rather than modulation of either kinases or phosphatases.

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

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.

IRAK-M regulates the inhibition of TLR-mediated macrophage immune response during late in vitro Leishmania donovani infection

Intramacrophage protozoan parasite Leishmania donovani, causative agent of visceral leishmaniasis, escapes Toll‐like receptor (TLR) dependent early host immune response by inducing the deubiquitinating enzyme A20, which is sustained up to 6 h postinfection only. Therefore, Leishmania must apply other means to deactivate late host responses. Here, we elucidated the role of IL‐1 receptor‐associated kinase M (IRAK‐M), a negative regulator of TLR signaling, in downregulating macrophage proinflammatory response during late hours of in vitro infection. Our data reveal a sharp decline in IRAK1 and IRAK4 phosphorylation at 24 h postinfection along with markedly reduced association of IRAK1–TNF receptor associated factor 6, which is mandatory for TLR activation. In contrast, IRAK‐M was induced after A20 levels decreased and reached a maximum at 24 h postinfection. IRAK‐M induction coincided with increased stimulation of TGF‐β, a hallmark cytokine of visceral infection. TGF‐β‐dependent signaling‐mediated induction of SMAD family of proteins, 2, 3, and 4 plays important roles in transcriptional upregulation of IRAK‐M. In infected macrophages, siRNA‐mediated silencing of IRAK‐M displayed enhanced IRAK1 and IRAK4 phosphorylation with a concomitant increase in downstream NF‐κB activity and reduced parasite survival. Taken together, the results suggest that IRAK‐M may be targeted by L. donovani to inhibit TLR‐mediated proinflammatory response late during in vitro infection.

The role of PD-1 in regulation of macrophage apoptosis and its subversion by Leishmania donovani

Programmed death‐1 receptor (PD‐1) expressed in many immune cells is known to trigger T‐cell exhaustion but the significance of macrophage‐associated PD‐1 in relevance to macrophage apoptosis is not known. This study is aimed to delineate whether PD‐1 pathway has any role in eliciting macrophage apoptosis and, if so, then how the intra‐macrophage parasite, Leishmania donovani modulates PD‐1 pathway for protecting its niche. Resting macrophages when treated with H2O2 showed increased PD‐1 expression and apoptosis, which was further enhanced on PD‐1 agonist treatment. The administration of either PD‐1 receptor or PD‐1 ligand‐blocking antibodies reversed the process thus documenting the involvement of PD‐1 in macrophage apoptosis. On the contrary, L. donovani‐infected macrophages showed decreased PD‐1 expression concurrent with inhibition of apoptosis. The activation of PD‐1 pathway was found to negatively regulate the phosphorylation of pro‐survival AKT, which was reversed during infection. Infection‐induced PD‐1 downregulation led to the activation of AKT resulting in phosphorylation and subsequent inhibition of proapoptotic protein BAD. Strong association of SHP2 (a SH2‐containing ubiquitously expressed tyrosine‐specific protein phosphatase) with PD‐1 along with AKT deactivation observed in H2O2‐treated macrophages was reversed by L. donovani infection. Kinetic analysis coupled with inhibitor‐based approach and knockdown experiments demonstrated that L. donovani infection actively downregulated the PD‐1 by deactivating NFATc1 as revealed by its reduced nuclear translocation. The study thus elucidates the detailed mechanism of the role of PD‐1 in macrophage apoptosis and its negative modulation by Leishmania for their intracellular survival.