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

Elucidation of the Signaling Mechanisms Involved in the Subversion of Host Immune Response by Intracellular Parasite Leishmania Donovani

The intracellular parasite L. donovani has the unique capacity to survive and replicate inside host macrophages. Since this cell type is specialized for the destruction of invading pathogens and priming of the host immune response, Leishmania has had to evolve a range of sophisticated mechanisms to subvert normal macrophage function. This enables the parasite to evade the innate immune response and to divide within the phagolysosome of the infected macrophage, from where it can spread and propagate the disease within the host. There are multiple ways by which intracellular pathogens like Leishmania make use of host cell’s machinery in order to survive and replicate. One such mechanism is the distortion of host macrophage’s own signaling pathways to selectively repress or enhance the expression of various cytokines and microbicidal molecules and antigen presentation. Within the scope of this work, an attempt has been made to focus on the molecular mechanisms by which Leishmania can subvert host immune surveillance by altering the macrophage signal transduction machinery, thereby modulating the macrophage environment in its favour. Toll-like receptors (TLRs), which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of pro-inflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens like Leishmania, can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan (LPG), a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated pro-inflammatory response is not understood. Although the ability of Leishmania to inhibit inflammatory signaling pathways has been proposed as a virulence mechanism, the molecular events underlying this process remain still to be explored. The aim of this study was to determine the mechanism used by Leishmania to modulate TLR signaling cascade for its own favor

Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-ß synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host

Effect of GRA on in vivo kinase/phosphatases balance, modulation of parasitemia and cytokine response.

<p><i>L. donovani</i>-infected mice was treated with GRA (50 mg/kg) i.p. for 3 times 5 days apart starting at 10 days post-infection. In a separate group, infected mice were treated with either p38 inhibitor SB203580-HCL (10 mg/kg) or ERK-inhibitor PD0325901 (20 mg/kg) over a 4 wk period along with GRA. (<b>A)</b> Spleen parasite burdens were determined weekly in each group and expressed as LDU ± SD. (<b>B</b>) Spleen parasite burden determined 2 and 6 wk following infection measured by limiting dilution assay expressed as log₁₀ parasite burden ± SD. MKP1, MKP3 and PP2A were immunoprecipitated from cell lysates of splenocytes isolated from different experimental groups with respective antibodies. MKP1 (<b>C</b>) and MKP3 (<b>D</b>) and PP2A (<b>E</b>) activity was assayed by pNPP hydrolysis and assay kit respectively. Expression of TNF-α (<b>F</b>) and IL-10 (<b>G</b>) at mRNA level were determined by Real time PCR in the splenocytes. (<b>H</b>) NF-κB DNA binding activity was determined in the splenocytes of different experimental groups as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029062#pone-0029062-g001" target="_blank"><b>Figure 1B</b></a>. Animal experiments were done with five animals/group and the results are representative of three individual experiments. ns, not significant; **<i>p</i><0.01, ***<i>p</i><0.001; Student's t-test.</p

Effect of GRA on the regulation of iNOS and Th1 cytokine by MAPK activation.

<p>(<b>A</b>) BMDM were treated with GRA (20 µM) for different time periods. The expression and phosphorylation of MAPKs were detected by Western blotting. (<b>B</b>) <i>L. donovani</i>-infected BMDM were treated with GRA (20 µM) for various time periods and the level of phosphorylated p38 was measured by Western blotting. (<b>C</b> and <b>D</b>) BMDM were treated (1 h) with either SB203580 (30 µM) or apigenin (40 µM) or both before stimulation with GRA (24 h). iNOS expression (<b>C</b>) by Taqman analysis and levels of IL-12 and TNF-α (<b>D</b>) by ELISA were determined. (<b>E</b>) RAW 264.7 cells were transiently transfected using Lipofectamine reagent with 1 µg of NF-κB luciferase reporter vector along with 0.5 µg pcMV-βgal. After 24 h, cells were stimulated with GRA (20 µM) for different time periods. In a separate set of experiment, transfected cells were pre-incubated with either SB203580 (30 µM) or apigenin (40 µM) or both for 1 h before stimulation with GRA (12 h). Cells were lysed and processed for luciferase activity. (<b>F</b>) BMDM were treated with GRA (20 µM) for different time periods or pre-incubated with either SB203580 (30 µM) or apigenin (40 µM) or both for 1 h before stimulation with GRA (2 h). Cell lysates were then immunoprecipitated with anti-MSK1 Ab and MSK1 activity was assayed using CREBTIDE as substrate. Bands were analyzed densitometrically. Error bars represent mean ± SD (n = 3). The data shown are representative of three independent experiments. ns, not significant; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Student's t-test.</p

Effect of GRA on cellular kinase/phosphatase balance.

<p>BMDM were infected with <i>L. donovani</i> promastigotes and treated with GRA (20 µM) for various time periods. (<b>A</b>) Induction of PTP activity was measured using a PTP assay kit. Results are expressed as the relative increase (n-fold) over PTP activity in control cells. Expression of MKP1, MKP3 (<b>B</b>), PP2A (<b>C</b>) and SHP-1 (<b>D</b>) at mRNA and at protein level (<b>E and F</b>) were determined by Real time PCR and immunoblot analysis respectively. SHP-1, MKP1, MKP3 and PP2A were immunoprecipitated from infected and GRA (20 µM, 4 h)-treated macrophages and assayed for dephosphorylation activity using either recombinant p-ERK or p-p38 as substrate and visualizing by immunoblotting with anti-p-ERK (<b>G and H</b>) and anti-p-p38 (<b>I</b>) antibody. The amount of individual phosphatases was measured by stripping the blot and reprobing with antibodies against SHP-1, MKP1, MKP3 or PP2A. Results are representative of three individual experiments. Data represent the mean ± SD, n = 3. ***<i>p</i><0.001; Student's t-test.</p

Antileishmanial activity of GRA.

<p>(<b>A</b>) Dose- and time-dependent expression of iNOS by Taqman analysis of the mRNA transcript in BMDM stimulated with GRA. mRNA levels were normalized to β-actin and expressed as a -fold change compared with control. (<b>B</b>) Percentages of infected cells and (<b>C</b>) number of parasites per macrophage were counted at various time periods following infection. (<b>D</b>) Effect of GRA on NO production (solid bar) and parasite suppression (open bar) in BMDM in presence or absence of AMT (10 µM). (<b>E</b>) Generation of NO (solid bar) and parasite suppression (open bar) in peritoneal macrophages isolated from mice which received GRA (10, 50 and 100 mg/kg) i.p. for 3 times 5 days apart. Macrophages were harvested 10 h after the last injection and infected with <i>L. donovani</i>. After 4 h infection and 20 h of incubation, the percentage of parasite suppression and the amount of NO₂⁻ in the medium were determined. (<b>F</b>) and (<b>G</b>) Levels of IL-12, TNF-α, IL-10 ,TGF-β, IL-6 and IL-1β in culture supernatants from infected and GRA (20 µM)-treated BMDM were determined by ELISA. (<b>H</b>) Mice were administered i.v. with 10⁷ promastigotes, and after 10 days of infection, were treated with GRA (25 or 50/mg/kg) i.p. 3 times 5 days apart. Spleen parasite burdens were determined 6 wk after infection and are expressed as LDU ± SD. (<b>I</b>) Spleen parasite burden determined 2 and 6 wk following infection measured by limiting dilution assay expressed as log₁₀ parasite burden ± SD. Cultures were set in triplicate and experiments were done a minimum of three times. Animal experiments were done with five animals/group and the results are representative of three independent experiments. Data represent the mean ± SD. ***<i>p</i><0.001; Student's t-test.</p

Involvement of NF-κB in GRA-mediated anti-leishmanial response.

<p>(<b>A</b>) <i>L. donovani</i>-infected and GRA (20 µM)-treated BMDM were stained with anti-p65 monoclonal antibody and APC-conjugated secondary antibody. Nuclei were stained with DAPI and cells were analyzed under fluorescence microscope as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029062#s4" target="_blank">Materials and Methods</a>. (<b>B</b> and <b>C</b>) <i>L. donovani</i>-infected mice were treated with GRA (50 mg/kg) for 3 times 5 days apart starting at 10 days after infection and splenocytes were isolated 3 days after last treatment. Labeled NF-κB probe was incubated with nuclear extracts of splenocytes and EMSA was performed (<b>B</b>). The bands were analyzed densitometrically and –fold changes are indicated. (<b>C</b>) For supershift assay, nuclear extracts from splenocytes were incubated with antibodies against individual components of NF-κB complex for 30 min. The results are representative of one of three separate experiments. (<b>D</b>) Western blot analysis shows a depletion of IκBα protein in splenocytes of GRA-treated mice as opposed to increased preservation in infected group. (<b>E</b>) IKKβ was immunoprecipitated from splenocyte cell lysate and IKK activity was assayed using GST-IκBα as substrate, and GST-phosphorylated IκBα was visualized by autoradiography. Relative amount of IKKβ was determined by Western blots. <i>L. donovani</i>-infected mice were treated either with GRA (50 mg/kg) as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029062#pone-0029062-g001" target="_blank"><b>Figure 1E</b></a> or with GRA plus BAY11-7085 (5 mg/kg/day) 3 times weekly for 4 wk starting at 2 wk after infection. Parasite burden in spleen (<b>F</b>) mRNA expression of iNOS (<b>G</b>) and the level of TNF-α (<b>H</b>) in the splenocytes were determined. Results are representative of three individual experiments and the error bars represent mean ± SD (n = 3). ***<i>p</i><0.001; Student's t-test.</p