Antileishmanial effect of 18β-glycyrrhetinic acid is mediated by Toll-like receptor-dependent canonical and noncanonical p38 activation - PubMed (original) (raw)

Antileishmanial effect of 18β-glycyrrhetinic acid is mediated by Toll-like receptor-dependent canonical and noncanonical p38 activation

Purnima Gupta et al. Antimicrob Agents Chemother. 2015 May.

Abstract

18β-Glycyrrhetinic acid (GRA), a natural immunomodulator, greatly reduced the parasite load in experimental visceral leishmaniasis through nitric oxide (NO) upregulation, proinflammatory cytokine expression, and NF-κB activation. For the GRA-mediated effect, the primary kinase responsible was found to be p38, and analysis of phosphorylation kinetics as well as studies with dominant-negative (DN) constructs revealed mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 as the immediate upstream regulators of p38. However, detection of remnant p38 kinase activity in the presence of both DN MKK3 and MKK6 suggested alternative pathways of p38 activation. That residual p38 activity was attributed to an autophosphorylation event ensured by the transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1)-p38 interaction and was completely abolished upon pretreatment with SB203580 in DN MKK3/6 double-transfected macrophage cells. Further upstream signaling evaluation by way of phosphorylation kinetics and transfection studies with DN constructs identified TAK1, myeloid differentiation factor 88 (MyD88), interleukin 1 receptor (IL-1R)-activated kinase 1 (IRAK1), and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) as important contributors to GRA-mediated macrophage activation. Finally, gene knockdown studies revealed Toll-like receptor 2 (TLR2) and TLR4 as the membrane receptors associated with GRA-mediated antileishmanial activity. Together, the results of this study brought mechanistic insight into the antileishmanial activity of GRA, which is dependent on the TLR2/4-MyD88 signaling axis, leading to MKK3/6-mediated canonical and TAB1-mediated noncanonical p38 activation.

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Figures

FIG 1

FIG 1

Analysis of MAPK pathway activation effect on infection and GRA treatment. (A, B, and C) RAW264.7 cells (2 × 106/ml) were infected with L. donovani promastigotes (macrophage/parasite ratio, 1:10) and then were either treated with 20 μM GRA for 4 h or left untreated. The levels of p38 and phosphorylated p38 (p-p38) (A), ERK and p-ERK (B), and JNK and p-JNK were detected by Western blotting. The bands were analyzed densitometrically, and fold changes are indicated below each blot. All experiments were repeated at least three times each, and one set of representative data is shown.

FIG 2

FIG 2

Determination of p38-mediated antileishmanial activity of GRA in vivo. Spleen (A) and liver (B) parasite burdens were determined every 2 weeks in each group and are expressed as LDU ± SD. Animal experiments were done with five animals per group. Error bars represent means ± SD; n = 5. Statistical significance was determined by Student’s t test and indicated by asterisks as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

FIG 3

FIG 3

Role of MKK3/6 in GRA-mediated p38 activation. (A and B) Infected cells as stated in the legend to Fig. 1 were either treated with GRA (20 μM) (A) or left untreated (B) for different times, and the total and phosphorylated levels of various MAP kinases were detected by Western blotting using the respective anti-MAP kinase antibodies. (B, C, and D) Cells were transiently transfected with the wild-type or dominant-negative constructs of MKK3 (B), the wild-type or dominant-negative constructs of MKK6 (C), and the wild-type or dominant-negative constructs of both MKK3 and MKK6 (D) for 24 h. Thereafter, they were infected and treated with GRA for 4 h, and in all the samples, the levels of total and phosphorylated p38 were determined by Western blot analysis. The bands were analyzed densitometrically, and the fold changes are indicated below each blot. All experiments were repeated at least three times each, and one set of representative data is shown.

FIG 4

FIG 4

p38 autophosphorylation in GRA-stimulated cells. (A) RAW 264.7 cells were transiently transfected with either WT or DN MKK3/6 expression plasmids (24 h) and treated with SB203580 (30 μM) for 1 h before infection and stimulation with GRA (4 h). The levels of total and phosphorylated p38 were determined by Western blotting. (B) Cells were treated as described for panel A. Cell lysates were then subjected to immunoprecipitation by anti-p-p38 antibody and Western blotting using anti-TAB1 antibody. (C) Cells were treated as stated for panel A. Cell lysates were then subjected to immunoprecipitation by anti-TAB1 antibody and Western blotting using anti-p-p38 antibody. The bands were analyzed densitometrically, and the fold changes are indicated below each blot. All experiments were repeated at least three times each, and one set of representative data is shown. IP, immunoprecipitation using the indicated antibody (Ab); IB, immunoblot analysis using the indicated Ab.

FIG 5

FIG 5

Role of TAK1 in GRA-mediated antileishmanial activity. (A) RAW 264.7 cells were infected with L. donovani and treated with 20 μM GRA for various time periods, and the levels of total and phosphorylated TAK1 were determined by Western blotting. (B and C) Cells were transiently transfected with WT or DN TAK1 expression plasmid (24 h) and then infected with L. donovani and treated with GRA (4 h). The levels of total and phosphorylated p38 (B) and MKK3/6 (C) were determined by Western blotting. (D) WT or DN TAK1-transfected cells were treated with SB203580 for 1 h before infection, and GRA stimulation and total and phosphorylated p38 levels were determined by Western blotting. The bands were analyzed densitometrically, and fold changes are indicated below each blot. All experiments were repeated at least three times each and one set of representative data is shown.

FIG 6

FIG 6

Importance of TLR-MyD88-IRAK1-TRAF6 axis in GRA-mediated antileishmanial response. (A) Cells were transiently transfected with the indicated constructs, infected with L. donovani, and stimulated with GRA for 4 h. Cells were lysed and processed for p38 expression and phosphorylation by Western blotting. (B to D) To determine the effect of TLR inhibition, macrophages were transfected (24 h) with either TLR2 siRNA (B) or TLR4 siRNA (C), and TLR expression was determined by Western blotting. Cells were transiently transfected with TLR2 or TLR4 siRNA, respectively, infected with L. donovani, stimulated with GRA for 4 h, and processed for determination of NF-κB luciferase activity (D). The bands were analyzed densitometrically, and fold changes are indicated below each blot. All experiments were repeated at least three times each, and one set of representative data is shown. Error bars represent means ± SD; n = 3. ***, P < 0.001; Student's t test.

FIG 7

FIG 7

Schematic representation depicting mechanistic details of GRA-mediated host cell activation. Leishmania inhibits proinflammatory cytokine production and host cell activation. GRA treatment resulted in both (i) TLR2/4-MyD88-IRAK-TRAF6-TAK1-dependent MKK3/6-mediated canonical p38 activation and (ii) TAB1-dependent noncanonical p38 activation. Overall, p38 activation by GRA thus leads to proinflammatory cytokine production and host cell activation.

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References

    1. Croft SL, Coombs GH. 2003. Leishmaniasis—current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 19:502–508. doi:10.1016/j.pt.2003.09.008. - DOI - PubMed
    1. Singh S, Sivakumar R. 2004. Challenges and new discoveries in the treatment of leishmaniasis. J Infect Chemother 10:307–315. doi:10.1007/s10156-004-0348-9. - DOI - PubMed
    1. Ukil A, Biswas A, Das T, Das PK. 2005. 18 Beta-glycyrrhetinic acid triggers curative Th1 response and nitric oxide up-regulation in experimental visceral leishmaniasis associated with the activation of NF-kappa B. J Immunol 175:1161–1169. doi:10.4049/jimmunol.175.2.1161. - DOI - PubMed
    1. Rossi T, Galatulas I, Bossa R, Tampieri A, Tartoni P, Baggio G, Ruberto AI, Castelli M. 1995. Influence of glycyrrhizin on the evolution and respiration of Ehrlich ascites tumour cells. In Vivo 9:183–186. - PubMed
    1. Jeong HG, You HJ, Park SJ, Moon AR, Chung YC, Kang SK, Chun HK. 2002. Hepatoprotective effects of 18beta-glycyrrhetinic acid on carbon tetrachloride-induced liver injury: inhibition of cytochrome P450 2E1 expression. Pharmacol Res 46:221–227. doi:10.1016/S1043-6618(02)00121-4. - DOI - PubMed

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