<i>Pedilanthus tithymaloides</i> Inhibits HSV Infection by Modulating NF-κB Signaling

<div><p><i>Pedilanthus tithymaloides</i> (PT), a widely used ethnomedicinal plant, has been employed to treat a number of skin conditions. To extend its utility and to fully exploit its medicinal potential, we have evaluated the <i>in vitro</i> antiviral activity of a methanolic extract of PT leaves and its isolated compounds against Herpes Simplex Virus type 2 (HSV-2). Bioactivity-guided studies revealed that the extract and one of its constituents, luteolin, had potent antiviral activity against wild-type and clinical isolates of HSV-2 (EC₅₀ 48.5–52.6 and 22.4–27.5 μg/ml, respectively), with nearly complete inhibition at 86.5–101.8 and 40.2–49.6 μg/ml, respectively. The inhibitory effect was significant (p<0.001) when the drug was added 2 h prior to infection, and was effective up to 4 h post-infection. As viral replication requires NF-κB activation, we examined whether the observed extract-induced inhibition of HSV-2 was related to NF-κB inhibition. Interestingly, we observed that treatment of HSV-2-infected cells with extract or luteolin suppressed NF-κB activation. Although NF-κB, JNK and MAPK activation was compromised during HSV replication, neither the extract nor luteolin affected HSV-2-induced JNK1/2 and MAPK activation. Moreover, the PT leaf extract and luteolin potently down-regulated the expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, NO and iNOS and the production of gamma interferon (IFN-γ), which are directly involved in controlling the NF-κB signaling pathway. Thus, our results indicate that both PT leaf extract and luteolin modulate the NF-κB signaling pathway, resulting in the inhibition of HSV-2 replication.</p></div

Chemical structure of the isolated compound(s) Luteolin (A) and Tetradecanediol (B) isolated from the antiviral fraction of <i>Pedilanthus tithymaloides</i> leave extracts.

<p>Chemical structure of the isolated compound(s) Luteolin (A) and Tetradecanediol (B) isolated from the antiviral fraction of <i>Pedilanthus tithymaloides</i> leave extracts.</p

Primers used in Real time-PCR assays.

<p>Primers used in Real time-PCR assays.</p

Effect of MEPT leaves and Luteolin on pro-inflammatory cytokine release in HSV-2-infected peritoneal macrophages.

<p>Peritoneal macrophages were cultured overnight and infected with HSV-2, washed after 1 h and then treated with MEPT leaves (100 μg/ml) or Luteolin (20 μg/ml). The cells were further incubated for 24 h, and the cell-free supernatants were subjected to sandwich ELISA to determine the level of (A) TNF-α, (C) IL-1β, (E) IL-6 and (G) IFN-γ (pg/mL). The ELISA data are expressed as Mean ± SD from triplicate experiments, yielding similar results. Asterisks indicate statistically significant (*, P<0.05; **, P<0.001) induction of TNF-α, IL-1ß, IL-6, and IFN-γ release, compared to the infected macrophages.</p

Effect of MEPT leaves and Luteolin on HSV-2-induced MAPK, JNK1/2, NF-kB and IkBα activation.

<p>Expression of MAPK (A), JNK (B), NF-kB (C) and IkBα (D) was determined by Western blot, using GAPDH as the internal control. The HSV-2-infected peritoneal macrophage(s) were treated with MEPT (86.5 μg/ml) or Luteolin (40.2 μg/ml) and after 4 h, equal amounts of protein (40 μg/sample) extract from whole cells were harvested in buffer (200 μl/well) containing 50 nM Tris-Cl, 150 mM NaCl, 1% NP-40, 1% Triton X-100, and 1% protease inhibitor cocktail. The soluble fraction was separated by centrifugation, subjected to SDS-PAGE and blotted to pre-equilibrated PVDF membrane. The membrane was then blocked in 5% NFDM in 1X TBST, rinsed and incubated with specific antibody at 4°C overnight. Immunoblotting was performed with peroxidase-labelled specific antibodies and visualized by ECL Western blot detection kit. The average expression of NF-kB was significantly higher in the HSV-2-induced macrophage, as compared to the control and MEPT or luteolin co-treated group (*, P<0.05; **, P<0.001). Lane 1, cells only control; Lane 2, cells + HSV-2; Lane 3, cells + HSV-2 + MEPT leaves; Lane 4, cells + HSV-2 + luteolin.</p

Effect of MEPT leaves and Luteolin on NO production and iNOS expression in HSV-2 infected murine macrophages.

<p>(A) Macrophages (10⁶ cells/ml) were infected with HSV-2 treated with the MEPT leaves (100 μg/ml) or Luteolin (20 μg/ml), and incubated for 24 h. The supernatant was removed and the concentration of NO was determined by Griess reagent. Data are expressed as Mean ± SD from triplicate experiments, yielding similar results (m moles of nitrite). Asterisks indicate a statistically significant increase (*, P<0.05; **, P<0.001) in nitrite generation, compared to the infected macrophages. (B) HSV-2-infected macrophages were treated with the MEPT leaves or Luteolin, incubated for 12 h, following which RNA was isolated and subjected to RT–PCR analysis for the expression of iNOS2 mRNA. The data are expressed as Mean ± SD from triplicate experiments yielding similar results. The asterisk indicates a statistically significant increase (*, P<0.001) in iNOS2 expression, compared to the infected macrophage.</p

TLR-2-deficient animals induce reduced levels of protective immune responses.

<p>For <i>in vivo</i> experiments, wild type and TLR-2^-/- mice were infected with H37Rv for 30 days, and subsequently treated with antibiotics for 4 weeks as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002378#s4" target="_blank">materials and methods</a>. Mice were then re-challenged with H37Rv. Groups of animals were sacrificed for the following studies. (<b>A</b>) CFU in lung homogenates of mice re-challenged with H37Rv. (<b>B</b>) Intracellular cytokines in CD4⁺ T lymphocytes derived from the lung. Results presented here are representative of three independent experiments.</p

Infection with H37Rv or BCG::RD1 induces both Th1 and Th17 immunity, whereas BCG and H37RvΔRD1 selectively induce Th1 cell responses in the lung.

<p>C57BL/6 mice were challenged with H37Rv, BCG, H37RvΔRD1 or BCG::RD1 by the aerosol route and lungs were harvested at different time points. (<b>A</b>) CFU from the lung homogenate of mice that were infected with H37Rv, BCG, H37RvΔRD1 or BCG::RD1 strains. (<b>B</b>) Intracellular staining for IFN-γ and IL-17 of CD4⁺ T lymphocytes isolated from the lungs of infected mice. (<b>C</b>) Presence of IFN-γ and IL-17 in BAL fluid was measured by Luminex. (<b>D</b>) miR146a expression profile of DCs infected with different strains of bacteria. miR146a expression was normalized with 5S rRNA control primer. (<b>E</b>) IL-6 mRNA expression profile after infection of DCs with different bacterial strains and compared after knock-down of miR146a with anti-miR146a miRCURY LNA knock-down probes. IL-6 mRNA expression increases after knock-down of miR146a increases. (<b>F</b>) IL-6 cytokine production increases after knock-down of miR146a increases. The results shown are representative of at least 3–4 independent experiments.</p

Anti-Inflammatory Activity of <i>Odina wodier</i> Roxb, an Indian Folk Remedy, through Inhibition of Toll-Like Receptor 4 Signaling Pathway

<div><p>Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of <i>Odina wodier</i> (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular <i>in vivo</i> antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1β, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.</p></div

H37Rv directs differentiation of both Th1 and Th17 cells.

<p>(<b>A</b>) Intracellular cytokine staining for IFN-γ and IL-17 in OT-II TCR Tg CD4⁺ T cells co-cultured with infected DCs. (<b>B</b>) IL-22 mRNA expression profile from OT-II TCR Tg CD4⁺ T cells co-cultured with DCs after infection of DCs with H37Rv, BCG, BCG::RD1 or H37RvΔRD1. Results are representative of at least three independent experiments.</p