Curcuphenol possesses an unusual histone deacetylase enhancing activity that counters immune escape in metastatic tumours

Curcuphenol, a common component of the culinary spices, naturally found in marine invertebrates and plants, has been identified as a novel candidate for reversing immune escape by restoring expression of the antigen presentation machinery (APM) in invasive cancers, thereby resurrecting the immune recognition of metastatic tumours. Two synthetic curcuphenol analogues, were prepared by informed design that demonstrated consistent induction of APM expression in metastatic prostate and lung carcinoma cells. Both analogues were subsequently found to possess a previously undescribed histone deacetylase (HDAC)-enhancing activity. Remarkably, the H3K27ac ChIPseq analysis of curcuphenol-treated cells reveals that the induced epigenomic marks closely resemble the changes in genome-wide pattern observed with interferon-γ, a cytokine instrumental for orchestrating innate and adaptive immunity. These observations link dietary components to modifying epigenetic programs that modulate gene expression guiding poised immunity

Validation of <i>N</i>-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of <i>Trypanosoma cruzi</i>

BACKGROUND:Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT) has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids. METHODOLOGY/PRINCIPAL FINDINGS:Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8) have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors. CONCLUSIONS/SIGNIFICANCE:Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy

Tweeters and Woofers: The Complex Orchestra of Calcium Currents in T Lymphocytes

Elevation of intracellular calcium ion (Ca2+) levels is a vital event that regulates T lymphocyte homeostasis, activation, proliferation, differentiation, and apoptosis. The mechanisms that regulate intracellular Ca2+ signalling in lymphocytes involve tightly controlled orchestration of multiple ion channels, membrane receptors, and signalling molecules. T cell receptor (TCR) engagement results in depletion of endoplasmic reticulum (ER) Ca2+ stores and subsequent sustained influx of extracellular Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane. This process termed store-operated Ca2+ entry (SOCE) involves the ER Ca2+ sensing molecule, stromal interaction molecule 1 (STIM1), and a pore-forming plasma membrane protein, ORAI1. However, several other important Ca2+ channels that are instrumental in T cell function also exist. In this review, we discuss the role of additional Ca2+ channel families expressed on the plasma membrane of T cells that likely contribute to Ca2+ influx following TCR engagement, which include the IP3 receptors, the P2X receptors, the NMDA receptors, and the TRP channels, with a focus on the voltage-dependent Ca2+ (CaV) channels

Immunomodulatory Effects of Serotype B Glucuronoxylomannan from Cryptococcus gattii Correlate with Polysaccharide Diameter▿

Glucuronoxylomannan (GXM), the major capsular component in the Cryptococcus complex, interacts with the immune system in multiple ways, which include the activation of Toll-like receptors (TLRs) and the modulation of nitric oxide (NO) production by phagocytes. In this study, we analyzed several structural parameters of GXM samples from Cryptococcus neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C) and correlated them with the production of NO by phagocytes and the activation of TLRs. GXM fractions were differentially recognized by TLR2/TLR1 (TLR2/1) and TLR2/6 heterodimers expressed on TLR-transfected HEK293A cells. Higher NF-κB luciferase reporter activity induced by GXM was observed in cells expressing TLR2/1 than in cells transfected with TLR2/6 constructs. A serotype B GXM from C. gattii was the most effective polysaccharide fraction activating the TLR-mediated response. This serotype B polysaccharide, which was also highly efficient at eliciting the production of NO by macrophages, was similar to the other GXM samples in monosaccharide composition, zeta potential, and electrophoretic mobility. However, immunofluorescence with four different monoclonal antibodies and dynamic light-scattering analysis revealed that the serotype B GXM showed particularities in serological reactivity and had the smallest effective diameter among the GXM samples analyzed in this study. Fractionation of additional serotype B GXMs, followed by exposure of these fractions to macrophages, revealed a correlation between NO production and reduced effective diameters. Our results demonstrate a great functional diversity in GXM samples from different isolates and establish their abilities to differentially activate cellular responses. We propose that serological properties as well as physical chemical parameters, such as the diameter of polysaccharide molecules, may potentially influence the inflammatory response against Cryptococcus spp. and may contribute to the differences in granulomatous inflammation between cryptococcal species

Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties

ArtinM induces the activation of TLR2/1- and TLR2/6-expressing cells.

<p>HEK293A cells were transfected with TLR2/1 (A and C) or TLR2/6 (B and D), co-receptors, an NF-κB reporter construct, and a <i>Renilla</i> luciferase reporter plasmid as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#pone-0098512-g002" target="_blank">Figure 2</a>. The transfected cells were stimulated with ArtinM (15.6, 156, and 780 nM) at 37°C for 4 h. Medium and cells transfected with an empty vector were used as the negative controls. The positive controls were P3C4 (1 nM) for TLR2/1 activation (A and C) and FSL1 (0.1 nM) for TLR2/6 activation (B and D). The luciferase activity (A and B) was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. IL-8 levels in the culture supernatants (C and D) were measured by ELISA. Statistical comparisons between the cells incubated with medium and the cells stimulated with ArtinM were performed with a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

Enhanced TLR2 relative expression by ArtinM-stimulated macrophages.

<p>Peritoneal macrophages from C57BL/6 mice were incubated with ArtinM (39 nM) for 5 h. Medium was used as a negative control and P3C4 (1 µg/mL) was used as a positive control. RNA from macrophages were isolated and used for qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">Materials and Methods</a>. The results are expressed as the relative expression of TLR2 after quantification using the ΔΔCt method and normalized to β-actin expression. Statistical comparisons between stimulated cells and unstimulated were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. ** p<0.01.</p

ArtinM binding to TLR2 depends on sugar recognition.

<p>(A and B) Peritoneal macrophages from C57BL/6 mice were incubated with biotinylated ArtinM after pre-incubation with anti-TLR2 antibody or non-specific IgG. ArtinM binding was detected with streptavidin-FITC and analyzed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Results are expressed as the percentage of cells positive for ArtinM binding (A) and MFI (median fluorescence intensity) (B). (C) The dependence of ArtinM-TLR2 binding on carbohydrate recognition used anti-TLR2 antibody coated onto 96-well microplates (5 µg/mL) to capture TLR2 from a cellular extract of peritoneal macrophages. Biotinylated ArtinM (40 µg/mL), previously incubated with the indicated concentrations of Manα1-3 [Manα1-6] Man or Galα(1,6)Galα(1,6)Gluα(1,2)Fru, was added to the wells. After washing, ArtinM binding was detected by neutravidin-AP, and signal was developed with <i>p</i>-nitrophenyl phosphate. Results are expressed in O.D as the mean ± SEM. Statistical comparisons between cells incubated or not with carbohydrates were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p