Glutaminolysis and fumarate accumulation integrate immunometabolic and epigenetic programs in trained immunity

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by ß-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to ß-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by ß-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.Netherlands Organization for Scientific Research (NWO). B.N. is supported by an NHMRC (Australia) CJ Martin Early Career Fellowship. N.P.R. Netherlands Heart Foundation (2012T051). N.P.R. and M.G.N. received a H2020 grant (H2020-PHC-2015-667873-2) from the European Union (grant agreement 667837). Fundação para a Ciência e Tecnologia, FCT (IF/00735/2014 to A.C., IF/00021/2014 to R.S., RECI/BBB-BQB/0230/2012 to L.G.G., and SFRH/BPD/96176/2013 to C. Cunha). The NMR spectrometers are part of the National NMR Facility supported by FCT (RECI/BBB-BQB/0230/2012). The research leading to these results received funding from the Fundação para a Ciência e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte); from the Quadro de Referência Estratégico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estratégico – LA 26 – 2013–2014 (PEst-C/SAU/LA0026/2013). NIH (DK43351 and DK097485) and Helmsley Trust. D.L.W. is supported, in part, by the NIH (GM53522, GM083016, GM119197, and C06RR0306551

(R)-[11C]Verapamil PET studies to assess changes in P-glycoprotein expression and functionality in rat blood-brain barrier after exposure to kainate-induced status epilepticus

<p>Abstract</p> <p>Background</p> <p>Increased functionality of efflux transporters at the blood-brain barrier may contribute to decreased drug concentrations at the target site in CNS diseases like epilepsy. In the rat, pharmacoresistant epilepsy can be mimicked by inducing status epilepticus by intraperitoneal injection of kainate, which leads to development of spontaneous seizures after 3 weeks to 3 months. The aim of this study was to investigate potential changes in P-glycoprotein (P-gp) expression and functionality at an early stage after induction of status epilepticus by kainate.</p> <p>Methods</p> <p><it>(R)</it>-[¹¹C]verapamil, which is currently the most frequently used positron emission tomography (PET) ligand for determining P-gp functionality at the blood-brain barrier, was used in kainate and saline (control) treated rats, at 7 days after treatment. To investigate the effect of P-gp on <it>(R)</it>-[¹¹C]verapamil brain distribution, both groups were studied without or with co-administration of the P-gp inhibitor tariquidar. P-gp expression was determined using immunohistochemistry in post mortem brains. <it>(R)</it>-[¹¹C]verapamil kinetics were analyzed with approaches common in PET research (Logan analysis, and compartmental modelling of individual profiles) as well as by population mixed effects modelling (NONMEM).</p> <p>Results</p> <p>All data analysis approaches indicated only modest differences in brain distribution of <it>(R)</it>-[¹¹C]verapamil between saline and kainate treated rats, while tariquidar treatment in both groups resulted in a more than 10-fold increase. NONMEM provided most precise parameter estimates. P-gp expression was found to be similar for kainate and saline treated rats.</p> <p>Conclusions</p> <p>P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.</p

ABLATING LEPTIN-MEDIATED CELL SURVIVAL WITH SMALL LEPTIN ANTAGONIST IN VARIOUS CANCERS

The risk for cancer development and progression continues to be linked to obesity via excessive circulating levels of the adipokine leptin, which is primarily secreted from white adipose tissue. In the context of cancer, circulating leptin binds to its receptor (OB-R), which has been shown to be overexpressed by cancer cells, leading to aberrant leptin/OB-R signaling. Activation of leptin signaling pathways in cancer cells is associated with S-phase progression, angiogenesis, apoptosis evasion, and cell invasion and migration. Therefore, blocking leptin signaling in highly aggressive cancers that lack specific treatment could be a novel therapeutic strategy. Our aim was to investigate the efficacy of leptin antagonism in abrogating the deleterious effects leptin signaling in triple negative breast cancer. To this end, we tested the hypothesis that leptin antagonists will prevent leptin-induced activation of proliferation and cell cycle progression in triple negative breast cancer cells (MDA-MB 231 and MDA-MB 468). Data generated show that the antagonists effectively blocked p-STAT3 and cyclin D induced by leptin in cancer cells. Leptin antagonism was shown to increase the effects of chemotherapeutic drugs. Additionally, the antagonists did not exert toxic effects in non-malignant cells (MCF-10A cell line). Leptin-mediated progression of S-phase was also reduced by the antagonists, which correlated with the abrogation of leptin-induced proliferation of breast cancer cells. PCR analysis demonstrated that blockade of leptin signaling decreased SOX3 gene expression (a transcription factor linked to proliferative and oncogenic signature). Taken together these data suggest that leptin signaling may be a therapeutic target in combating obesity-related chemotherapeutic resistance

Differential gene and microRNA expression between etoposide resistant and etoposide sensitive MCF7 breast cancer cell lines.

In order to develop targeted strategies for combating drug resistance it is essential to understand it's basic molecular mechanisms. In an exploratory study we have found several possible indicators of etoposide resistance operating in MCF7VP cells, including up-regulation of ABC transporter genes, modulation of miRNA, and alteration in copy numbers of genes

GSEA analysis of microarray data showing enrichment of the JAK-STAT and MAP Kinase pathways and upregulation of ECM structural component genes along with qRT-PCR validation of the down-regulation of topoisomerase 2 gene expression in MCF7VP cells.

<p>Fig. 5A. Gene set enrichment analysis of microrray data depicting the enrichment of genes in the JAK-STAT signaling pathway. The GSEA software was used to calculate the enrichment levels. Fig. 5B. Gene set enrichment analysis of microrray data depicting the enrichment of genes in the MAP kinase signaling pathway. The GSEA software was used to calculate the enrichment levels. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045268#pone-0045268-g005" target="_blank">Figure 5C</a>. Gene set enrichment analysis depicting enrichment of ECM (extra-cellular matrix) genes in MCF7VP cells. The GSEA algorithm was used to calculate the enrichment levels. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045268#pone-0045268-g005" target="_blank">Figure 5D</a>. Down-regulation of TOPO2A (the drug target of etoposide) in MCF7VP cells. The microarray data was validated by qRTPCR as depicted in the bar chart which shows differences in fold change.</p