Project description:Abscisic acid (ABA) has shown efficacy in the treatment of diabetes and inflammation; however, its molecular targets and the mechanisms of action underlying its immunomodulatory effects remain unclear. This study investigates the role of peroxisome proliferator-activated receptor ? (PPAR ?) and lanthionine synthetase C-like 2 (LANCL2) as molecular targets for ABA. We demonstrate that ABA increases PPAR ? reporter activity in RAW 264.7 macrophages and increases ppar ? expression in vivo, although it does not bind to the ligand-binding domain of PPAR ?. LANCL2 knockdown studies provide evidence that ABA-mediated activation of macrophage PPAR ? is dependent on lancl2 expression. Consistent with the association of LANCL2 with G proteins, we provide evidence that ABA increases cAMP accumulation in immune cells. ABA suppresses LPS-induced prostaglandin E(2) and MCP-1 production via a PPAR ?-dependent mechanism possibly involving activation of PPAR ? and suppression of NF-?B and nuclear factor of activated T cells. LPS challenge studies in PPAR ?-expressing and immune cell-specific PPAR ? null mice demonstrate that ABA down-regulates toll-like receptor 4 expression in macrophages and T cells in vivo through a PPAR ?-dependent mechanism. Global transcriptomic profiling and confirmatory quantitative RT-PCR suggest novel candidate targets and demonstrate that ABA treatment mitigates the effect of LPS on the expression of genes involved in inflammation, metabolism, and cell signaling, in part, through PPAR ?. In conclusion, ABA decreases LPS-mediated inflammation and regulates innate immune responses through a bifurcating pathway involving LANCL2 and an alternative, ligand-binding domain-independent mechanism of PPAR ? activation.
Project description:OBJECTIVE:M1-like inflammatory phenotype of macrophages plays a critical role in tissue damage in chronic inflammatory diseases. Previously, we found that the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) dampens lipopolysaccharide (LPS)-triggered inflammatory priming of RAW 264.7 cells. Herein, we tested whether DMPO by itself can induce changes in macrophage transcriptome, and that these effects may prevent LPS-induced activation of macrophages. MATERIALS AND METHODS:To test our hypothesis, we performed a transcriptomic and bioinformatics analysis in RAW 264.7 cells incubated with or without LPS, in the presence or in the absence of DMPO. RESULTS:Functional data analysis showed 79 differentially expressed genes (DEGs) when comparing DMPO vs Control. We used DAVID databases for identifying enriched gene ontology terms and Ingenuity Pathway Analysis for functional analysis. Our data showed that DMPO vs Control comparison of DEGs is related to downregulation immune-system processes among others. Functional analysis indicated that interferon-response factor 7 and toll-like receptor were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses of the DMPO?+?LPS vs LPS DEGs were consistent with DMPO-dampening LPS-induced inflammatory transcriptomic profile in RAW 264.7. These changes were confirmed using Nanostring technology. CONCLUSIONS:Taking together our data, surprisingly, indicate that DMPO by itself affects gene expression related to regulation of immune system and that DMPO dampens LPS-triggered MyD88- and TRIF-dependent signaling pathways. Our research provides critical data for further studies on the possible use of DMPO as a structural platform for the design of novel mechanism-based anti-inflammatory drugs.
Project description:BACKGROUND: Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. RESULTS: For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. CONCLUSIONS: These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.
Project description:Changes in the nuclear positioning of specific genes, depending on their expression status, have been observed in a large diversity of physiological processes. However, gene position is poorly documented in immune cells subjected to activation upon bacterial infection. Using the pig as a model organism, we focused our study on monocytes-derived macrophages and neutrophils, the first lines of defense against pathogens. We examine whether changes in gene expression due to LPS-activation imply genes repositioning in the nuclear space. A transcriptomic analysis was first performed to identify the genes differentially expressed and analyse the networks implicated during LPS-IFNγ activation in monocytes-derived macrophages. This allowed us to select 4 up-regulated (IL1β, IL8, CXCL10 and TNFα) and 4 down-regulated (VIM, LGALS3, TUBA3, and IGF2) genes to be further studied by 3D-FISH for their behavior in the nuclear space, particularly towards their chromosome territories (CT) during macrophages activation. Among the 4 up-regulated genes, 3 changed their position during the activation process while the 4 down-regulated ones did not. The analysis of gene behaviors towards their CT was extended to neutrophils for 3 up- and 2 down-regulated genes and similar results were obtained. Our data suggest that relocation in the nuclear space of genes differentially expressed in activated immune cells is gene specific and concern mostly up-regulated ones. Porcine monocyte-derived macrophages: Control vs. LPS-IFNγ activated vs. T-2 toxin/LPS-IFNγ activated. A common reference hybridization scheme was used to compare the three-condition experiment, CM vs. AM vs. TAM. The reference consisted of a pool of RNA from all samples. Biological replicates: 5 control, 5 LPS-IFNγ activated, 5 T-2 toxin/LPS-IFNγ activated independently. One replicate per array.
Project description:BACKGROUND:In animal breeding, identification of causative genetic variants is of major importance and high economical value. Usually, the number of candidate variants exceeds the number of variants that can be validated. One way of prioritizing probable candidates is by evaluating their potential to have a deleterious effect, e.g. by predicting their consequence. Due to experimental difficulties to evaluate variants that do not cause an amino-acid substitution, other prioritization methods are needed. For human genomes, the prediction of deleterious genomic variants has taken a step forward with the introduction of the combined annotation dependent depletion (CADD) method. In theory, this approach can be applied to any species. Here, we present pCADD (p for pig), a model to score single nucleotide variants (SNVs) in pig genomes. RESULTS:To evaluate whether pCADD captures sites with biological meaning, we used transcripts from miRNAs and introns, sequences from genes that are specific for a particular tissue, and the different sites of codons, to test how well pCADD scores differentiate between functional and non-functional elements. Furthermore, we conducted an assessment of examples of non-coding and coding SNVs, which are causal for changes in phenotypes. Our results show that pCADD scores discriminate between functional and non-functional sequences and prioritize functional SNVs, and that pCADD is able to score the different positions in a codon relative to their redundancy. Taken together, these results indicate that based on pCADD scores, regions with biological relevance can be identified and distinguished according to their rate of adaptation. CONCLUSIONS:We present the ability of pCADD to prioritize SNVs in the pig genome with respect to their putative deleteriousness, in accordance to the biological significance of the region in which they are located. We created scores for all possible SNVs, coding and non-coding, for all autosomes and the X chromosome of the pig reference sequence Sscrofa11.1, proposing a toolbox to prioritize variants and evaluate sequences to highlight new sites of interest to explain biological functions that are relevant to animal breeding.
Project description:Microglial cells become rapidly activated through interactions with pathogens, and the persistent activation of these cells is associated with various neurodegenerative diseases. Previous studies have investigated the transcriptomic signatures in microglia or macrophages using microarray technologies. However, this method has numerous restrictions, such as spatial biases, uneven probe properties, low sensitivity, and dependency on the probes spotted. To overcome this limitation and identify novel transcribed genes in response to LPS, we used RNA Sequencing (RNA-Seq) to determine the novel transcriptomic signatures in BV-2 microglial cells. Sequencing assessment and quality evaluation showed that approximately 263 and 319 genes (? 1.5 log2-fold), such as cytokines and chemokines, were strongly induced after 2 and 4 h, respectively, and the induction of several genes with unknown immunological functions was also observed. Importantly, we observed that previously unidentified transcription factors (TFs) (irf1, irf7, and irf9), histone demethylases (kdm4a) and DNA methyltransferases (dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. The gene expression levels, transcription start sites (TSS), isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with LPS. In addition, gene ontology, molecular networks and pathway analyses identified the top significantly regulated functional classification, canonical pathways and network functions at each activation status. Moreover, we further analyzed differentially expressed genes to identify transcription factor (TF) motifs (-950 to +50 bp of the 5' upstream promoters) and epigenetic mechanisms. Furthermore, we confirmed that the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This transcriptomic analysis is the first to show a comparison of the family-wide differential expression of most known immune genes and also reveal transcription evidence of multiple gene families in BV-2 microglial cells. Collectively, these findings reveal unique transcriptomic signatures in BV-2 microglial cells required for homeostasis and effective immune responses.
Project description:Large White and Meishan pigs were either non-treated or injected with mammalian 1-24 ACTH (Immediate Synachten, Novartis France) at the dose of 250 µg per animal. Pigs were sacrificed either immediately after capture from their home cage (non-treated animals) or 1 hour following ACTH injection. Adrenal glands were immediately collected from pigs and frozen on dry ice and then stored at -80°C until RNA isolation. Keywords: stress response, adrenal, gene expression, pig Overall design: 47 samples
Project description:IL-17 is a cytokine mainly from IL-17-producing T cells, which are one of subsets of CD4+ T cells and play a role in adaptive immune system. Recent studies have demonstrated that IL-17A can act rapidly as an innate immune responder during infection before the onset of its classic adaptive immune response. This role of IL-17A in innate immune response is implicated in lipopolysaccharide (LPS)-induced lung inflammation. Very recently, we have reported that endoplasmic reticulum (ER) stress is involved in LPS-induced lung inflammation in vivo and in vitro. This study aimed to elucidate the role of IL-17A in LPS-induced lung injury, focusing on the link with ER stress. We treated a murine model of LPS-induced lung injury with IL-17A neutralizing antibody and 4-phenylbutyrate (4-PBA), a representative ER stress inhibitor. In addition, we evaluated the effects of IL-17A on ER stress in LPS-stimulated bronchial epithelial cells. Our results showed that inhibition of IL-17A decreased LPS-induced pulmonary neutrophilia, vascular leakage, nuclear translocation of nuclear factor-?B (NF-?B), infiltration of dendritic cells, increased expression of Toll-like receptor 4 (TLR4), activation of NLRP3 inflammasome, and increased ER stress in the lung. 4-PBA or TAK-242, a TLR4 inhibitor attenuated expression of IL-17A thereby improving LPS-induced lung inflammation. Intriguingly, we observed that stimulation with LPS increased expression of IL-17A in airway epithelial cells and co-stimulation with IL-17A further increased ER stress and NF-?B activation. This study indicates that the interrelationship between IL-17A and ER stress plays an important role in LPS-induced injury showing a positive feedback in airway epithelial cells and suggests that targeting their interaction can be a potential therapeutic approach to overcome one of severe refractory pulmonary disorders.
Project description:Zearalenone (ZEA) is a secondary metabolite produced by Fusarium species. ZEA was proved to exert a wide range of unwanted side effects, but its mechanism of action, particularly at duodenum levels, remains unclear. In our study based on the microarray technology we assessed the alteration of gene expression pattern Sus scrofa duodenum which has been previously exposed to ZEA. Gene expression data was validated by qRT-PCR and ELISA. The gene expression data were further extrapolated the results to their human orthologues and analyzed the data in the context of human health using IPA (Ingenuity Pathways Analysis).Using Agilent microarray technology, we found that gene expression pattern was significantly affected by ZEA exposure, considering a 2-fold expression difference as a cut-off level and a p-value < 0.05. In total, we found 1576 upregulated and 2446 downregulated transcripts. About 1084 genes (764 downregulated and 751 overexpressed) were extrapolated to their human orthologues. IPA analysis showed various altered key cellular and molecular pathways. As expected, we observed a significant alteration of immune response related genes, MAPK (mitogen activate protein kinases) pathways or Toll-Like Receptors (TLRs). What captured our attention was the modulation of pathways related to the activation of early carcinogenesis.Our data demonstrate that ZEA has a complex effect at duodenum level. ZEA is able to activate not only the immune response related genes, but also those relate to colorectal carcinogenesis. The effects can be more dramatic when connected with the exposure to other environmental toxic agents or co-occurrence with different microorganisms.