Immunolipidomics Reveals a Globoside Network During the Resolution of Pro-Inflammatory Response in Human Macrophages.

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages.

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP-compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells.

Vector Copy Distribution at a Single-Cell Level Enhances Analytical Characterization of Gene-Modified Cell Therapies.

The ability to deliver transgenes into the human genome using viral vectors is a major enabler of the gene-modified cell therapy field. However, the control of viral transduction is difficult and can lead to product heterogeneity, impacting efficacy and safety, as well as increasing the risk of batch failure during manufacturing. To address this, we generated a novel analytical method to measure vector copy distribution at the single-cell level in a gene-modified, lentiviral-based immunotherapy model. As the limited amount of genomic DNA in a single cell hinders reliable quantification, we implemented a preamplification strategy on selected lentiviral and human gene targets in isolated live single cells, followed by quantification of amplified material by droplet digital PCR. Using a bespoke probability framework based on Bayesian statistics, we show that we can estimate vector copy number (VCN) integers with maximum likelihood scores. Notably, single-cell data are consistent with population analysis and also provide an overall measurement of transduction efficiency by discriminating transduced (VCN ≥ 1) from nontransduced (VCN = 0) cells. The ability to characterize cell-to-cell variability provides a powerful high-resolution approach for product characterization, which could ultimately allow improved control over product quality and safety.

Identification of a subset of trace amine-associated receptors and ligands as potential modulators of insulin secretion.

The worldwide prevalence of diabetes has reached 8.5% among adults, and this is characterised by elevated glucose concentrations and failing insulin secretion. Furthermore, most people with type 2 diabetes are either obese or overweight, with the associated dyslipidaemia contributing to the development of insulin resistance and increased cardiovascular risk. Here we incubated INS-1 pancreatic ß-cells for 72 h in RPMI-1640 media, or media supplemented with 28 mM glucose, 200 µM palmitic acid, and 200 µM oleic acid as a cellular model of diabetic glucolipotoxicity. Illumina HiSeq gene expression analysis showed the trace amine-associated receptor (TAAR) family to be among the most highly downregulated by glucolipotoxicity. Importantly, MetaCore integrated knowledge database, from Clarivate Analytics, indicated potential TAAR impact on insulin secretion through adenylyl cyclase signalling pathways. We therefore investigated the effect of TAAR ligands on cAMP signalling and insulin secretion, and found that only the branch of the TAAR family tree that is activated by isopentylamine, 2-phenylethylamine, p-tyramine, and agmatine significantly increased intracellular cAMP and resulted in increased insulin secretion from INS-1 cells and primary mouse islets under normal conditions. Crucially however, this enhancement was not evident when the receptor family was downregulated by glucolipotoxic conditions. This data indicates that a subset of TAARs are regulators of insulin secretion in pancreatic ß-cells, and that their downregulation contributes to glucolipotoxic inhibition of insulin secretion. As such they may be potential targets for treatment of type 2 diabetes.

Systems genetics identifies a macrophage cholesterol network associated with physiological wound healing.

Among other cells, macrophages regulate the inflammatory and reparative phases during wound healing but genetic determinants and detailed molecular pathways that modulate these processes are not fully elucidated. Here, we took advantage of normal variation in wound healing in 1,378 genetically outbred mice, and carried out macrophage RNA-sequencing profiling of mice with extreme wound healing phenotypes (i.e., slow and fast healers, n = 146 in total). The resulting macrophage coexpression networks were genetically mapped and led to the identification of a unique module under strong trans-acting genetic control by the Runx2 locus. This macrophage-mediated healing network was specifically enriched for cholesterol and fatty acid biosynthetic processes. Pharmacological blockage of fatty acid synthesis with cerulenin resulted in delayed wound healing in vivo, and increased macrophage infiltration in the wounded skin, suggesting the persistence of an unresolved inflammation. We show how naturally occurring sequence variation controls transcriptional networks in macrophages, which in turn regulate specific metabolic pathways that could be targeted in wound healing.

Changes in macrophage transcriptome associate with systemic sclerosis and mediate GSDMA contribution to disease risk.

OBJECTIVES: Several common and rare risk variants have been reported for systemic sclerosis (SSc), but the effector cell(s) mediating the function of these genetic variants remains to be elucidated. While innate immune cells have been proposed as the critical targets to interfere with the disease process underlying SSc, no studies have comprehensively established their effector role. Here we investigated the contribution of monocyte-derived macrophages (MDMs) in mediating genetic susceptibility to SSc. METHODS: We carried out RNA sequencing and genome-wide genotyping in MDMs from 57 patients with SSc and 15 controls. Our differential expression and expression quantitative trait locus (eQTL) analysis in SSc was further integrated with epigenetic, expression and eQTL data from skin, monocytes, neutrophils and lymphocytes. RESULTS: We identified 602 genes upregulated and downregulated in SSc macrophages that were significantly enriched for genes previously implicated in SSc susceptibility (P=5×10-4), and 270 cis-regulated genes in MDMs. Among these, GSDMA was reported to carry an SSc risk variant (rs3894194) regulating expression of neighbouring genes in blood. We show that GSDMA is upregulated in SSc MDMs (P=8.4×10-4) but not in the skin, and is a significant eQTL in SSc macrophages and lipopolysaccharide/interferon gamma (IFNγ)-stimulated monocytes. Furthermore, we identify an SSc macrophage transcriptome signature characterised by upregulation of glycolysis, hypoxia and mTOR signalling and a downregulation of IFNγ response pathways. CONCLUSIONS: Our data further establish the link between macrophages and SSc, and suggest that the contribution of the rs3894194 risk variant to SSc susceptibility can be mediated by GSDMA expression in macrophages.

BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases.

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions.

Identification of Ceruloplasmin as a Gene that Affects Susceptibility to Glomerulonephritis Through Macrophage Function.

Crescentic glomerulonephritis (Crgn) is a complex disorder where macrophage activity and infiltration are significant effector causes. In previous linkage studies using the uniquely susceptible Wistar Kyoto (WKY) rat strain, we have identified multiple crescentic glomerulonephritis QTL (Crgn) and positionally cloned genes underlying Crgn1 and Crgn2, which accounted for 40% of total variance in glomerular inflammation. Here, we have generated a backcross (BC) population (n = 166) where Crgn1 and Crgn2 were genetically fixed and found significant linkage to glomerular crescents on chromosome 2 (Crgn8, LOD = 3.8). Fine mapping analysis by integration with genome-wide expression QTLs (eQTLs) from the same BC population identified ceruloplasmin (Cp) as a positional eQTL in macrophages but not in serum. Liquid chromatography-tandem mass spectrometry confirmed Cp as a protein QTL in rat macrophages. WKY macrophages overexpress Cp and its downregulation by RNA interference decreases markers of glomerular proinflammatory macrophage activation. Similarly, short incubation with Cp results in a strain-dependent macrophage polarization in the rat. These results suggest that genetically determined Cp levels can alter susceptibility to Crgn through macrophage function and propose a new role for Cp in early macrophage activation.

Genome-wide analysis of differential RNA editing in epilepsy.

The recoding of genetic information through RNA editing contributes to proteomic diversity, but the extent and significance of RNA editing in disease is poorly understood. In particular, few studies have investigated the relationship between RNA editing and disease at a genome-wide level. Here, we developed a framework for the genome-wide detection of RNA sites that are differentially edited in disease. Using RNA-sequencing data from 100 hippocampi from mice with epilepsy (pilocarpine-temporal lobe epilepsy model) and 100 healthy control hippocampi, we identified 256 RNA sites (overlapping with 87 genes) that were significantly differentially edited between epileptic cases and controls. The degree of differential RNA editing in epileptic mice correlated with frequency of seizures, and the set of genes differentially RNA-edited between case and control mice were enriched for functional terms highly relevant to epilepsy, including "neuron projection" and "seizures." Genes with differential RNA editing were preferentially enriched for genes with a genetic association to epilepsy. Indeed, we found that they are significantly enriched for genes that harbor nonsynonymous de novo mutations in patients with epileptic encephalopathy and for common susceptibility variants associated with generalized epilepsy. These analyses reveal a functional convergence between genes that are differentially RNA-edited in acquired symptomatic epilepsy and those that contribute risk for genetic epilepsy. Taken together, our results suggest a potential role for RNA editing in the epileptic hippocampus in the occurrence and severity of epileptic seizures.

Glucolipotoxicity initiates pancreatic ß-cell death through TNFR5/CD40-mediated STAT1 and NF-κB activation.

Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing ß-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the ß-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in ß-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death.

Overcoming mass transport limitations to achieve femtomolar detection limits on silicon protein microarrays.

Here we combine the use of fluorescence-enhancing silicon substrates coated by copoly(DMA-NAS-MAPS), a ter-copolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), with an efficient dynamic incubation to overcome mass transport limitations and obtain femtomolar limits of detection. The high sensitivity was obtained with a conventional microarray scanner without the use of any sophisticated detection strategy or protocol. When the method was applied, an improvement of the analytical sensitivity of approximately three orders of magnitude was achieved for antibody detection when compared with the same assay performed on regular glass slides and static conditions. Moreover, limits of detection of 45 and 54 pg/ml were obtained for hepatitis B superficial antigen and HIV p24 antigen, respectively.

Silicon biochips for dual label-free and fluorescence detection: Application to protein microarray development.

A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500 nm SiO2 layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100 nm SiO2 providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fluorescence detection of the molecular interaction counterparts. Such a combined chip is of high practical utility during assay development process to image arrays, check consistency and quality of the protein array, quantify the amount of immobilized probes and finally detect fluorescence of bioassays.