Identifying transcript-level differential expression in primary human immune cells.

BACKGROUND: Multipotential hematopoietic stem cells differentiate into a wide variety of immune cells with a diversity of functions, including the ability to respond to a variety of stimuli. Importantly, numerous studies have demonstrated the importance of gene transcription in defining cell identity and functions. While these studies have primarily been performed at the level of the gene, it is known that key immune genes such as CD44 and CD45 generate multiple different transcripts that are differentially expressed across different immune cells, and that encode proteins with different sequences and functions. Prior genomic surveys have shown that the mechanisms for generating diversity in expressed transcripts (alternate splicing, alternate transcription start sites, etc.) are very active in immune cells, but have been lacking in terms of identifying genes with multiple transcripts, that are differentially expressed, and likely to affect cell functions. METHODS: We first identified the set of genes that had at least two transcripts expressed in our RNA sequencing dataset generated from purified populations of neutrophils, monocytes and five lymphocyte populations (B, NK, γδ T, CD4 + T and CD8 + T) from twelve healthy donors. Next, we developed a heuristic approach to identify genes where two or more transcripts have distinct expression patterns across lymphoid and/or myeloid populations. We then focused our annotation and interpretation on differentially expressed transcripts that affect the coding sequence. This process was repeated to identify transcripts that were differentially expressed between monocytes and populations of macrophages and LPS-stimulated macrophages derived from these monocytes in vitro. RESULTS: We found that over 55 % of genes had two or more expressed transcripts, with an average ∼3 transcripts per gene, and that 70 % of these had at least two of the transcripts that encoded proteins with different sequences. As expected, we identified a complex pattern of differential expression for multiple transcripts encoding the CD45 transmembrane protein, but we also found similar evidence for ten other genes (CD300A, FYB1, GPI, LITAF, PSMA1, PTMA, RPL32, SEPTIN9, SH3BP2, SH3KBP1) when comparing the expression patterns of transcripts within myeloid and lymphoid cells. We also identified five genes with differentially expressed transcripts associated with the transition from monocytes to macrophages (FNBP1, KLF6, and SEPTIN9) or between macrophages and LPS-stimulated macrophages (CD44, OAZ2, and SEPTIN9). For the most part, we found that the different transcripts of these genes are expected to impact specific biological functions, for example the different transcripts of SEPTIN9 likely regulate the cytoskeleton in immune cells via their interactions with actins filaments and microtubules. CONCLUSIONS: This analytic approach successfully identified multi-transcript genes that are differentially expressed across immune cells and could be applied to other transcriptomic data. DATA AVAILABILITY STATEMENT: Researchers can request access to the individual-level data from the current study by contacting the Montreal Heart Institute ethics committee at the following institutional email address: cer.icm@icm-mhi.org.

Prostaglandins and calprotectin are genetically and functionally linked to the Inflammatory Bowel Diseases.

BACKGROUND: Genome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage. METHODS: The open reading frames (ORF) of 42 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cell's transcriptome was analyzed using a RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes. RESULTS: This functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 gene expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, and diminished by their antagonists. CONCLUSION: Our results support a causal role for the PTGIR, PTGER4, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all five genes regulating the expression of CP in myeloid cells, as well as potential roles for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. METHODS: We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. RESULTS: We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1ß secretion in response to an inflammatory stimulus. CONCLUSIONS: We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

Functional screen of inflammatory bowel disease genes reveals key epithelial functions.

BACKGROUND: Genetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts has been more limited. METHODS: We identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. By comparing the genes in which expression was modulated by each ORF, as well as the functions enriched within these gene lists, we identified ORFs with shared impacts and their putative disease-relevant biological functions. RESULTS: Analysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1, and SMAD3 identified functions consistent with what is already known for these genes. These analyses also identified two major clusters of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1, and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates can impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16 acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota. CONCLUSIONS: This functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cell's transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

A transcriptome-based approach to identify functional modules within and across primary human immune cells.

Genome-wide transcriptomic analyses have provided valuable insight into fundamental biology and disease pathophysiology. Many studies have taken advantage of the correlation in the expression patterns of the transcriptome to infer a potential biologic function of uncharacterized genes, and multiple groups have examined the relationship between co-expression, co-regulation, and gene function on a broader scale. Given the unique characteristics of immune cells circulating in the blood, we were interested in determining whether it was possible to identify functional co-expression modules in human immune cells. Specifically, we sequenced the transcriptome of nine immune cell types from peripheral blood cells of healthy donors and, using a combination of global and targeted analyses of genes within co-expression modules, we were able to determine functions for these modules that were cell lineage-specific or shared among multiple cell lineages. In addition, our analyses identified transcription factors likely important for immune cell lineage commitment and/or maintenance.

BioJava: an open-source framework for bioinformatics in 2012.

UNLABELLED: BioJava is an open-source project for processing of biological data in the Java programming language. We have recently released a new version (3.0.5), which is a major update to the code base that greatly extends its functionality. RESULTS: BioJava now consists of several independent modules that provide state-of-the-art tools for protein structure comparison, pairwise and multiple sequence alignments, working with DNA and protein sequences, analysis of amino acid properties, detection of protein modifications and prediction of disordered regions in proteins as well as parsers for common file formats using a biologically meaningful data model. AVAILABILITY: BioJava is an open-source project distributed under the Lesser GPL (LGPL). BioJava can be downloaded from the BioJava website (http://www.biojava.org). BioJava requires Java 1.6 or higher. All inquiries should be directed to the BioJava mailing lists. Details are available at http://biojava.org/wiki/BioJava:MailingLists.

Genome-wide expression profiling implicates a MAST3-regulated gene set in colonic mucosal inflammation of ulcerative colitis patients.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBDs) presumably caused by dysregulated immune responses to the gut microbiota. Genetic association studies have implicated dozens of chromosomal regions or loci in IBD susceptibility. The next challenge is to explain the individual role of each of these modest effect loci in the disease state. We have previously identified MAST3 as an IBD susceptibility gene through genetic fine-mapping of the 19p linkage region. Testing MAST3 in a reporter assay provided preliminary evidence that MAST3 modulates the activity of inflammation-related transcription factor nuclear factor kappa B. METHODS: Here we characterized the function of MAST3 through an examination of the influence of the modulation of MAST3 expression on endogenous genome-wide expression patterns. More specifically, we looked at differential gene expression resulting from overexpression and knockdown of the MAST3 gene in epithelial and macrophage cell lines. From we highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-jB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. RESULTS: We highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-κB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. These MAST3-regulated genes are central to mucosal immune responses. Among them are proinflammatory cytokines (e.g., CCL20, IL8), regulators of NF-κB (e.g., TNFAIP3, LY96, NFKBIA), genes involved in interferon-induced defense against pathogen invasion (e.g., IFIT1, ISG15), and genes involved in cell adhesion and/or migration (e.g., CD44, TMOD1). CONCLUSIONS: Taken together, these results confirm MAST3 as a modulator of the inflammatory response through regulation of immune gene expression in the gut of IBD patients.

Post-translational modifications, a key process in CD36 function: lessons from the spontaneously hypertensive rat heart.

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of (13)C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to ß-oxidation (-40%) and triglycerides (+2.8 folds), which was not explained by transcriptional changes or malonyl-CoA level, a recognized ß-oxidation inhibitor, and (ii) membrane-associated CD36 protein level, but unchanged distribution. Other results demonstrate alterations in CD36 PTM in SHR hearts, specifically by N-glycosylation, and the importance of O-linked-ß-N-acetylglucosamine for its membrane recruitment and role in LCFA use in the heart.

Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases.

The human MHC represents the strongest susceptibility locus for autoimmune diseases. However, the identification of the true predisposing gene(s) has been handicapped by the strong linkage disequilibrium across the region. Furthermore, most studies to date have been limited to the examination of a subset of the HLA and non-HLA genes with a marker density and sample size insufficient for mapping all independent association signals. We genotyped a panel of 1,472 SNPs to capture the common genomic variation across the 3.44 megabase (Mb) classic MHC region in 10,576 DNA samples derived from patients with systemic lupus erythematosus, Crohn's disease, ulcerative colitis, rheumatoid arthritis, myasthenia gravis, selective IgA deficiency, multiple sclerosis, and appropriate control samples. We identified the primary association signals for each disease and performed conditional regression to identify independent secondary signals. The data demonstrate that MHC associations with autoimmune diseases result from complex, multilocus effects that span the entire region.

Metabolic diapause in pancreatic beta-cells expressing a gain-of-function mutant of the forkhead protein Foxo1.

Diabetes is associated with decreased pancreatic beta-cell function and mass. It is unclear whether diabetes treatment should aim at restoring beta-cell performance/mass or at inducing "beta-cell rest" to prevent further deterioration. The transcription factor Foxo1 protects beta-cells against oxidative stress induced by hyperglycemia and prevents beta-cell replication in insulin-resistant states. Here we show that these combined effects are associated with a concerted repression of genes involved in glycolysis, nitric-oxide synthesis, G protein-coupled receptor signaling, and ion transport. Conversely, Foxo1 increases expression of several neurotransmitter receptors and fails to regulate target genes predicted from Caenorhabditis elegans and Drosophila studies. Functional analyses show decreased glucose utilization and insulin secretion in beta-cells overexpressing Foxo1. We propose the definition of "metabolic diapause" for the changes induced by Foxo1 to protect beta-cells against oxidative stress. The data provide genetic underpinning for the concept of beta-cell rest as a treatment goal in diabetes.

Decreased cardiac mitochondrial NADP+-isocitrate dehydrogenase activity and expression: a marker of oxidative stress in hypertrophy development.

Mitochondrial dysfunction subsequent to increased oxidative stress and alterations in energy metabolism is considered to play a role in the development of cardiac hypertrophy and its progression to failure, although the sequence of events remains to be elucidated. This study aimed at characterizing the impact of hypertrophy development on the activity and expression of mitochondrial NADP+-isocitrate dehydrogenase (mNADP+-ICDH), a metabolic enzyme that controls redox and energy status. We expanded on our previous finding of its inactivation through posttranslational modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in 7-wk-old spontaneously hypertensive rat (SHR) hearts before hypertrophy development (Benderdour et al. J Biol Chem 278: 45154-45159, 2003). In this study, we used 7-, 15-, and 30-wk-old SHR and Sprague-Dawley (SD) rats with abdominal aortic coarctation. Compared with age-matched control Wistar-Kyoto (WKY) rats, SHR hearts showed a significant 25% decrease of mNADP+-ICDH activity, which preceded in time 1) the decline in its protein and mRNA expression levels (between 10% and 35%) and 2) the increase in hypertrophy markers. The chronic and persistent loss of mNADP+-ICDH activity in SHR was associated with enhanced tissue accumulation of HNE-mNADP+-ICDH and total HNE-protein adducts at all ages and contrasted with the profile of changes in the activity of other mitochondrial enzymes involved in antioxidant or energy metabolism. Two-way ANOVA of the data also revealed a significant effect of age on most parameters measured in SHR and WKY hearts. The mNADP+-ICDH activity, protein, and mRNA expression were reduced between 25% and 35% in coarctated SD rats and were normalized by treatment of SHR or coarctated SD rats with renin-angiotensin system inhibitors, which prevented or attenuated hypertrophy. Altogether, our data show that cardiac mNADP+-ICDH activity and expression are differentially and sequentially affected in hypertrophy development and, to a lesser extent, with aging. Decreased cardiac mNADP+-ICDH activity, which is attributed at least in part to HNE adduct formation, appears to be a relevant early and persistent marker of mitochondrial oxidative stress-related alterations in hypertrophy development. Potentially, this could also contribute to the aetiology of cardiomyopathy.

Glucagon-like peptide 1 induces pancreatic beta-cell proliferation via transactivation of the epidermal growth factor receptor.

We previously provided evidence that glucagon-like peptide 1 (GLP-1) induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol (PI) 3-kinase- and protein kinase C zeta-dependent manner. However, the exact mechanism by which the GLP-1 receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, activates the PI 3-kinase signaling pathway to promote beta-cell growth remains unknown. We hypothesized that the GLP-1R could activate PI 3-kinase and promote beta-cell proliferation through transactivation of the epidermal growth factor (EGF) receptor (EGFR), an event possibly linked to GPCRs via activation of c-Src and the production of putative endogenous EGF-like ligands. Both the c-Src inhibitor PP1 and the EGFR-specific inhibitor AG1478 blocked GLP-1-induced [(3)H]thymidine incorporation in INS(832/13) cells as well as in isolated rat islets, while only AG1478 inhibited the proliferative action of betacellulin (BTC), an EGFR agonist. Both compounds also suppressed GLP-1-induced PI 3-kinase activation. A time-dependent increase in tyrosine phosphorylation of the EGFR in response to GLP-1 was observed in INS(832/13) cells. This transactivation of the EGFR was sensitive to both the pharmacological agents PP1 and AG1478. The action of GLP-1 and BTC on INS cell proliferation was found to be not additive. Overexpression of a dominant-negative EGFR in INS cells with a retroviral expression vector curtailed GLP-1-induced beta-cell proliferation. GLP-1 treatment of INS cells caused a decrease in cell surface-associated BTC, as shown by FACS analysis. Also, the metalloproteinase inhibitor GM6001 and an anti-BTC neutralizing antibody suppressed the GLP-1 proliferative effect. Finally, coculturing the prostatic cancer cell line LNCaP that lacks GLP-1 responsiveness with INS cells increased LNCaP cell proliferation in the presence of GLP-1, thus revealing that INS cells secrete a growth factor in response to GLP-1. GM6001 and an anti-BTC neutralizing antibody suppressed increased LNCaP cell proliferation in the presence of GLP-1 in the coculture experiments. The results are consistent with a model in which GLP-1 increases PI 3-kinase activity and enhances beta-cell proliferation via transactivation of the EGFR that would require the proteolytic processing of membrane-anchored BTC or other EGF-like ligands.

EGF receptor transactivation is obligatory for protein synthesis stimulation by G protein-coupled receptors.

The epidermal growth factor receptor (EGFR) was recently identified as a signal transducer of G protein-coupled receptors (GPCRs). In this study, we have examined the contribution of EGFR transactivation to the growth-promoting effect of GPCRs on vascular smooth muscle cells. Activation of the G(q)-coupled ANG II receptor or G(i)-coupled lysophosphatidic acid receptor resulted in increased tyrosine phosphorylation and activation of EGFR. Specific inhibition of EGFR kinase activity by tyrphostin AG-1478 or expression of a dominant-negative EGFR mutant abolished this response. Importantly, inhibition of EGFR function strongly attenuated the global stimulation of protein synthesis by GPCR agonists in vitro in cultured aortic smooth muscle cells and in vivo in the rat aorta and in small resistance arteries. The growth inhibition was associated with a marked reduction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathway activity and the resulting suppression of eukaryotic translation initiation factor 4E and 4E binding protein 1 phosphorylation. Our results demonstrate that EGFR transactivation is a physiologically relevant action of GPCRs linked to translational control and protein synthesis.