Cell Type-specific Expression Patterns Research Articles

The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells

What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster? The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis. The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report. We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes. We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression. We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes. While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary. The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.

The Expression of HDAC1 and HDAC2 During Cerebellar Cortical Development

Histone deacetylases (HDACs) are epigenetic regulatory proteins that repress gene transcription by changing DNA conformation. The regulation of gene expression through histone deacetylation is an important mechanism for the development of the central nervous system. Although the disruption of the balance in epigenetic gene regulation has been implicated in many CNS developmental abnormalities and diseases, the expression pattern of HDACs in various cell types in the brain during its maturation process has had limited exploration. Therefore, in this study, we investigate the cell type-specific and developmental stage-specific expression pattern of HDAC1 and HDAC2 in the mouse cerebellum. Our experimental results show that the cerebellar progenitors and glial cells express high levels of HDAC1 and low levels of HDAC2. On the other hand, the post-mitotic migrating neuronal cells of the cerebellar cortex show strong HDAC2 and weak HDAC1 expressions. In more differentiated neuronal cells, including Purkinje cells, granule cells, unipolar brush cells, and GABAergic interneurons, we found a consistent expression pattern, high levels of HDAC2 and low levels of HDAC1. Therefore, our data provide support for the potential important roles of HDAC1 in cell proliferation and HDAC2 in migration and differentiation.

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Epigenetic Activation and Silencing of the Gene that Encodes IFN-γ

Transcriptional activation and repression of genes that are developmentally regulated or exhibit cell-type specific expression patterns is largely achieved by modifying the chromatin template at a gene locus. Complex formation of stable epigenetic histone marks, loss or gain of DNA methylation, alterations in chromosome conformation, and specific utilization of both proximal and distal transcriptional enhancers and repressors all contribute to this process. In addition, long non-coding RNAs are a new species of regulatory RNAs that either positively or negatively regulate transcription of target gene loci. IFN-γ is a pro-inflammatory cytokine with critical functions in both innate and adaptive arms of the immune system. This review focuses on our current understanding of how the chromatin template is modified at the IFNG locus during developmental processes leading to its transcriptional activation and silencing.

The methylmalonic aciduria related genes, Mmaa, Mmab, and Mut, are broadly expressed in placental and embryonic tissues during mouse organogenesis

Organ-specific birth defects are seen in patients with some inborn errors of vitamin B12 metabolism. To determine whether three mouse genes, whose human counterparts are associated with isolated methylmalonic aciduria (Mmaa, Mmab and Mut), show tissue-specific expression during organogenesis, we used in situ hybridization to characterize their pattern of expression in wild type embryos and placentas at embryonic days (E) E10.5, E11.5 and E12.5. These three genes are ubiquitously expressed in the placenta and in embryos at E10.5. At E11.5, we observed tissue specific expression patterns for these three genes in lung, head and Rathke's pouch. At E12.5, although Mut expression was ubiquitous, we found cell-type specific expression patterns for Mmaa and Mmab in the developing craniofacial region, the lung, the liver, and the gut. These results suggest that during organogenesis the proteins encoded by these three genes may interact in only a subset of cells.

Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function

Kainate-type glutamate receptors (KARs) regulate synaptic transmission and neuronal excitability via multiple mechanisms, depending on their subunit composition. Presynaptic KARs tonically depress glutamatergic transmission during restricted period of synapse development; however, the molecular basis behind this effect is unknown. Here, we show that the developmental and cell-type specific expression pattern of a KAR subunit splice variant, GluK1c, corresponds to the immature-type KAR activity in the hippocampus. GluK1c localizes to dendritic contact sites at distal axons, the distal targeting being promoted by heteromerization with the subunit GluK4. Presynaptic expression of GluK1c strongly suppresses glutamatergic transmission in cell-pairs in vitro and mimics the immature-type KAR activity at CA3-CA1 synapses in vivo, at a developmental stage when the endogenous expression is already downregulated. These data support a central role for GluK1c in mediating tonic inhibition of glutamate release and the consequent effects on excitability and activity-dependent fine-tuning of the developing hippocampal circuitry.

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns.

Hepatic Cell–Type Specific Gene Expression Better Predicts HCV Treatment Outcome Than IL28B Genotype

Cell-type specific expression patterns of hepatic interferon-stimulated genes (ISGs) and single nucleotide polymorphisms (SNPs) near the IL28B gene are associated with response to interferon-based therapy in patients with chronic hepatitis C virus (HCV) infection. It is not known how the IL28B genotype influences the ISG expression pattern and which is a better predictor of treatment response. Patients at the Toronto Western Hospital Liver Centre with known outcome to interferon-based treatment for HCV infection were evaluated. Analysis included hepatic gene expression profile using complementary DNA microarrays, genotype at the IL28B SNP rs12979860, and immunostaining for human myxovirus A protein 1 (MxA) in hepatocytes and macrophages. The level of ISG immunostaining in hepatic macrophages correlated inversely with that of hepatocytes and was strongly associated with treatment outcome. Gene expression profiles and the IL28B genotype were associated with treatment response, but only absence of MxA staining in macrophages accurately predicted nonresponse to treatment. The positive predictive value (PPV) of the IL28B genotype was 94% and the negative predictive value (NPV) was 51% (n = 209). For messenger RNA expression, the PPV was 94% and the NPV was 54% (n = 65). For detection of MxA in macrophages, the PPV was 60% and the NPV was 98% (n = 110). Of 53 patients with undetectable macrophage MxA staining, only one had a sustained virologic response. IL28B genotype was strongly associated with cell-type specific staining for MxA. There was a stepwise increase in macrophage staining and decrease in hepatocyte staining from the TT (lack of response) to CC SNP (associated with response) in IL28B. By logistic regression, after controlling for the presence of macrophage MxA staining, the IL28B genotype was no longer associated with treatment response. The cell-type-specific expression pattern of ISGs varies among patients with different IL28B genotypes and is a strong predictor of response to interferon-based treatment.

Regional distribution and cell type-specific subcellular localization of Prothymosin alpha in brain.

Prothymosin alpha (ProTα) is an acidic nuclear protein implicated in several cellular functions including cell survival. ProTα is found in the central nervous system, but the regional and cell type-specific expression patterns are not known. In this study, our immunohistochemical analysis demonstrated that ProTα is expressed ubiquitously throughout adult brain with difference in the intensity of region-specific protein reactivity. Interestingly, the highest ProTα signals were observed in the brain regions relevant to neurogenesis, such as sub-ventricular zone, granular cell layer of dentate gyrus, as well as granule cell layer of olfactory bulb. Strong immunoreactivity was also found in habenula, ependymal cells lining the dorsal third and fourth ventricle, and in neurons in the Purkinje cell layer of cerebellum. We showed that ProTα was strictly localized in the nuclei of neurons, while it was found in the cytosolic space of astroglial and microglial processes and cell body in the adult brain. To clarify the phenomenon underlying cytosolic localization of ProTα in non-neuronal cells, ZVAD-fmk, a caspase-3 inhibitor, was delivered intracerebroventricularly in the brain. At the follow-up 24h after ZVAD-fmk injection, we found that nuclear intensity of ProTα was significantly increased in astrocytes, whereas the ProTα expression was not affected in microglia. The present study would contribute toward better understanding of physiological and pathophysiological roles of ProTα in the brain.

Epigenetic Regulation of Cell Type–Specific Expression Patterns in the Human Mammary Epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type–specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type–specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.

Tracking in vivo IL-17A production during infection and autoimmunity (114.11)

Abstract Interleukin (IL)-17A plays a crucial role in host defense against a variety of pathogens and also contributes to the pathogenesis of autoimmune diseases. Various studies have suggested that IL-17A can be produced by multiple cell types, including CD4+ T cells, CD8+ T cells, γδ T cells, NKT cells, dendritic cells, macrophages and granulocytes. However, since most studies rely on ex-vivo restimulation to assay IL-17A production, it is unclear which cell types are the predominant sources of IL-17A during the actual course of an immune response in vivo. To address this issue, we generated IL-17A reporter mice in which IL-17A-expressing cells are marked by the presence of a surface marker. We verified the ability of these reporter mice to mark the activation of IL-17A-producing Th17 cells in vitro. We then used these mice to gain a better understanding of the temporal and cell-type specific patterns of IL-17A expression during infections with Citrobacter rodentium and Klebsiella pneumoniae and in the mouse model of experimental autoimmune encephalomyelitis (EAE). In all of these models, we found that a significant portion of γδ T cells produced IL-17A, while smaller fractions of CD4+ T cells and NKT cells produced this cytokine. These mice should prove to be a valuable tool for understanding the in vivo biology of IL-17A.

Epigenetic regulation of transposable element derived human gene promoters

It was previously thought that epigenetic histone modifications of mammalian transposable elements (TEs) serve primarily to defend the genome against deleterious effects associated with their activity. However, we recently showed that, genome-wide, human TEs can also be epigenetically modified in a manner consistent with their ability to regulate host genes. Here, we explore the ability of TE sequences to epigenetically regulate individual human genes by focusing on the histone modifications of promoter sequences derived from TEs. We found 1520 human genes that initiate transcription from within TE-derived promoter sequences. We evaluated the distributions of eight histone modifications across these TE-promoters, within and between the GM12878 and K562 cell lines, and related their modification status with the cell-type specific expression patterns of the genes that they regulate. TE-derived promoters are significantly enriched for active histone modifications, and depleted for repressive modifications, relative to the genomic background. Active histone modifications of TE-promoters peak at transcription start sites and are positively correlated with increasing expression within cell lines. Furthermore, differential modification of TE-derived promoters between cell lines is significantly correlated with differential gene expression. LTR-retrotransposon derived promoters in particular play a prominent role in mediating cell-type specific gene regulation, and a number of these LTR-promoter genes are implicated in lineage-specific cellular functions. The regulation of human genes mediated by histone modifications targeted to TE-derived promoters is consistent with the ability of TEs to contribute to the epigenomic landscape in a way that provides functional utility to the host genome.

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

DNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃(+) groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci. We analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin(+)) and progenitor lineage marker-negative (Lin⁻) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence. We observed that Lin⁻ and Lin(+) cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4(-/-) mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4. Overall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.

HDL3, but not HDL2, stimulates plasminogen activator inhibitor-1 release from adipocytes: the role of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that regulates numerous key cardiovascular functions. High-density lipoproteins (HDLs) are the major plasma lipoprotein carriers of S1P. Fibrinolysis is a physiological process that allows fibrin clot dissolution, and decreased fibrinolytic capacity may result from increased circulating levels of plasminogen activator inhibitor-1 (PAI-1). We examined the effect of S1P associated with HDL subfractions on PAI-1 secretion from 3T3 adipocytes. S1P concentration in HDL3 averaged twice that in HDL2. Incubation of adipocytes with increasing concentrations of S1P in HDL3, but not HDL2, or with S1P complexed to albumin stimulated PAI-I secretion in a concentration-dependent manner. Quantitative RT-PCR revealed that S1P(1-3) are expressed in 3T3 adipocytes, with S1P(2) expressed in the greatest amount. Treatment of adipocytes with the S1P(1) and S1P(3) antagonist VPC23019 did not block PAI-1 secretion. Inhibiting S1P(2) with JTE-013 or reducing the expression of the gene coding for S1P(2) using silencing RNA (siRNA) technology blocked PAI-1 secretion, suggesting that the S1P(2) receptor mediates PAI-1 secretion from adipocytes exposed to HDL3 or S1P. Treatment with the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor RO-318425, or the Rho-associated protein kinase (ROCK) inhibitor Y27632 all significantly inhibited HDL3- and S1P-mediated PAI-1 release, suggesting that HDL3- and/or S1P-stimulated PAI-1 secretion from 3T3 cells is mediated by activation of multiple, downstream signaling pathways of S1P(2).

Gene Expression Programs of Mouse Endothelial Cells in Kidney Development and Disease

Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.

Sumoylation of bZIP Transcription Factor NRL Modulates Target Gene Expression during Photoreceptor Differentiation

Development of rod photoreceptors in the mammalian retina is critically dependent on the basic motif-leucine zipper transcription factor NRL (neural retina leucine zipper). In the absence of NRL, photoreceptor precursors in mouse retina produce only cones that primarily express S-opsin. Conversely, ectopic expression of NRL in post-mitotic precursors leads to a rod-only retina. To explore the role of signaling molecules in modulating NRL function, we identified putative sites of post-translational modification in the NRL protein by in silico analysis. Here, we demonstrate the sumoylation of NRL in vivo and in vitro, with two small ubiquitin-like modifier (SUMO) molecules attached to the Lys-20 residue. NRL-K20R and NRL-K20R/K24R sumoylation mutants show reduced transcriptional activation of Nr2e3 and rhodopsin promoters (two direct targets of NRL) in reporter assays when compared with wild-type NRL. Consistent with this, in vivo electroporation of the NRL-K20R/K24R mutant into newborn Nrl(-/-) mouse retina leads to reduced Nr2e3 activation and only a partial rescue of the Nrl(-/-) phenotype in contrast to the wild-type NRL that is able to convert cones to rod photoreceptors. Although PIAS3 (protein inhibitor of activated STAT3), an E3-SUMO ligase implicated in photoreceptor differentiation, can be immunoprecipitated with NRL, there appears to be redundancy in E3 ligases, and PIAS3 does not seem to be essential for NRL sumoylation. Our studies suggest an important role of sumoylation in fine-tuning the activity of NRL and thereby incorporating yet another layer of control in gene regulatory networks involved in photoreceptor development and homeostasis.

Myeloid DAP12-associating lectin (MDL)-1 regulates synovial inflammation and bone erosion associated with autoimmune arthritis

DNAX adaptor protein 12 (DAP12) is a trans-membrane adaptor molecule that transduces activating signals in NK and myeloid cells. Absence of functional Dap12 results in osteoclast defects and bone abnormalities. Because DAP12 has no extracelluar binding domains, it must pair with cell surface receptors for signal transduction. There are at least 15 known DAP12-associating cell surface receptors with distinct temporal and cell type–specific expression patterns. Our aim was to determine which receptors may be important in DAP12-associated bone pathologies. Here, we identify myeloid DAP12-associating lectin (MDL)-1 receptor (also known as CLEC5A) as a key regulator of synovial injury and bone erosion during autoimmune joint inflammation. Activation of MDL-1 leads to enhanced recruitment of inflammatory macrophages and neutrophils to the joint and promotes bone erosion. Functional blockade of MDL-1 receptor via Mdl1 deletion or treatment with MDL-1-Ig fusion protein reduces the clinical signs of autoimmune joint inflammation. These findings suggest that MDL-1 receptor may be a therapeutic target for treatment of immune-mediated skeletal disorders.

Existence of seven human IL-1R1 promoters

Previous studies have reported the existence of three promoters for the human type I interleukin-1 receptor (hIL-1R1) gene. These promoters were discovered by identifying discrete transcription start sites (TSS) from limited human cell lines. In this study, we examined the TSSs of hIL-1R1 mRNA from 24 different tissues and identified several novel TSSs in hIL-1R1 that suggest the existence of seven hIL-1R1 promoters: three of them are the same as those reported previously and four are putative novel promoters. Using a promoter-reporter assay, we show here that these promoters can drive the transcription of the reporter gene. In addition, these promoters exhibit cell type specific expression patterns and they can be regulated by enhancer elements in a cell type specific manner. Only one of the promoters was found to be sensitive to the stimulation by glucocorticoids. Similar to our recent report on murine IL-1R1, two of the hIL-1R1 promoters appear to be the dominant promoters, one of which was published previously and the other is identified in the present study. We also found an internal promoter that drives the expression of IL-1R1 after the conventional translation start codon, suggesting that a truncated hIL-1R1 may be expressed by this promoter. These results provide additional information regarding the transcription of hIL-1R1.

Selective changes in GABAA receptor subtypes in white matter neurons of patients with focal epilepsy

Mapping the distribution of GABAA receptor subtypes represents a promising approach to characterize alterations in cortical circuitry associated with neurological disorders. We previously reported subtype-selective changes in GABAA receptor expression in the grey matter of patients with focal epilepsy. In the present follow-up study, we focused on the subcortical white matter in the same tissue specimens obtained at surgery from 9 patients with temporal lobe epilepsy (TLE) and hippocampal sclerosis, 12 patients with TLE associated with neocortical lesions and 5 patients with frontal lobe epilepsy; post-mortem tissue from 4 subjects served as controls. The subunit composition and distribution of three major GABAA receptor subtypes were determined immunohistochemically with subunit-specific antibodies. In all cases, a majority of neurons in the white matter was distinctly labelled, allowing detailed visualization of their dendritic arborization and revealing a differential, cell type-specific expression pattern of alpha-subunit variants. In controls, alpha1-subunit staining was most prominent, displaying a gradient that decreased with depth, in parallel with the density of NeuN-positive cells. Subsets of pyramidal cells were alpha3-subunit-positive, and alpha2-subunit-labelled neurons were rare. In 19 of the 26 patients with focal epilepsy, no changes were detected as compared with controls. In five patients with TLE, striking changes in the dendritic arborization of a subset of white matter neurons were seen with the alpha1-subunit antibody. In two further patients with TLE, we observed a disorganized dendritic network immuno-positive for the alpha1-subunit, cell clusters selectively expressing the alpha2-subunit and small neuronal aggregates that expressed all subunits and appeared to connect to neighbouring white matter neurons. All seven patients with anomalies in the white matter had a selective reduction in alpha3-containing GABAA receptors in the superficial layers of the grey matter. These results demonstrate a distinct organization of GABAA receptors in human white matter neurons, consistent with an inhibitory network that is likely to be integrated functionally with the overlying grey matter. The altered dendritic morphology and changes in GABAA receptor expression in the white matter of a subset of patients with focal epilepsy are suggestive for a rewiring of neuronal circuits.

Generation of IL-17A reporter mice to track IL-17A expression in vivo (38.19)

Abstract IL-17A plays a crucial role in host defense against a variety of pathogens and also contributes to the pathogenesis of autoimmune diseases. Various studies have suggested that IL-17A can be produced by a multiple cell types, including CD4+ T cells, CD8+ T cells, γδ T cells, NKT cells, dendritic cells, macrophages and granulocytes. However, since most studies rely on ex-vivo restimulation to assay IL-17A production, it is unclear which cell types are the predominant sources of IL-17A during the actual course of an immune response in vivo. To address this issue, we generated IL-17A reporter mice in which IL-17A-expressing cells are marked by the presence of a surface marker. We verified the ability of these reporter mice to mark the activation of IL-17A-producing Th17 cells in vitro. We then used these mice to gain a better understanding of the temporal and cell-type specific patterns of IL-17A expression in the mouse model of experimental autoimmune encephalomyelitis (EAE). We demonstrated that a small subset of CD4+ T cells and a larger fraction of γδ T cells express IL-17A in the CNS of mice with EAE, and that IL-17A expression levels peak in the CNS before the onset of paralysis. We have thus far been unable to detect IL-17A expression during infection with bacterial or fungal pathogens, which raises questions as to the actual contribution of IL-17A in these models.