Retinoic acid receptor α as a novel contributor to adrenal cortex structure and function through interactions with Wnt and Vegfa signalling.

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.

Temporal evolution of anti-Clostridium antibody responses in sheep after vaccination with polyvalent clostridial vaccines.

Polyvalent clostridial vaccines, composed of a complex mixture of toxoids from up to 9 different species, are highly effective in controlling clostridial diseases in cattle and sheep. Commercially available vaccines usually state that in normal field conditions two doses administered 4 to 6 weeks apart elicit protective antibody levels that will last for one year. However, studies on the development and duration of the antibody response against the different Clostridium species in target animals are scarce and only partial. Evaluating the temporal evolution of the antibody responses upon vaccination in target species is relevant to understand the bases of protective immunity induced by these vaccines and to develop new optimized vaccines. Here, we assessed the antibody response in sheep against each Clostridium component of two different 9-valent Clostridial vaccines over the period of one year. One vaccine was a commercially available vaccine and the other was an experimental vaccine prepared by us with the same antigens that we used to set up a specific ELISA for each Clostridium species. Both vaccines showed similar results, irrespectively of the origin of the antigens used for the ELISAs, with antibody titers that peaked at day 36 after vaccination and large inter individual variations in the magnitude of the response. Antibody titers were maintained up to 90 days and then markedly decreased, becoming even undetectable in some animals 6 months after vaccination. Given that the current scheme of yearly revaccination has largely shown to be effective at controlling the burden of disease, our results strongly suggest that circulating antibody levels cannot completely explain the protective immunity elicited by these vaccines, and prompt for further studies into the correlates of protection of clostridial vaccines.

The siRNA-mediated knockdown of GluN3A in 46C-derived neural stem cells affects mRNA expression levels of neural genes, including known iGluR interactors.

For years, GluN3A was solely considered to be a dominant-negative modulator of NMDARs, since its incorporation into receptors alters hallmark features of conventional NMDARs composed of GluN1/GluN2 subunits. Only recently, increasing evidence has accumulated that GluN3A plays a more diversified role. It is considered to be critically involved in the maturation of glutamatergic synapses, and it might act as a molecular brake to prevent premature synaptic strengthening. Its expression pattern supports a putative role during neural development, since GluN3A is predominantly expressed in early pre- and postnatal stages. In this study, we used RNA interference to efficiently knock down GluN3A in 46C-derived neural stem cells (NSCs) both at the mRNA and at the protein level. Global gene expression profiling upon GluN3A knockdown revealed significantly altered expression of a multitude of neural genes, including genes encoding small GTPases, retinal proteins, and cytoskeletal proteins, some of which have been previously shown to interact with GluN3A or other iGluR subunits. Canonical pathway enrichment studies point at important roles of GluN3A affecting key cellular pathways involved in cell growth, proliferation, motility, and survival, such as the mTOR pathway. This study for the first time provides insights into transcriptome changes upon the specific knockdown of an NMDAR subunit in NSCs, which may help to identify additional functions and downstream pathways of GluN3A and GluN3A-containing NMDARs.

BIM and NOXA are mitochondrial effectors of TAF6δ-driven apoptosis.

TAF6δ is a pro-apoptotic splice variant of the RNA polymerase II general transcription factor, TAF6, that can dictate life vs. death decisions in animal cells. TAF6δ stands out from classical pro-apoptotic proteins because it is encoded by a gene that is essential at the cellular level, and because it functions as a component of the basal transcription machinery. TAF6δ has been shown to modulate the transcriptome landscape, but it is not known if changes in gene expression trigger apoptosis nor which TAF6δ-regulated genes contribute to cell death. Here we used microarrays to interrogate the genome-wide impact of TAF6δ on transcriptome dynamics at temporal resolution. The results revealed changes in pro-apoptotic BH3-only mitochondrial genes that correlate tightly with the onset of cell death. These results prompted us to test and validate a role for the mitochondrial pathway by showing that TAF6δ expression causes cytochrome c release into the cytoplasm. To further dissect the mechanism by which TAF6δ drives apoptosis, we pinpointed BIM and NOXA as candidate effectors. siRNA experiments showed that both BIM and NOXA contribute to TAF6δ-dependent cell death. Our results identify mitochondrial effectors of TAF6δ-driven apoptosis, thereby providing the first of mechanistic framework underlying the atypical TAF6δ apoptotic pathway's capacity to intersect with the classically defined apoptotic machinery to trigger cell death.

Latent HIV-1 TAR Regulates 7SK-responsive P-TEFb Target Genes and Targets Cellular Immune Responses in the Absence of Tat.

The transactivating response element (TAR) structure of the nascent HIV-1 transcript is critically involved in the recruitment of inactive positive transcription elongation factor b (P-TEFb) to the promoter proximal paused RNA polymerase II. The viral transactivator Tat is responsible for subsequent P-TEFb activation in order to start efficient viral transcription elongation. In the absence of the viral transactivator of transcription (Tat), e.g., during latency or in early stages of HIV transcription, TAR mediates an interaction of P-TEFb with its inhibitor hexamethylene bis-acetamide-inducible protein 1 (HEXIM1), keeping P-TEFb in its inactive form. In this study, we address the function of HIV-1 TAR in the absence of Tat by analyzing consequences of HIV-1 TAR overexpression on host cellular gene expression. An RNA chimera consisting of Epstein-Barr virus-expressed RNA 2 (EBER2) and HIV-1 TAR was developed to assure robust overexpression of TAR in HEK293 cells. The overexpression results in differential expression of more than 800 human genes. A significant proportion of these genes is involved in the suppression of cellular immune responses, including a significant set of 7SK-responsive P-TEFb target genes. Our findings identify a novel role for HIV-1 TAR in the absence of Tat, involving the interference with host cellular immune responses by targeting 7SK RNA-mediated gene expression and P-TEFb inactivation.

HIC1 controls cellular- and HIV-1- gene transcription via interactions with CTIP2 and HMGA1.

Among many cellular transcriptional regulators, Bcl11b/CTIP2 and HGMA1 have been described to control the establishment and the persistence of HIV-1 latency in microglial cells, the main viral reservoir in the brain. In this present work, we identify and characterize a transcription factor i.e. HIC1, which physically interacts with both Bcl11b/CTIP2 and HMGA1 to co-regulate specific subsets of cellular genes and the viral HIV-1 gene. Our results suggest that HIC1 represses Tat dependent HIV-1 transcription. Interestingly, this repression of Tat function is linked to HIC1 K314 acetylation status and to SIRT1 deacetylase activity. Finally, we show that HIC1 interacts and cooperates with HGMA1 to regulate Tat dependent HIV-1 transcription. Our results also suggest that HIC1 repression of Tat function happens in a TAR dependent manner and that this TAR element may serve as HIC1 reservoir at the viral promoter to facilitate HIC1/TAT interaction.

RNA-Mediated Regulation of HMGA1 Function.

The high mobility group protein A1 (HMGA1) is a master regulator of chromatin structure mediating its major gene regulatory activity by direct interactions with A/T-rich DNA sequences located in the promoter and enhancer regions of a large variety of genes. HMGA1 DNA-binding through three AT-hook motifs results in an open chromatin structure and subsequently leads to changes in gene expression. Apart from its significant expression during development, HMGA1 is over-expressed in virtually every cancer, where HMGA1 expression levels correlate with tumor malignancy. The exogenous overexpression of HMGA1 can lead to malignant cell transformation, assigning the protein a key role during cancerogenesis. Recent studies have unveiled highly specific competitive interactions of HMGA1 with cellular and viral RNAs also through an AT-hook domain of the protein, significantly impacting the HMGA1-dependent gene expression. In this review, we discuss the structure and function of HMGA1-RNA complexes during transcription and epigenomic regulation and their implications in HMGA1-related diseases.

Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract.

The Toll-like receptor 5 (TLR5) agonist flagellin is an effective adjuvant for vaccination. Recently, we demonstrated that the adaptive responses stimulated by intranasal administration of flagellin and antigen were linked to TLR5 signaling in the lung epithelium. The present study sought to identify the antigen presenting cells involved in this adjuvant activity. We first found that the lung dendritic cells captured antigen very efficiently in a process independent of TLR5. However, TLR5-mediated signaling specifically enhanced the maturation of lung dendritic cells. Afterward, the number of antigen-bound and activated conventional dendritic cells (both CD11b(+) and CD103(+)) increased in the mediastinal lymph nodes in contrast to monocyte-derived dendritic cells. These data suggested that flagellin-activated lung conventional dendritic cells migrate to the draining lymph nodes. The lymph node dendritic cells, in particular CD11b(+) cells, were essential for induction of CD4 T-cell response. Lastly, neutrophils and monocytes were recruited into the lungs by flagellin administration but did not contribute to the adjuvant activity. The functional activation of conventional dendritic cells was independent of direct TLR5 signaling, thereby supporting the contribution of maturation signals produced by flagellin-stimulated airway epithelium. In conclusion, our results demonstrated that indirect TLR5-dependent stimulation of airway conventional dendritic cells is essential to flagellin's mucosal adjuvant activity.

Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas.

CONTEXT: Adrenal mast cells can stimulate aldosterone secretion through the local release of serotonin (5-HT) and activation of the 5-HT4 receptor (5-HT4). In aldosterone-producing adenomas (APAs), 5-HT4 receptor is overexpressed and the administration of 5-HT4 receptor agonists to patients with APA increases plasma aldosterone levels. These data and the well-documented role of mast cells in tumorigenesis suggest that mast cells may be involved in the pathophysiology of APA. OBJECTIVE: The study aimed at investigating the occurrence of mast cells in a series of APA tissues and to examine the influence of mast cells on aldosterone secretion. DESIGN: The occurrence of mast cells in APAs was investigated by immunohistochemistry. Mast cell densities were compared with clinical data. The influence of mast cells on aldosterone production was studied by using cultures of human mast cell and adrenocortical cell lines. RESULTS: In APA tissues, the density of mast cells was found to be increased in comparison with normal adrenals. Mast cells were primarily observed in adrenal cortex adjacent to adenomas or in the adenomas themselves, distinguishing two groups of APAs. A subset of adenomas was found to contain a high density of intratumoral mast cells, which was correlated with aldosterone synthase expression and in vivo aldosterone secretory parameters. Administration of conditioned medium from cultures of human mast cell lines to human adrenocortical cells induced a significant increase in aldosterone synthase (CYP11B2) mRNA expression and aldosterone production. CONCLUSION: APA tissues commonly contain numerous mast cells that may influence aldosterone secretion through the local release of regulatory factors.

Alternative splicing of TAF6: downstream transcriptome impacts and upstream RNA splice control elements.

The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ.

MicroRNAs establish robustness and adaptability of a critical gene network to regulate progenitor fate decisions during cortical neurogenesis.

Over the course of cortical neurogenesis, the transition of progenitors from proliferation to differentiation requires a precise regulation of involved gene networks under varying environmental conditions. In order to identify such regulatory mechanisms, we analyzed microRNA (miRNA) target networks in progenitors during early and late stages of neurogenesis. We found that cyclin D1 is a network hub whose expression is miRNA-dosage sensitive. Experimental validation revealed a feedback regulation between cyclin D1 and its regulating miRNAs miR-20a, miR-20b, and miR-23a. Cyclin D1 induces expression of miR-20a and miR-20b, whereas it represses miR-23a. Inhibition of any of these miRNAs increases the developmental stage-specific mean and dynamic expression range (variance) of cyclin D1 protein in progenitors, leading to reduced neuronal differentiation. Thus, miRNAs establish robustness and stage-specific adaptability to a critical dosage-sensitive gene network during cortical neurogenesis. Understanding such network regulatory mechanisms for key developmental events can provide insights into individual susceptibilities for genetically complex neuropsychiatric disorders.

HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters.

Active positive transcription elongation factor b (P-TEFb) is essential for cellular and human immunodeficiency virus type 1 (HIV-1) transcription elongation. CTIP2 represses P-TEFb activity in a complex containing 7SK RNA and HEXIM1. Recently, the inactive 7SK/P-TEFb small nuclear RNP (snRNP) has been detected at the HIV-1 core promoter as well as at the promoters of cellular genes, but a recruiting mechanism still remains unknown to date. Here we show global synergy between CTIP2 and the 7SK-binding chromatin master-regulator HMGA1 in terms of P-TEFb-dependent endogenous and HIV-1 gene expression regulation. While CTIP2 and HMGA1 concordingly repress the expression of cellular 7SK-dependent P-TEFb targets, the simultaneous knock-down of CTIP2 and HMGA1 also results in a boost in Tat-dependent and independent HIV-1 promoter activity. Chromatin immunoprecipitation experiments reveal a significant loss of CTIP2/7SK/P-TEFb snRNP recruitment to cellular gene promoters and the HIV-1 promoter on HMGA1 knock-down. Our findings not only provide insights into a recruiting mechanism for the inactive 7SK/P-TEFb snRNP, but may also contribute to a better understanding of viral latency.

Activation of Type 3 innate lymphoid cells and interleukin 22 secretion in the lungs during Streptococcus pneumoniae infection.

Mucosal sites are continuously exposed to pathogenic microorganisms and are therefore equipped to control respiratory infections. Type 3 innate lymphoid cells (ILC3) are key players in antimicrobial defense in intestinal mucosa, through interleukin 17 and interleukin 22 (IL-22) production. The present study aimed at analyzing the distribution and function of ILC3 in the respiratory tract. We first observed that lung mucosa harbors a discrete population of ILC3 expressing CD127, CD90, CCR6, and the transcriptional factor RORγt. In addition, lung ILC3 were identified as a major source of IL-22 in response to interleukin 23 stimulation. During Streptococcus pneumoniae infection, ILC3 rapidly accumulated in the lung tissue to produce IL-22. In response to S. pneumoniae, dendritic cells and MyD88, an important adaptor of innate immunity, play critical functions in IL-22 production by ILC3. Finally, administration of the Toll-like receptor 5 agonist flagellin during S. pneumoniae challenge exacerbated IL-22 production by ILC3, a process that protects against lethal infection. In conclusion, boosting lung ILC3 might represent an interesting strategy to fight respiratory bacterial infections.

Diastrophic dysplasia sulfate transporter (SLC26A2) is expressed in the adrenal cortex and regulates aldosterone secretion.

Elucidation of the molecular mechanisms leading to autonomous aldosterone secretion is a prerequisite to define potential targets and biomarkers in the context of primary aldosteronism. After a genome-wide association study with subjects from the population-based Cooperative Health Research in the Region of Augsburg F4 survey, we observed a highly significant association (P=6.78×10(-11)) between the aldosterone to renin ratio and a locus at 5q32. Hypothesizing that this locus may contain genes of relevance for the pathogenesis of primary aldosteronism, we investigated solute carrier family 26 member 2 (SLC26A2), a protein with known transport activity for sulfate and other cations. Within murine tissues, adrenal glands showed the highest expression levels for SLC26A2, which was significantly downregulated on in vivo stimulation with angiotensin II and potassium. SLC26A2 expression was found to be significantly lower in aldosterone-producing adenomas in comparison with normal adrenal glands. In adrenocortical NCI-H295R cells, specific knockdown of SLC26A2 resulted in a highly significant increase in aldosterone secretion. Concomitantly, expression of steroidogenic enzymes, as well as upstream effectors including transcription factors such as NR4A1, CAMK1, and intracellular Ca(2+) content, was upregulated in knockdown cells. To substantiate further these findings in an SLC26A2 mutant mouse model, aldosterone output proved to be increased in a sex-specific manner. In summary, these findings point toward a possible effect of SLC26A2 in the regulation of aldosterone secretion potentially involved in the pathogenesis of primary aldosteronism.

A TDG/CBP/RARα ternary complex mediates the retinoic acid-dependent expression of DNA methylation-sensitive genes.

The thymine DNA glycosylase (TDG) is a multifunctional enzyme, which is essential for embryonic development. It mediates the base excision repair (BER) of G:T and G:U DNA mismatches arising from the deamination of 5-methyl cytosine (5-MeC) and cytosine, respectively. Recent studies have pointed at a role of TDG during the active demethylation of 5-MeC within CpG islands. TDG interacts with the histone acetylase CREB-binding protein (CBP) to activate CBP-dependent transcription. In addition, TDG also interacts with the retinoic acid receptor α (RARα), resulting in the activation of RARα target genes. Here we provide evidence for the existence of a functional ternary complex containing TDG, CBP and activated RARα. Using global transcriptome profiling, we uncover a coupling of de novo methylation-sensitive and RA-dependent transcription, which coincides with a significant subset of CBP target genes. The introduction of a point mutation in TDG, which neither affects overall protein structure nor BER activity, leads to a significant loss in ternary complex stability, resulting in the deregulation of RA targets involved in cellular networks associated with DNA replication, recombination and repair. We thus demonstrate for the first time a direct coupling of TDG's epigenomic and transcription regulatory function through ternary complexes with CBP and RARα.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques.

In 2012, a novel betacoronavirus, designated Middle East respiratory syndrome coronavirus or MERS-CoV and associated with severe respiratory disease in humans, emerged in the Arabian Peninsula. To date, 108 human cases have been reported, including cases of human-to-human transmission. The availability of an animal disease model is essential for understanding pathogenesis and developing effective countermeasures. Upon a combination of intratracheal, ocular, oral, and intranasal inoculation with 7 × 10(6) 50% tissue culture infectious dose of the MERS-CoV isolate HCoV-EMC/2012, rhesus macaques developed a transient lower respiratory tract infection. Clinical signs, virus shedding, virus replication in respiratory tissues, gene expression, and cytokine and chemokine profiles peaked early in infection and decreased over time. MERS-CoV caused a multifocal, mild to marked interstitial pneumonia, with virus replication occurring mainly in alveolar pneumocytes. This tropism of MERS-CoV for the lower respiratory tract may explain the severity of the disease observed in humans and the, up to now, limited human-to-human transmission.

Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques.

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) is of global concern: the virus has caused severe respiratory illness, with 111 confirmed cases and 52 deaths at the time of this article's publication. Therapeutic interventions have not been evaluated in vivo; thus, patient management relies exclusively on supportive care, which, given the high case-fatality rate, is not highly effective. The rhesus macaque is the only known model organism for MERS-CoV infection, developing an acute localized to widespread pneumonia with transient clinical disease that recapitulates mild to moderate human MERS-CoV cases. The combination of interferon-α2b and ribavirin was effective in reducing MERS-CoV replication in vitro; therefore, we initiated this treatment 8 h after inoculation of rhesus macaques. In contrast to untreated, infected macaques, treated animals did not develop breathing abnormalities and showed no or very mild radiographic evidence of pneumonia. Moreover, treated animals showed lower levels of systemic (serum) and local (lung) proinflammatory markers, in addition to fewer viral genome copies, distinct gene expression and less severe histopathological changes in the lungs. Taken together, these data suggest that treatment of MERS-CoV infected rhesus macaques with IFN-α2b and ribavirin reduces virus replication, moderates the host response and improves clinical outcome. As these two drugs are already used in combination in the clinic for other infections, IFN-α2b and ribavirin should be considered for the management of MERS-CoV cases.

A network integration approach to predict conserved regulators related to pathogenicity of influenza and SARS-CoV respiratory viruses.

Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel "crowd-based" approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse 'omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models.

Multiparameter single-cell profiling of human CD4+FOXP3+ regulatory T-cell populations in homeostatic conditions and during graft-versus-host disease.

Understanding the heterogeneity of human CD4+FOXP3+ regulatory T cells (Tregs) and their potential for lineage reprogramming is of critical importance for moving Treg therapy into the clinics. Using multiparameter single-cell analysis techniques, we explored the heterogeneity and functional diversity of human Tregs in healthy donors and in patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). Human Tregs displayed a level of complexity similar to conventional CD4+ effector T cells with respect to the expression of transcription factors, homing receptors and inflammatory cytokines. Single-cell profiling of the rare Treg producing interleukin-17A or interferon-γ showed an overlap of gene expression signatures of Th17 or Th1 cells and of Tregs. To assess whether Treg homeostasis is affected by an inflammatory and lymphopenic environment, we characterized the Treg compartment in patients early after alloHSCT. This analysis suggested a marked depletion of Treg with a naive phenotype in patients developing acute graft-versus-host disease, compared with tolerant patients. However, single-cell profiling showed that CD4+FOXP3+ T cells maintain the Treg gene expression signature and Treg-suppressive activity was preserved. Our study establishes that heterogeneity at the single-cell level, rather than lineage reprogramming of CD4+FOXP3+ T cells, explains the remarkable complexity and functional diversity of human Tregs.