Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.

ADNP dysregulates methylation and mitochondrial gene expression in the cerebellum of a Helsmoortel-Van der Aa syndrome autopsy case.

BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation. RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain. CONCLUSION: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.

Two regulatory T cell populations in the visceral adipose tissue shape systemic metabolism.

Visceral adipose tissue (VAT) is an energy store and endocrine organ critical for metabolic homeostasis. Regulatory T (Treg) cells restrain inflammation to preserve VAT homeostasis and glucose tolerance. Here, we show that the VAT harbors two distinct Treg cell populations: prototypical serum stimulation 2-positive (ST2+) Treg cells that are enriched in males and a previously uncharacterized population of C-X-C motif chemokine receptor 3-positive (CXCR3+) Treg cells that are enriched in females. We show that the transcription factors GATA-binding protein 3 and peroxisome proliferator-activated receptor-γ, together with the cytokine interleukin-33, promote the differentiation of ST2+ VAT Treg cells but repress CXCR3+ Treg cells. Conversely, the differentiation of CXCR3+ Treg cells is mediated by the cytokine interferon-γ and the transcription factor T-bet, which also antagonize ST2+ Treg cells. Finally, we demonstrate that ST2+ Treg cells preserve glucose homeostasis, whereas CXCR3+ Treg cells restrain inflammation in lean VAT and prevent glucose intolerance under high-fat diet conditions. Overall, this study defines two molecularly and developmentally distinct VAT Treg cell types with unique context- and sex-specific functions.

Chromatin remodeler Activity-Dependent Neuroprotective Protein (ADNP) contributes to syndromic autism.

BACKGROUND: Individuals affected with autism often suffer additional co-morbidities such as intellectual disability. The genes contributing to autism cluster on a relatively limited number of cellular pathways, including chromatin remodeling. However, limited information is available on how mutations in single genes can result in such pleiotropic clinical features in affected individuals. In this review, we summarize available information on one of the most frequently mutated genes in syndromic autism the Activity-Dependent Neuroprotective Protein (ADNP). RESULTS: Heterozygous and predicted loss-of-function ADNP mutations in individuals inevitably result in the clinical presentation with the Helsmoortel-Van der Aa syndrome, a frequent form of syndromic autism. ADNP, a zinc finger DNA-binding protein has a role in chromatin remodeling: The protein is associated with the pericentromeric protein HP1, the SWI/SNF core complex protein BRG1, and other members of this chromatin remodeling complex and, in murine stem cells, with the chromodomain helicase CHD4 in a ChAHP complex. ADNP has recently been shown to possess R-loop processing activity. In addition, many additional functions, for instance, in association with cytoskeletal proteins have been linked to ADNP. CONCLUSIONS: We here present an integrated evaluation of all current aspects of gene function and evaluate how abnormalities in chromatin remodeling might relate to the pleiotropic clinical presentation in individual"s" with Helsmoortel-Van der Aa syndrome.

Unlocking autofluorescence in the era of full spectrum analysis: Implications for immunophenotype discovery projects.

Understanding the complex elements affecting signal resolution in cytometry is key for quality experimental design and data. In this study, we incorporate autofluorescence as a contributing factor to our understanding of resolution in cytometry and corroborate its impact in fluorescence signal detection through mathematical predictions supported by empirical evidence. Our findings illustrate the critical importance of autofluorescence extraction via full spectrum unmixing in unmasking dim signals and delineating the expression and subset distribution of low abundance markers in discovery projects. We apply our findings to the precise definition of the tissue and cellular distribution of a weakly expressed fluorescent protein that reports on a low-abundance immunological gene. Exploiting the full spectrum coverage enabled by Aurora 5L, we describe a novel approach to the isolation of pure cell subset-specific autofluorescence profiles based on high dimensionality reduction algorithms. This method can also be used to unveil differences in the autofluorescent fingerprints of tissues in homeostasis and after immunological challenges.

Resident and migratory adipose immune cells control systemic metabolism and thermogenesis.

Glucose is a vital source of energy for all mammals. The balance between glucose uptake, metabolism and storage determines the energy status of an individual, and perturbations in this balance can lead to metabolic diseases. The maintenance of organismal glucose metabolism is a complex process that involves multiple tissues, including adipose tissue, which is an endocrine and energy storage organ that is critical for the regulation of systemic metabolism. Adipose tissue consists of an array of different cell types, including specialized adipocytes and stromal and endothelial cells. In addition, adipose tissue harbors a wide range of immune cells that play vital roles in adipose tissue homeostasis and function. These cells contribute to the regulation of systemic metabolism by modulating the inflammatory tone of adipose tissue, which is directly linked to insulin sensitivity and signaling. Furthermore, these cells affect the control of thermogenesis. While lean adipose tissue is rich in type 2 and anti-inflammatory cytokines such as IL-10, obesity tips the balance in favor of a proinflammatory milieu, leading to the development of insulin resistance and the dysregulation of systemic metabolism. Notably, anti-inflammatory immune cells, including regulatory T cells and innate lymphocytes, protect against insulin resistance and have the characteristics of tissue-resident cells, while proinflammatory immune cells are recruited from the circulation to obese adipose tissue. Here, we review the key findings that have shaped our understanding of how immune cells regulate adipose tissue homeostasis to control organismal metabolism.

A thermogenic fat-epithelium cell axis regulates intestinal disease tolerance.

Disease tolerance, the capacity of tissues to withstand damage caused by a stimulus without a decline in host fitness, varies across tissues, environmental conditions, and physiologic states. While disease tolerance is a known strategy of host defense, its role in noninfectious diseases has been understudied. Here, we provide evidence that a thermogenic fat-epithelial cell axis regulates intestinal disease tolerance during experimental colitis. We find that intestinal disease tolerance is a metabolically expensive trait, whose expression is restricted to thermoneutral mice and is not transferable by the microbiota. Instead, disease tolerance is dependent on the adrenergic state of thermogenic adipocytes, which indirectly regulate tolerogenic responses in intestinal epithelial cells. Our work has identified an unexpected mechanism that controls intestinal disease tolerance with implications for colitogenic diseases.

Complications of cervical total disc replacement and their associations with heterotopic ossification: a systematic review and meta-analysis.

PURPOSE: Although cervical total disc replacement (CTDR) is perceived as a safe procedure, no review to date has quantified the complication rates. Of note, heterotopic ossification (HO), one of the complications of CTDR, is hypothesised to cause adjacent segment degeneration (ASDegeneration). This association has not been proven in meta-analysis. Hence, this systematic review and meta-analysis aims to investigate the pooled prevalence of complications following CTDR among studies that concomitantly reported the rate of HO, and the associations between HO and other complications, including ASDegeneration. METHODS: Literatures search was conducted in Embase, MEDLINE, PubMed, and the Cochrane Central Register of Controlled Trials. Complications were stratified into ≥ 1 and < 2 years, ≥ 2 and < 5 years, and ≥ 5 years follow-up. Subgroup and meta-regression analyses were performed. RESULTS: Fifty-three studies were included, composed of 3223 patients in total. The pooled prevalence of post-operative complications following CTDR was low, ranging from 0.8% in vascular adverse events to 4.7% in dysphagia at short-term follow-up. The rate of ASDegeneration was significantly higher at long-term follow-up (pooled prevalence 36.0%, 95% confidence interval [CI] 22.8-49.1%) than that at mid-term follow-up (pooled prevalence 7.3%, 95% CI 2.8-11.8%). Multivariate meta-regression analysis demonstrated that ASDegeneration was independently and inversely correlated with age (p = 0.007) and positively correlated with HO (p = 0.010) at mid-term follow-up. At long-term follow-up, ASDegeneration was still positively correlated with HO (p = 0.011), but not age. Furthermore, dysphagia was inversely associated with HO (p = 0.016), after adjustment for age and length of follow-up. CONCLUSION: In conclusion, HO is associated with ASDegeneration and dysphagia.

Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance.

Host defenses against pathogens are energetically expensive, leading ecological immunologists to postulate that they might participate in energetic trade-offs with other maintenance programs. However, the metabolic costs of immunity and the nature of physiologic trade-offs it engages are largely unknown. We report here that activation of immunity causes an energetic trade-off with the homeothermy (the stable maintenance of core temperature), resulting in hypometabolism and hypothermia. This immunity-induced physiologic trade-off was independent of sickness behaviors but required hematopoietic sensing of lipopolysaccharide (LPS) via the toll-like receptor 4 (TLR4). Metabolomics and genome-wide expression profiling revealed that distinct metabolic programs supported entry and recovery from the energy-conserving hypometabolic state. During bacterial infections, hypometabolic states, which could be elicited by competition for energy between maintenance programs or energy restriction, promoted disease tolerance. Together, our findings suggest that energy-conserving hypometabolic states, such as dormancy, might have evolved as a mechanism of tissue tolerance.

PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia.

Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and ß receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.

Transcription Factor IRF4 Promotes CD8+ T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

During chronic stimulation, CD8+ T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection.

Tissue Immunometabolism: Development, Physiology, and Pathobiology.

Evolution of metazoans resulted in the specialization of cellular and tissue function. This was accomplished by division of labor, which allowed tissue parenchymal cells to prioritize their core functions while ancillary functions were delegated to tissue accessory cells, such as immune, stromal, and endothelial cells. In metabolic organs, the accessory cells communicate with their clients, the tissue parenchymal cells, to optimize cellular processes, allowing organisms to adapt to changes in their environment. Here, we discuss tissue immunometabolism from this vantage point and use examples from adipose tissues (white, beige, and brown) and liver to outline the general principles by which accessory cells support metabolic homeostasis in parenchymal cells. A corollary of this model is that disruption of communication between client and accessory cells might predispose metabolic organs to the development of disease.

Immunity around the clock.

Immunity is a high-cost, high-benefit trait that defends against pathogens and noxious stimuli but whose overactivation can result in immunopathologies and sometimes even death. Because many immune parameters oscillate rhythmically with the time of day, the circadian clock has emerged as an important gatekeeper for reducing immunity-associated costs, which, in turn, enhances organismal fitness. This is mediated by interactions between extrinsic environmental cues and the intrinsic oscillators of immune cells, which together optimize immune responses throughout the circadian cycle. The elucidation of these clock-controlled immunomodulatory mechanisms might uncover new approaches for treating infections and chronic inflammatory diseases.

CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes.

Tissue-resident memory T (Trm) cells permanently localize to portals of pathogen entry, where they provide immediate protection against reinfection. To enforce tissue retention, Trm cells up-regulate CD69 and down-regulate molecules associated with tissue egress; however, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically up-regulated in Trm cells and, together with related Blimp1, mediates the development of Trm cells in skin, gut, liver, and kidney in mice. The Hobit-Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes, including natural killer T (NKT) cells and liver-resident NK cells, all of which share a common transcriptional program. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations.

The role of endoscopy in the management of recurrent pyogenic cholangitis: a review.

Recurrent pyogenic cholangitis (RPC) is a clinical syndrome characterized by repeated episodes of suppurative cholangitis due to hepatolithiasis and extrahepatic stones in the biliary ducts. It is now recognized as a distinct syndrome with a different natural history and pathoetiology than spontaneously occurring liver abscesses. Most commonly seen in East Asian populations, this syndrome is growing increasingly common in Western Nations due to migration patterns. The exact pathogenesis of RPC remains elusive; although colonization of the biliary tract with particular enteric bacterial species, in combination with possible dietary factors, has been attributed as causative factors. Hepatobiliary surgery, in particular segmental hepatectomy, is often described as the definitive treatment of choice for RPC. The exact role of endoscopic intervention has been less clearly described in the literature. This review focuses on the management of RPC while highlighting situations in which endoscopic retrograde cholangiopancreatography may be preferred over surgery as an initial or salvage therapeutic measure.

Synchronizing transcriptional control of T cell metabolism and function.

During an immune response, cytokines and transcription factors regulate the differentiation and function of effector and memory T cells. At the same time, T cell metabolism undergoes dynamic and differentiation-stage-specific changes that are required for initial T cell activation, rapid proliferation and the acquisition of effector functions. Similarly, during the resolution of an immune response, metabolic regulation is crucial for restraining inflammatory responses and promoting peripheral tolerance, and it is required for the long-term maintenance of memory T cells. T cell receptor (TCR)-induced transcription factors, in particular MYC and interferon-regulatory factor 4 (IRF4), cooperate with canonical nutrient-sensing pathways to integrate antigen-specific and metabolic signals to appropriately modulate adaptive immune responses. In this Review, we focus on the emerging evidence that T cell differentiation and metabolism are closely linked and synchronized by immune cell-specific cytokines and transcription factors that are induced by TCR signalling.

The interplay between the intestinal microbiota and the immune system.

The relationship between commensal microbes and their hosts has been studied for many years. Commensal microorganisms are known to have a significant role in regulating the physiology of their hosts and preventing pathogenic infections while the hosts' immune system is important in determining the composition of the microbiota. More recently, specific effects of the intestinal microbiota on the local and distal immune systems have been uncovered with important consequences for health and disease, and alterations in intestinal microbial composition has been associated with various disease states. Here, we will review the current understanding of the microbiota/immune system crosstalk, highlight the clinical consequences of changes in the microbiota and consider how to harness this symbiotic relationship to improve public health.