ABSTRACT
BACKGROUND: Clinical interpretation of genetic variants in the context of the patient's phenotype is becoming the largest component of cost and time expenditure for genome-based diagnosis of rare genetic diseases. Artificial intelligence (AI) holds promise to greatly simplify and speed genome interpretation by integrating predictive methods with the growing knowledge of genetic disease. Here we assess the diagnostic performance of Fabric GEM, a new, AI-based, clinical decision support tool for expediting genome interpretation. METHODS: We benchmarked GEM in a retrospective cohort of 119 probands, mostly NICU infants, diagnosed with rare genetic diseases, who received whole-genome or whole-exome sequencing (WGS, WES). We replicated our analyses in a separate cohort of 60 cases collected from five academic medical centers. For comparison, we also analyzed these cases with current state-of-the-art variant prioritization tools. Included in the comparisons were trio, duo, and singleton cases. Variants underpinning diagnoses spanned diverse modes of inheritance and types, including structural variants (SVs). Patient phenotypes were extracted from clinical notes by two means: manually and using an automated clinical natural language processing (CNLP) tool. Finally, 14 previously unsolved cases were reanalyzed. RESULTS: GEM ranked over 90% of the causal genes among the top or second candidate and prioritized for review a median of 3 candidate genes per case, using either manually curated or CNLP-derived phenotype descriptions. Ranking of trios and duos was unchanged when analyzed as singletons. In 17 of 20 cases with diagnostic SVs, GEM identified the causal SVs as the top candidate and in 19/20 within the top five, irrespective of whether SV calls were provided or inferred ab initio by GEM using its own internal SV detection algorithm. GEM showed similar performance in absence of parental genotypes. Analysis of 14 previously unsolved cases resulted in a novel finding for one case, candidates ultimately not advanced upon manual review for 3 cases, and no new findings for 10 cases. CONCLUSIONS: GEM enabled diagnostic interpretation inclusive of all variant types through automated nomination of a very short list of candidate genes and disorders for final review and reporting. In combination with deep phenotyping by CNLP, GEM enables substantial automation of genetic disease diagnosis, potentially decreasing cost and expediting case review.
Subject(s)
Artificial Intelligence , Rare Diseases/genetics , Databases, Genetic , Female , Genomics/methods , Genotype , Humans , Male , Phenotype , Retrospective Studies , Exome SequencingABSTRACT
Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, -1306, -345, -132, and -26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future.
ABSTRACT
Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and ß cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and ß cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (ß cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing ß cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and ß cells predicts highly functional, mature subpopulations.
Subject(s)
Glucagon-Secreting Cells/metabolism , Insulin-Secreting Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Adult , Electrophysiological Phenomena , Gene Expression , Glucagon-Secreting Cells/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Insulin/metabolism , Insulin-Secreting Cells/physiology , Maf Transcription Factors, Large/genetics , Maf Transcription Factors, Large/metabolism , MafB Transcription Factor/genetics , MafB Transcription Factor/metabolism , Middle Aged , Sequence Analysis, RNA , Single-Cell Analysis , Transcriptome , Young AdultABSTRACT
Endogenous ß cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that ß cell recovery following hypervascularization-induced ß cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during ß cell loss, and recruited MΦs are essential for ß cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during ß cell recovery and leads to increased ß cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of ß cells, ECs, and MΦs reveals that ß cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in ß cells. Collectively, these findings suggest a new ß cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate ß cell self-renewal.
ABSTRACT
Bipolar disorder (BD) is a serious mental illness with substantial common variant heritability. However, the role of rare coding variation in BD is not well established. We examined the protein-coding (exonic) sequences of 3,987 unrelated individuals with BD and 5,322 controls of predominantly European ancestry across four cohorts from the Bipolar Sequencing Consortium (BSC). We assessed the burden of rare, protein-altering, single nucleotide variants classified as pathogenic or likely pathogenic (P-LP) both exome-wide and within several groups of genes with phenotypic or biologic plausibility in BD. While we observed an increased burden of rare coding P-LP variants within 165 genes identified as BD GWAS regions in 3,987 BD cases (meta-analysis OR = 1.9, 95% CI = 1.3-2.8, one-sided p = 6.0 × 10-4), this enrichment did not replicate in an additional 9,929 BD cases and 14,018 controls (OR = 0.9, one-side p = 0.70). Although BD shares common variant heritability with schizophrenia, in the BSC sample we did not observe a significant enrichment of P-LP variants in SCZ GWAS genes, in two classes of neuronal synaptic genes (RBFOX2 and FMRP) associated with SCZ or in loss-of-function intolerant genes. In this study, the largest analysis of exonic variation in BD, individuals with BD do not carry a replicable enrichment of rare P-LP variants across the exome or in any of several groups of genes with biologic plausibility. Moreover, despite a strong shared susceptibility between BD and SCZ through common genetic variation, we do not observe an association between BD risk and rare P-LP coding variants in genes known to modulate risk for SCZ.
Subject(s)
Bipolar Disorder , Schizophrenia , Bipolar Disorder/genetics , Exome/genetics , Genetic Predisposition to Disease/genetics , Genetic Variation/genetics , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide/genetics , Schizophrenia/geneticsABSTRACT
Novel treatments for Huntington's disease (HD), a progressive neurodegenerative disorder, include selective targeting of the mutant allele of the huntingtin gene (mHTT) carrying the abnormally expanded disease-causing cytosine-adenine-guanine (CAG) repeat. WVE-120101 and WVE-120102 are investigational stereopure antisense oligonucleotides that enable selective suppression of mHTT by targeting single-nucleotide polymorphisms (SNPs) that are in haplotype phase with the CAG repeat expansion. Recently developed long-read sequencing technologies can capture CAG expansions and distant SNPs of interest and potentially facilitate haplotype-based identification of patients for clinical trials of oligonucleotide therapies. However, improved methods are needed to phase SNPs with CAG repeat expansions directly and reliably without need for familial genotype/haplotype data. Our haplotype phasing method uses single-molecule real-time sequencing and a custom algorithm to determine with confidence bases at SNPs on mutant alleles, even without familial data. Herein, we summarize this methodology and validate the approach using patient-derived samples with known phasing results. Comparison of experimentally measured CAG repeat lengths, heterozygosity, and phasing with previously determined results showed improved performance. Our methodology enables the haplotype phasing of SNPs of interest and the disease-causing, expanded CAG repeat of the huntingtin gene, enabling accurate identification of patients with HD eligible for allele-selective clinical studies.
ABSTRACT
The human sperm is one of the smallest cells in the body, but also one of the most important, as it serves as the entire paternal genetic contribution to a child. Investigating RNA and mutations in sperm is especially relevant for diseases such as autism spectrum disorders (ASD), which have been correlated with advanced paternal age. Historically, studies have focused on the assessment of bulk sperm, wherein millions of individual sperm are present and only high-frequency variants can be detected. Using 10× Chromium single-cell sequencing technology, we assessed the transcriptome from >65,000 single spermatozoa across six sperm donors (scSperm-RNA-seq), including two who fathered multiple children with ASD and four fathers of neurotypical children. Using RNA-seq methods for differential expression and variant analysis, we found clusters of sperm mutations in each donor that are indicative of the sperm being produced by different stem cell pools. Finally, we have shown that genetic variations can be found in single sperm.
ABSTRACT
Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of ß cell mass. Interestingly, these ß cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced ß cell dysfunction and partially prevented SIR-induced ß cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human ß cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.
Subject(s)
Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Sirolimus/pharmacology , Tacrolimus/pharmacology , Animals , Calcineurin/metabolism , Diabetes Mellitus , Graft Rejection , Humans , Immunosuppressive Agents/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans Transplantation , Male , Mice , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/drug effectsABSTRACT
BACKGROUND: Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood. METHODS: Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types. RESULTS: AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs. CONCLUSIONS: Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches.
Subject(s)
Immunity, Innate , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Squalene/immunology , alpha-Tocopherol/immunology , Adaptive Immunity , Adjuvants, Immunologic/therapeutic use , Adult , Double-Blind Method , Drug Combinations , Gene Expression Profiling , Humans , Influenza, Human/immunology , Influenza, Human/virology , Leukocytes/immunology , Polysorbates , Signal Transduction , Young AdultABSTRACT
Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained ß cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/metabolism , Genetic Variation , Hepatocyte Nuclear Factor 1-alpha , Insulin-Secreting Cells/metabolism , Transcription, Genetic , Adolescent , Adult , Diabetes Mellitus, Type 1/pathology , Hepatocyte Nuclear Factor 1-alpha/biosynthesis , Hepatocyte Nuclear Factor 1-alpha/genetics , Heterozygote , Humans , Insulin-Secreting Cells/pathology , MaleABSTRACT
Identification of cell-surface markers specific to human pancreatic ß cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human ß cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet ß cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human ß cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human ß cells. Thus, NTPDase3 is a cell-surface biomarker of adult human ß cells, and the antibody directed to this protein should be a useful new reagent for ß cell sorting, in vivo imaging, and targeting.
Subject(s)
Adenosine Triphosphatases/metabolism , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 2/metabolism , Insulin-Secreting Cells/metabolism , Islets of Langerhans/metabolism , Pancreas/metabolism , Adult , Animals , Biomarkers/metabolism , Cells, Cultured , Humans , Insulin-Secreting Cells/pathology , Islets of Langerhans/pathology , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred NOD , Pancreas/pathology , Young AdultABSTRACT
More than a decade ago, the term "next-generation" sequencing was coined to describe what was, at the time, revolutionary new methods to sequence RNA and DNA at a faster pace and cheaper cost than could be performed by standard bench-top protocols. Since then, the field of DNA sequencing has evolved at a rapid pace, with new breakthroughs allowing capacity to exponentially increase and cost to dramatically decrease. As genome-scale sequencing has become routine, a paradigm shift is occurring in genomics, which uses the power of high-throughput, rapid sequencing power with large-scale studies. These new approaches to genetic discovery will provide direct impact to fields such as personalized medicine, evolution, and biodiversity. This work reviews recent technology advances and methods in next-generation sequencing and highlights current large-scale sequencing efforts driving the evolution of the genomics space.
Subject(s)
Genome, Human , Genomics/trends , High-Throughput Nucleotide Sequencing/trends , High-Throughput Nucleotide Sequencing/methods , Humans , Sequence Analysis, DNA/methodsABSTRACT
Deficiency in diacylglycerol acyltransferase (DGAT1) is a rare cause of neonatal diarrhea, without a known mechanism or in vitro model. A patient presenting at our institution at 7 weeks of life with failure to thrive and diarrhea was found by whole-exome sequencing to have a homozygous DGAT1 truncation mutation. Duodenal biopsies showed loss of DGAT1 and deficits in apical membrane transporters and junctional proteins in enterocytes. When placed on a very low-fat diet, the patient's diarrhea resolved with normalization of brush border transporter localization in endoscopic biopsies. DGAT1 knockdown in Caco2-BBe cells modeled the deficits in apical trafficking, with loss of apical DPPIV and junctional occludin. Elevation in cellular lipid levels, including diacylglycerol (DAG) and phospholipid metabolites of DAG, was documented by lipid analysis in DGAT1 knockdown cells. Culture of the DGAT1 knockdown cells in lipid-depleted media led to re-establishment of occludin and return of apical DPPIV. DGAT1 loss appears to elicit global changes in enterocyte polarized trafficking that could account for deficits in absorption seen in the patient. The in vitro modeling of this disease should allow for investigation of possible therapeutic targets.
Subject(s)
Diacylglycerol O-Acyltransferase/genetics , Diarrhea, Infantile/genetics , Digestive System Diseases/genetics , Caco-2 Cells , Child, Preschool , Diacylglycerol O-Acyltransferase/deficiency , Diacylglycerol O-Acyltransferase/metabolism , Diarrhea, Infantile/pathology , Digestive System Diseases/pathology , Humans , Infant , Intestinal Absorption , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Protein TransportABSTRACT
It is estimated that â¼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant-expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.
Subject(s)
Cell Lineage , Embryonic Stem Cells/metabolism , Genes, X-Linked , Genetic Markers , Intellectual Disability/genetics , Mutation, Missense , N-Acetylglucosaminyltransferases/genetics , Cell Differentiation , Child , Crystallography, X-Ray , Embryonic Stem Cells/pathology , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Humans , Infant, Newborn , Intellectual Disability/enzymology , Intellectual Disability/pathology , Male , N-Acetylglucosaminyltransferases/chemistry , N-Acetylglucosaminyltransferases/metabolism , Pedigree , Protein Conformation , Signal TransductionABSTRACT
PURPOSE: Clinically relevant secondary variants were identified in parents enrolled with a child with developmental delay and intellectual disability. METHODS: Exome/genome sequencing and analysis of 789 "unaffected" parents was performed. RESULTS: Pathogenic/likely pathogenic variants were identified in 21 genes within 25 individuals (3.2%), with 11 (1.4%) participants harboring variation in a gene defined as clinically actionable by the American College of Medical Genetics and Genomics. These 25 individuals self-reported either relevant clinical diagnoses (5); relevant family history or symptoms (13); or no relevant family history, symptoms, or clinical diagnoses (7). A limited carrier screen was performed yielding 15 variants in 48 (6.1%) parents. Parents were also analyzed as mate pairs (n = 365) to identify cases in which both parents were carriers for the same recessive disease, yielding three such cases (0.8%), two of which had children with the relevant recessive disease. Four participants had two findings (one carrier and one noncarrier variant). In total, 71 of the 789 enrolled parents (9.0%) received secondary findings. CONCLUSION: We provide an overview of the rates and types of clinically relevant secondary findings, which may be useful in the design and implementation of research and clinical sequencing efforts to identify such findings.
Subject(s)
Exome Sequencing , Exome/genetics , Genetic Diseases, Inborn/genetics , Genetic Testing , Adult , Chromosome Mapping , Female , Genetic Carrier Screening , Genetic Diseases, Inborn/classification , Genetic Diseases, Inborn/physiopathology , Genetic Variation , Genome, Human/genetics , Humans , Male , Middle Aged , Mutation , Parents , Whole Genome SequencingABSTRACT
Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.
ABSTRACT
Many patients with type 1 diabetes (T1D) have residual ß cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual ß cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant ß cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and ß cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-ß cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Gene Expression Regulation , Glucagon-Secreting Cells/metabolism , Adolescent , Adult , Animals , Case-Control Studies , Cellular Reprogramming , Child , Female , Glucagon/metabolism , Glucagon-Secreting Cells/pathology , Humans , Insulin Secretion , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Male , Mice , Middle Aged , Phenotype , Tissue Donors , Transcription Factors/metabolism , Young AdultABSTRACT
OBJECTIVE: Alternatively activated macrophages (M2) are associated with the progression of spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach. However, the precise mechanism(s) and critical mediators that induce SPEM are unknown. DESIGN: To determine candidate genes important in these processes, macrophages from the stomach corpus of mice with SPEM (DMP-777-treated) or advanced SPEM (L635-treated) were isolated and RNA sequenced. Effects on metaplasia development after acute parietal cell loss induced by L635 were evaluated in interleukin (IL)-33, IL-33 receptor (ST2) and IL-13 knockout (KO) mice. RESULTS: Profiling of metaplasia-associated macrophages in the stomach identified an M2a-polarised macrophage population. Expression of IL-33 was significantly upregulated in macrophages associated with advanced SPEM. L635 induced metaplasia in the stomachs of wild-type mice, but not in the stomachs of IL-33 and ST2 KO mice. While IL-5 and IL-9 were not required for metaplasia induction, IL-13 KO mice did not develop metaplasia in response to L635. Administration of IL-13 to ST2 KO mice re-established the induction of metaplasia following acute parietal cell loss. CONCLUSIONS: Metaplasia induction and macrophage polarisation after parietal cell loss is coordinated through a cytokine signalling network of IL-33 and IL-13, linking a combined response to injury by both intrinsic mucosal mechanisms and infiltrating M2 macrophages.
Subject(s)
Interleukin-13/metabolism , Interleukin-33/metabolism , Macrophages/metabolism , Metaplasia/metabolism , Stomach/cytology , Animals , Flow Cytometry , Gastric Mucosa/metabolism , Immunohistochemistry , Intercellular Signaling Peptides and Proteins , Interleukin-1 Receptor-Like 1 Protein , Interleukin-13/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Parietal Cells, Gastric/cytology , Peptides/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Interleukin/genetics , Signal TransductionABSTRACT
Inadequate pancreatic ß cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human ß cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of ß cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human ß cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human ß cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet ß cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human ß cell proliferation, and identify elements that could be adapted for therapeutic expansion of human ß cells.