RESUMO
Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS)-known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Acute infection led to marked imbalance in the CNS CD4/CD8 ratio which persisted into the chronic phase. These observations underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our findings offer insights for potential T cell-focused interventions while underscoring the challenges in eradicating HIV from the CNS, particularly in the context of sub-optimal ART.
Assuntos
Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Humanos , Linfócitos T CD4-Positivos , Vírus da Imunodeficiência Símia/fisiologia , Macaca mulatta , Sistema Nervoso Central , Carga ViralRESUMO
Mutations in the Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. Here we report that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. We demonstrate that depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, we found two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, we report different modes of action of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, our research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs.
Assuntos
Proteínas de Drosophila , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , MicroRNAs , Ribonuclease III , Animais , Esclerose Lateral Amiotrófica/metabolismo , Drosophila/genética , Drosophila/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Doenças Neurodegenerativas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Ribonuclease III/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.
Assuntos
Encefalite Viral , Neurônios , Infecções por Reoviridae , Reoviridae , Animais , Camundongos , Apoptose/genética , Apoptose/imunologia , Quimiocinas/imunologia , Encefalite Viral/imunologia , Encefalite Viral/virologia , Neurônios/imunologia , NF-kappa B/genética , NF-kappa B/metabolismo , Reoviridae/imunologia , Reoviridae/patogenicidade , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologiaRESUMO
Cell-based quadrivalent inactivated influenza vaccine has been shown to have higher vaccine effectiveness than traditional egg-based quadrivalent inactivated influenza vaccine. This is observed despite similar levels of serum hemagglutinin antibodies induced by each vaccine. Here, we examine peripheral immune activation following egg-based or cell-based influenza vaccination in a clinical trial in children. Peripheral blood mononuclear cells were isolated and RNA sequenced from 81 study participants (41 Fluzone, egg-based and 40 Flucelvax, cell based) pre- and 7 days post- vaccination. Seroconversion was assessed by hemagglutinin inhibition assay. Differential gene expression was determined and pathway analysis was conducted. Cell-based influenza vaccine induced greater interferon stimulated and innate immune gene activation compared with egg-based influenza vaccine. Participants who seroconverted had increased interferon signaling activation versus those who did not seroconvert. These data suggest that cell-based influenza vaccine stimulates immune activation differently from egg-based influenza vaccine, shedding light on reported differences in vaccine effectiveness.
RESUMO
BACKGROUND & AIMS: Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. METHODS: We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. RESULTS: A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. CONCLUSIONS: BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes. IMPACT AND IMPLICATIONS: Liver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects.
Assuntos
Atresia Biliar , Criança , Animais , Camundongos , Humanos , Atresia Biliar/genética , Estudo de Associação Genômica Ampla , Predisposição Genética para Doença , Peixe-Zebra/genética , CanadáRESUMO
GEMIN5 is essential for core assembly of small nuclear Ribonucleoproteins (snRNPs), the building blocks of spliceosome formation. Loss-of-function mutations in GEMIN5 lead to a neurodevelopmental syndrome among patients presenting with developmental delay, motor dysfunction, and cerebellar atrophy by perturbing SMN complex protein expression and assembly. Currently, molecular determinants of GEMIN5-mediated disease have yet to be explored. Here, we identified SMN as a genetic suppressor of GEMIN5-mediated neurodegeneration in vivo. We discovered that an increase in SMN expression by either SMN gene therapy replacement or the antisense oligonucleotide (ASO), Nusinersen, significantly upregulated the endogenous levels of GEMIN5 in mammalian cells and mutant GEMIN5-derived iPSC neurons. Further, we identified a strong functional association between the expression patterns of SMN and GEMIN5 in patient Spinal Muscular Atrophy (SMA)-derived motor neurons harboring loss-of-function mutations in the SMN gene. Interestingly, SMN binds to the C-terminus of GEMIN5 and requires the Tudor domain for GEMIN5 binding and expression regulation. Finally, we show that SMN upregulation ameliorates defective snRNP biogenesis and alternative splicing defects caused by loss of GEMIN5 in iPSC neurons and in vivo. Collectively, these studies indicate that SMN acts as a regulator of GEMIN5 expression and neuropathologies.
Assuntos
Atrofia Muscular Espinal , Proteínas de Ligação a RNA , Humanos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/genética , Domínio TudorRESUMO
Flooding and drought are serious constraints that reduce crop productivity worldwide. Previous studies identified genes conferring tolerance to both water extremes in various plants. However, overlapping responses to flooding and drought at the genome-scale remain obscure. Here, we defined overlapping and stress-specific transcriptomic and hormonal responses to submergence, drought and recovery from these stresses in soybean (Glycine max). We performed comparative RNA-sequencing and hormone profiling, identifying genes, hormones and biological processes that are differentially regulated in an overlapping or stress-specific manner. Overlapping responses included positive regulation of trehalose and sucrose metabolism and negative regulation of cellulose, tubulin, photosystem II and I, and chlorophyll biosynthesis, facilitating the economization of energy reserves under both submergence and drought. Additional energy-consuming pathways were restricted in a stress-specific manner. Downregulation of distinct pathways for energy saving under each stress suggests energy-consuming processes that are relatively unnecessary for each stress adaptation are turned down. Our newly developed transcriptomic-response analysis revealed that abscisic acid and ethylene responses were activated in common under both stresses, whereas stimulated auxin response was submergence-specific. The energy-saving strategy is the key overlapping mechanism that underpins adaptation to both submergence and drought in soybean. Abscisic acid and ethylene are candidate hormones that coordinate transcriptomic energy-saving processes under both stresses. Auxin may be a signaling component that distinguishes submergence-specific regulation of the stress response.
Assuntos
Regulação da Expressão Gênica de Plantas , Glycine max/fisiologia , Reguladores de Crescimento de Plantas/genética , Estresse Fisiológico/fisiologia , Ácido Abscísico/metabolismo , Secas , Etilenos/metabolismo , Inundações , Ontologia Genética , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glycine max/genéticaRESUMO
Inherited errors of mitochondrial fatty acid ß-oxidation (FAO) are life threatening, even with optimum care. FAO is the major source of energy for heart and is critical for skeletal muscles especially during physiologic stress. Clinical trials revealed that triheptanoin (commercially known as Dojolvi; C7G), improved heart function and decreased hypoglycemia in long chain FAO disorders, but other symptoms including rhabdomyolysis persisted, suggesting suboptimal tissue distribution/utilization of heptanoic acid (C7) conjugates and/or rapid liver breakdown. In this study, medium branched chain fatty acids were tested as potential anaplerotic treatments in fibroblasts from patients deficient in very long chain acyl-CoA dehydrogenase (VLCAD), long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), trifunctional protein (TFP), and carnitine palmitoyltransferase II (CPT II). Cells were cultured to near confluency and treated with C7, 2,6-dimethylheptanoic acid (dMC7), 6-amino-2,4-dimethylheptanoic acid (AdMC7), or 4,8-dimethylnonanoic acid (dMC9) for 72 h and targeted metabolomics performed. The profile of TCA cycle intermediates was improved in cells treated with these branched chain fatty acids compared with C7. Intracellular propionate was higher in AdMC7 treated cells compared with C7 in VLCAD, LCHAD, and TFP deficient cell lines. With AdMC7 treatment, succinate was higher in CPT II and VLCAD deficient cells, compared with C7. Malate and glutamate were consistently higher in AdMC7 treated VLCAD, LCHAD, TFP, and CPT II deficient cells compared with the C7 treatment. The results provide the impetus to further evaluate and consider branched chain fatty acids as viable anaplerotic therapy for fatty acid oxidation disorders and other diseases.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa , Erros Inatos do Metabolismo Lipídico , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Ciclo do Ácido Cítrico , Ácidos Graxos/metabolismo , Humanos , Erros Inatos do Metabolismo Lipídico/metabolismo , OxirreduçãoRESUMO
Acute myocardial infarction (MI) is one of the leading causes of death worldwide. Early identification of ischemia and establishing reperfusion remain cornerstones in the treatment of MI, as mortality and morbidity can be significantly reduced by establishing reperfusion to the affected areas. The aim of the current study was to investigate the metabolomic changes in the serum in a swine model of MI induced by ischemia and reperfusion (I/R) injury, and to identify circulating metabolomic biomarkers for myocardial injury at different phases. Female Yucatan minipigs were subjected to 60 min of ischemia followed by reperfusion, and serum samples were collected at baseline, 60 min of ischemia, 4 h of reperfusion, and 24 h of reperfusion. Circulating metabolites were analyzed using an untargeted metabolomic approach. A bioinformatic approach revealed that serum metabolites show distinct profiles during ischemia and during early and late reperfusion. Some notable changes during ischemia include accumulation of metabolites that indicate impaired mitochondrial function and N-terminally modified amino acids. Changes in branched-chain amino-acid metabolites were noted during early reperfusion, while bile acid pathway derivatives and intermediates predominated in the late reperfusion phases. This indicates a potential for such an approach toward identification of the distinct phases of ischemia and reperfusion in clinical situations.
Assuntos
Doença da Artéria Coronariana , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Doença da Artéria Coronariana/complicações , Feminino , Isquemia/complicações , Metabolômica , Isquemia Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Reperfusão/efeitos adversos , Suínos , Porco MiniaturaRESUMO
Mutations in the RNA binding protein, Fused in Sarcoma (FUS), lead to amyotrophic lateral sclerosis (ALS), the most frequent form of motor neuron disease. Cytoplasmic aggregation and defective DNA repair machinery are etiologically linked to mutant FUS-associated ALS. Although FUS is involved in numerous aspects of RNA processing, little is understood about the pathophysiological mechanisms of mutant FUS. Here, we employed RNA-sequencing technology in Drosophila brains expressing FUS to identify significantly altered genes and pathways involved in FUS-mediated neurodegeneration. We observed the expression levels of DEAD-Box Helicase 17 (DDX17) to be significantly downregulated in response to mutant FUS in Drosophila and human cell lines. Mutant FUS recruits nuclear DDX17 into cytoplasmic stress granules and physically interacts with DDX17 through the RGG1 domain of FUS. Ectopic expression of DDX17 reduces cytoplasmic mislocalization and sequestration of mutant FUS into cytoplasmic stress granules. We identified DDX17 as a novel regulator of the DNA damage response pathway whose upregulation repairs defective DNA damage repair machinery caused by mutant neuronal FUS ALS. In addition, we show DDX17 is a novel modifier of FUS-mediated neurodegeneration in vivo. Our findings indicate DDX17 is downregulated in response to mutant FUS, and restoration of DDX17 levels suppresses FUS-mediated neuropathogenesis and toxicity in vivo.
Assuntos
Esclerose Lateral Amiotrófica/genética , RNA Helicases DEAD-box/genética , Reparo do DNA/genética , Proteína FUS de Ligação a RNA/toxicidade , Animais , Linhagem Celular , Grânulos Citoplasmáticos/química , Dano ao DNA , Drosophila , Feminino , Humanos , Masculino , Doenças Neurodegenerativas/genética , Análise de Sequência de RNARESUMO
BACKGROUND AND AIMS: A key challenge in biology is to systematically investigate and integrate the different levels of information available at the global and single-cell level. Recent studies have elucidated spatiotemporal expression patterns of root cell types in Arabidopsis thaliana, and genome-wide quantification of polysome-associated mRNA levels, i.e. the translatome, has also been obtained for corresponding cell types. Translational control has been increasingly recognized as an important regulatory step in protein synthesis. The aim of this study was to investigate coupled transcription and translation by use of publicly available root datasets. METHODS: Using cell-type-specific datasets of the root transcriptome and translatome of arabidopsis, a systematic assessment was made of the degree of co-ordination and divergence between these two levels of cellular organization. The computational analysis considered correlation and variation of expression across cell types at both system levels, and also provided insights into the degree of co-regulatory relationships that are preserved between the two processes. KEY RESULTS: The overall correlation of expression and translation levels of genes resemble an almost bimodal distribution (mean/median value of 0·08/0·12), with a second, less strongly pronounced 'mode' for negative Pearson's correlation coefficient values. The analysis conducted also confirms that previously identified key transcriptional activators of secondary cell wall development display highly conserved patterns of transcription and translation across the investigated cell types. Moreover, the biological processes that display conserved and divergent patterns based on the cell-type-specific expression and translation levels were identified. CONCLUSIONS: In agreement with previous studies in animal cells, a large degree of uncoupling was found between the transcriptome and translatome. However, components and processes were also identified that are under co-ordinated transcriptional and translational control in plant root cells.
Assuntos
Arabidopsis/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Proteoma , Transcriptoma , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biologia Computacional , Especificidade de Órgãos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genéticaRESUMO
Neuronal cells are highly specialized cells and have a specific metabolic profile to support their function. It has been demonstrated that the metabolic profiles of different cells/tissues undergo significant reprogramming with advancing age, which has often been considered a contributing factor towards aging-related diseases including Alzheimer's (AD) and Parkinson's (PD) diseases. However, it is unclear if the metabolic changes associated with normal aging predispose neurons to disease conditions or a distinct set of metabolic alterations happen in neurons in AD or PD which might contribute to disease pathologies. To decipher the changes in neuronal metabolism with age, in AD, or in PD, we performed high-throughput steady-state metabolite profiling on heads in wildtype Drosophila and in Drosophila models relevant to AD and PD. Intriguingly, we found that the spectrum of affected metabolic pathways is dramatically different between normal aging, Tau, or Synuclein overexpressing neurons. Genetic targeting of the purine and glutamate metabolism pathways, which were dysregulated in both old age and disease conditions partially rescued the neurodegenerative phenotype associated with the overexpression of wildtype and mutant tau. Our findings support a "two-hit model" to explain the pathological manifestations associated with AD where both aging- and Tau/Synuclein- driven metabolic reprogramming events cooperate with each other, and targeting both could be a potent therapeutic strategy.
RESUMO
OBJECTIVES: Despite surgical resection, chemoradiation and targeted therapy, brain tumors remain a leading cause of cancer related death in children. Immunotherapy has shown some promise and is actively being investigated for treating childhood brain tumors. However, a critical step in advancing immunotherapy for these patients is to uncover targets that can be effectively translated into therapeutic interventions. METHODS: In this study, our team performed a transcriptomic analysis across pediatric brain tumor types to identify potential targets for immunotherapy. Additionally, we assessed components that may impact patient response to immunotherapy, including the expression of genes essential for antigen processing and presentation, inhibitory ligands and receptors, interferon signature, and overall predicted T cell infiltration. RESULTS: We observed distinct expression patterns across tumor types. These included elevated expression of antigen genes and antigen processing machinery in some tumor types while other tumors had elevated inhibitory checkpoint receptors, known to be associated with response to checkpoint inhibitor immunotherapy. CONCLUSION: These findings suggest that pediatric brain tumors exhibit distinct potential for specific immunotherapies. We believe our findings can guide investigators in their assessment of appropriate immunotherapy classes and targets in pediatric brain tumors.
RESUMO
Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.
Assuntos
Células Acinares , Diabetes Mellitus Experimental , Células Secretoras de Insulina , Animais , Células Secretoras de Insulina/metabolismo , Camundongos , Células Acinares/metabolismo , Masculino , Insulina/metabolismo , Transdiferenciação Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacologia , Ilhotas Pancreáticas/metabolismoRESUMO
CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Insights into Tfh subsets that promote antibody persistence and functional capacity can critically inform vaccine design. Based on the Tfh profiles evoked by the live attenuated measles virus vaccine, renowned for its ability to establish durable humoral immunity, we investigated the potential of a Tfh1/17 recall response during the boost phase to enhance persistence of HIV-1 Envelope (Env) antibodies in rhesus macaques. Using a DNA-prime encoding gp160 antigen and Tfh polarizing cytokines (interferon protein-10 (IP-10) and interleukin-6 (IL-6)), followed by a gp140 protein boost formulated in a cationic liposome-based adjuvant (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells. In contrast, a similar DNA-prime (including IP-10) followed by gp140 formulated with monophosphoryl lipid A (MPLA) +QS-21 adjuvant predominantly induced GC Tfh1 cells. While the generation of GC Tfh1/17 cells with CAF01 and GC Tfh1 cells with MPLA +QS-21 induced comparable peak Env antibodies, the latter group demonstrated significantly greater antibody concentrations at week 8 after final immunization which persisted up to 30 weeks (gp140 IgG ng/ml- MPLA; 5500; CAF01, 2155; p<0.05). Notably, interferon γ+Env-specific Tfh responses were consistently higher with gp140 in MPLA +QS-21 and positively correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction promote persistent Env antibodies, important for protective immunity against HIV.
Assuntos
Vacinas contra a AIDS , HIV-1 , Animais , Macaca mulatta , Quimiocina CXCL10 , Anticorpos Anti-HIV , DNARESUMO
Maternal secretor status is one of the determinants of human milk oligosaccharides (HMOs) composition, which, in turn, influences the gut microbiota composition of infants. To understand if this change in gut microbiota impacts immune cell composition, intestinal morphology, and gene expression, 21-day-old germ-free C57BL/6 mice were transplanted with fecal microbiota from infants whose mothers were either secretors (SMM) or non-secretors (NSM) or from infants consuming dairy-based formula (MFM). For each group, one set of mice was supplemented with HMOs. HMO supplementation did not significantly impact the microbiota diversity; however, SMM mice had a higher abundance of genus Bacteroides, Bifidobacterium, and Blautia, whereas, in the NSM group, there was a higher abundance of Akkermansia, Enterocloster, and Klebsiella. In MFM, gut microbiota was represented mainly by Parabacteroides, Ruminococcaceae_unclassified, and Clostrodium_sensu_stricto. In mesenteric lymph node, Foxp3+ T cells and innate lymphoid cells type 2 were increased in MFM mice supplemented with HMOs, while in the spleen, they were increased in SMM + HMOs mice. Similarly, serum immunoglobulin A was also elevated in MFM + HMOs group. Distinct global gene expression of the gut was observed in each microbiota group, which was enhanced with HMOs supplementation. Overall, our data show that distinct infant gut microbiota due to maternal secretor status or consumption of dairy-based formula and HMO supplementation impacts immune cell composition, antibody response, and intestinal gene expression in a mouse model. IMPORTANCE: Early life factors like neonatal diet modulate gut microbiota, which is important for the optimal gut and immune function. One such factor, human milk oligosaccharides (HMOs), the composition of which is determined by maternal secretor status, has a profound effect on infant gut microbiota. However, how the infant gut microbiota composition determined by maternal secretor status or consumption of infant formula devoid of HMOs impacts infant intestinal ammorphology, gene expression, and immune signature is not well explored. This study provides insights into the differential establishment of infant microbiota derived from infants fed by secretor or non-secretor mothers milk or those consuming infant formula and demonstrates that the secretor status of mothers promotes Bifidobacteria and Bacteroides sps. establishment. This study also shows that supplementation of pooled HMOs in mice changed immune cell composition in the spleen and mesenteric lymph nodes and immunoglobulins in circulation. Hence, this study highlights that maternal secretor status has a role in infant gut microbiota composition, and this, in turn, can impact host gut and immune system.
Assuntos
Imunidade Inata , Microbiota , Lactente , Feminino , Humanos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos/metabolismo , Leite Humano/química , Sistema Imunitário/metabolismo , Oligossacarídeos/análise , Bifidobacterium/genéticaRESUMO
Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure.
Assuntos
Galectina 3 , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Neutrófilos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Galectina 3/metabolismo , Galectina 3/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Humanos , Camundongos , Infecções por Pseudomonas/imunologia , Masculino , Feminino , Insuficiência Respiratória/metabolismo , Camundongos Knockout , Fagocitose , Imunidade Inata , Galectinas/metabolismo , Galectinas/genéticaRESUMO
CD4+ T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-Seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4+ T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4+ T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4+ T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL757 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4+ T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4+ T cells in CNS immune surveillance, and their decline during chronic SIV highlights their responsiveness to neuroinflammation.
Assuntos
Encéfalo , Linfócitos T CD4-Positivos , Macaca mulatta , Receptores CCR7 , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T CD4-Positivos/imunologia , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores CCR7/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Encéfalo/patologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/patologia , Vigilância ImunológicaRESUMO
Traumatic brain injury (TBI) heterogeneity remains a critical barrier to translating therapies. Identifying final common pathways/molecular signatures that integrate this heterogeneity informs biomarker and therapeutic-target development. We present the first large-scale murine single-cell atlas of the transcriptomic response to TBI (334,376 cells) across clinically relevant models, sex, brain region, and time as a foundational step in molecularly deconstructing TBI heterogeneity. Results were unique to cell populations, injury models, sex, brain regions, and time, highlighting the importance of cell-level resolution. We identify cell-specific targets and previously unrecognized roles for microglial and ependymal subtypes. Ependymal-4 was a hub of neuroinflammatory signaling. A distinct microglial lineage shared features with disease-associated microglia at 24 h, with persistent gene-expression changes in microglia-4 even 6 months after contusional TBI, contrasting all other cell types that mostly returned to naive levels. Regional and sexual dimorphism were noted. CEREBRI, our searchable atlas (https://shiny.crc.pitt.edu/cerebri/), identifies previously unrecognized cell subtypes/molecular targets and is a leverageable platform for future efforts in TBI and other diseases with overlapping pathophysiology.
RESUMO
Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.