RESUMO
SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.
Assuntos
Lesão Pulmonar Aguda , COVID-19 , Animais , Criança , Humanos , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , COVID-19/complicações , Células Endoteliais/metabolismo , Glucocorticoides , Lipopolissacarídeos/efeitos adversos , Camundongos Endogâmicos C57BL , SARS-CoV-2/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like , Fator de Crescimento Transformador beta , Viremia/complicações , Envelope Viral/metabolismoRESUMO
Hyperoxia disrupts lung development in mice and causes bronchopulmonary dysplasia (BPD) in neonates. To investigate sex-dependent molecular and cellular programming involved in hyperoxia, we surveyed the mouse lung using single cell RNA sequencing (scRNA-seq), and validated our findings in human neonatal lung cells in vitro. Hyperoxia-induced inflammation in alveolar type (AT) 2 cells gave rise to damage-associated transient progenitors (DATPs). It also induced a new subpopulation of AT1 cells with reduced expression of growth factors normally secreted by AT1 cells, but increased mitochondrial gene expression. Female alveolar epithelial cells had less EMT and pulmonary fibrosis signaling in hyperoxia. In the endothelium, expansion of Car4+ EC (Cap2) was seen in hyperoxia along with an emergent subpopulation of Cap2 with repressed VEGF signaling. This regenerative response was increased in females exposed to hyperoxia. Mesenchymal cells had inflammatory signatures in hyperoxia, with a new distal interstitial fibroblast subcluster characterized by repressed lipid biosynthesis and a transcriptomic signature resembling myofibroblasts. Hyperoxia-induced gene expression signatures in human neonatal fibroblasts and alveolar epithelial cells in vitro resembled mouse scRNA-seq data. These findings suggest that neonatal exposure to hyperoxia programs distinct sex-specific stem cell progenitor and cellular reparative responses that underpin lung remodeling in BPD.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Recém-Nascido , Masculino , Feminino , Animais , Camundongos , Humanos , Displasia Broncopulmonar/metabolismo , Transcriptoma/genética , Hiperóxia/metabolismo , Animais Recém-Nascidos , Pulmão/metabolismo , Modelos Animais de DoençasRESUMO
Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.
Assuntos
Enterocolite Necrosante , Recém-Nascido , Animais , Camundongos , Humanos , Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/prevenção & controle , Enterocolite Necrosante/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Recém-Nascido Prematuro , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Ácidos Graxos Voláteis/farmacologia , Ácidos Graxos Voláteis/metabolismo , Butiratos/metabolismo , Imunoglobulinas/metabolismo , Interleucina-1/metabolismo , Receptor Notch1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
BACKGROUND & AIMS: Single immunoglobulin interleukin-1-related receptor (SIGIRR) is a major inhibitor of Toll-like receptor signaling. Our laboratory identified a novel SIGIRR stop mutation (p.Y168X) in an infant who died of severe necrotizing enterocolitis (NEC). Herein, we investigated the mechanisms by which SIGIRR mutations induce Toll-like receptor hyper-responsiveness in the neonatal gut, disrupting postnatal intestinal adaptation. METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate transgenic mice encoding the SIGIRR p.Y168X mutation. Ileal lysates, mouse intestinal epithelial cell (IEC) lysates, and intestinal sections were used to assess inflammation, signal transducer and activator of transcription 3 (STAT3) phosphorylation, microRNA (miRNA), and interleukin-1-related-associated kinase 1 (IRAK1) expression. Western blot, quantitative reverse-transcription polymerase chain reaction(qRT-PCR), and luciferase assays were performed to investigate SIGIRR-STAT3 signaling in human intestinal epithelial cells (HIEC) expressing wild-type or SIGIRR (p.Y168X) plasmids. RESULTS: SigirrTg mice showed increased intestinal inflammation and nuclear factor-κB activation concomitant with decreased IEC expression of miR-146a and miR-155. Mechanistic studies in HIECs showed that although SIGIRR induced STAT3-mediated expression of miR-146a and miR-155, the p.Y168X mutation disrupted SIGIRR-mediated STAT3-dependent miRNA expression. Chromatin immunoprecipitation and luciferase assays showed that SIGIRR activation of STAT3-induced miRNA expression is dependent on IRAK1. Both in HIECs and in the mouse intestine, decreased expression of miR-146a observed with the p.Y168X mutation increased expression of IRAK1, a protein whose down-regulation is important for postnatal gut adaptation. CONCLUSIONS: Our results uncover a novel pathway (SIGIRR-STAT3-miRNA-IRAK1 repression) by which SIGIRR regulates postnatal intestine adaptation, which is disrupted by a SIGIRR mutation identified in human NEC. These data provide new insights into how human genetic mutations in SIGIRR identified in NEC result in loss of postnatal intestinal immune tolerance.
Assuntos
Enterocolite Necrosante , MicroRNAs , Animais , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Camundongos , MicroRNAs/genética , Mutação/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
The intestine is extremely dynamic and the epithelial cells that line the intestine get replaced every 3-5 days by highly proliferative intestinal stem cells (ISCs). The instructions for ISCs to self-renew or to differentiate come as cues from their surrounding microenvironment or their niche. A small number of evolutionarily conserved signaling pathways act as a critical regulator of the stem cells in the adult intestine, and these pathways are well characterized. However, the mechanisms, nutritional, and environmental signals that help establish the stem cell niche in the neonatal intestine are less studied. Deciphering the key signaling pathways that regulate the development and maintenance of the stem cells is particularly important to understanding how the intestine regenerates from necrotizing enterocolitis, a devastating disease in newborn infants characterized by inflammation, tissues necrosis, and stem cell injury. In this review, we piece together current knowledge on morphogenetic and immune pathways that regulate intestinal stem cell in neonates and highlight how the cross talk among these pathways affect tissue regeneration. We further discuss how these key pathways are perturbed in NEC and review the scientific knowledge relating to options for stem cell therapy in NEC gleaned from pre-clinical experimental models of NEC.
Assuntos
Enterocolite Necrosante/genética , Células-Tronco Mesenquimais/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , HumanosRESUMO
Although immunohistochemistry of tissue sections has been the gold standard for analyzing tissue structure and cellular localization, this approach has significant shortcomings when it comes to analyzing complex and heterogeneous tissues such as the bone marrow with rare cells like hematopoietic stem cells (HSCs). Hence, studying rare cells and their relationship with the surrounding heterogenous microenvironment requires visualization of specifically labeled cells within large intact tissues in three dimensions. Here, we describe a whole mount sternal bone marrow imaging method which has enabled detailed quantitative and qualitative analysis of rare HSCs within the sternal tissue. The methodology is broadly applicable for examining the 3D architecture of niche cells in relation to HSCs.
Assuntos
Medula Óssea , Diagnóstico por Imagem , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Animais , Feminino , Masculino , CamundongosRESUMO
Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. ß-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of ß-catenin in restraining germline and somatic lineage differentiation genes. We show that ß-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help ß-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of ß-catenin in preserving lineage differentiation integrity in ground state ESCs.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , beta Catenina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação para Baixo/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.
Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cleistanthus collinus is a poisonous shrub used for deliberate self-harm in rural areas of South India and intake of boiled decoction of leaves is a common method of self-harm. Distal renal tubular acidosis (dRTA) is an important clinical symptom observed in C. collinus poisoning, and renal V-ATPases may be potential targets of damage. However, a lack of understanding of molecular mediators involved hampers medical management, which is mainly supportive. We hypothesized that C. collinus poisoning induces renal oxidative stress; probably by inducing mitochondrial uncoupling, which compromises V-ATPase activity to ultimately produce dRTA. This was tested by exposing renal BBMV, kidney cells in culture, and Wistar rats to C. collinus poisoning. Exposure to C. collinus aqueous extract resulted in significant elevations in the lipid peroxidation marker, conjugated dienes, in cell culture and in vivo. A significant decrease in mitochondrial respiratory control ratio was observed in kidneys from C. collinus-treated animals suggesting that mitochondrial oxidative phosphorylation is uncoupled. This was accompanied by significant increase in ADP levels and a decrease in proton pump activity. Thus, these results demonstrate that C. collinus poisoning induces oxidative stress which influences proton pump activity, probably due to feedback inhibition by elevated ADP levels because of mitochondrial dysfunction in the rat kidney.
Assuntos
Acidose Tubular Renal/induzido quimicamente , Euphorbiaceae/intoxicação , Rim/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Acidose Tubular Renal/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Mitocôndrias Musculares/metabolismo , Fosforilação Oxidativa , Extratos Vegetais/intoxicação , Ratos WistarRESUMO
Regulation of hematopoietic stem cells (HSCs) by bone marrow (BM) niches has been extensively studied; however, whether and how HSC subpopulations are distinctively regulated by BM niches remain unclear. Here, we functionally distinguished reserve HSCs (rHSCs) from primed HSCs (pHSCs) based on their response to chemotherapy and examined how they are dichotomously regulated by BM niches. Both pHSCs and rHSCs supported long-term hematopoiesis in homeostasis; however, pHSCs were sensitive but rHSCs were resistant to chemotherapy. Surviving rHSCs restored the HSC pool and supported hematopoietic regeneration after chemotherapy. The rHSCs were preferentially maintained in the endosteal region that enriches N-cadherin+ (N-cad+) bone-lining cells in homeostasis and post-chemotherapy. N-cad+ cells were functional bone and marrow stromal progenitor cells (BMSPCs), giving rise to osteoblasts, adipocytes, and chondrocytes in vitro and in vivo. Finally, ablation of N-cad+ niche cells or deletion of SCF from N-cad+ niche cells impaired rHSC maintenance during homeostasis and regeneration.
Assuntos
Caderinas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco/metabolismo , Células Estromais/metabolismo , HumanosRESUMO
Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.
Assuntos
Epigênese Genética/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Proteínas de Homeodomínio/antagonistas & inibidores , Retinoides/farmacologia , Animais , Elementos Facilitadores Genéticos/efeitos dos fármacos , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Células HEK293 , Hematopoese/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retinoides/químicaRESUMO
BACKGROUND: Non-cirrhotic intrahepatic portal hypertension (NCIPH) is characterized by thrombotic microangiopathy of the portal venous system, low ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13), and high vWF (von Willebrand factor) levels. This study aimed to screen for ADAMTS13 mutations, focusing on the CUB domain, in these patients. METHODS: Prospectively recruited NCIPH patients and healthy volunteers underwent tests for plasma vWF-ADAMTS13 balance. Sanger sequencing of the CUB domain of ADAMTS13 was done in a subset of the NCIPH patients, and the detected mutation was screened for in all the study participants. Next-generation sequencing of clinically relevant exome and liver immunostaining for ADAMTS13 was done in patients with detected ADAMTS13 mutation. RESULTS: Plasma vWF-ADAMTS13 balance was significantly altered in 24 NCIPH patients (Child's class A:23, B:1) as compared to 22 controls. On initial sequencing of the CUB domain (17 cases and 3 controls), one NCIPH patient showed a rare missense variant (SNV) at position c.3829C >T resulting in p.R1277W (rs14045669). Subsequent RFLP analysis targeted to the R1277W variant did not detect this in any other NCIPH patient, nor in any of the 22 controls. The NCIPH patient with the R1277W variant had severe ADAMTS13 deficiency, consistently high vWF, other missense SNVs in ADAMTS13, vWF, and complement genes. Immunostaining of his liver biopsy revealed globules of ADAMTS13 within stellate cells. CONCLUSIONS: We report missense variants in ADAMTS13, vWF, and complement genes in a patient with NCIPH who had decreased secretion and activity of ADAMTS13 protein. Further studies are needed in NCIPH patients in this regard.
Assuntos
Proteína ADAMTS13/genética , Proteína ADAMTS13/metabolismo , Estudos de Associação Genética , Hipertensão Portal/genética , Hipertensão Portal/fisiopatologia , Mutação de Sentido Incorreto/genética , Proteína ADAMTS13/fisiologia , Adolescente , Adulto , Idoso , Estudos de Coortes , Proteínas do Sistema Complemento/genética , Feminino , Humanos , Hipertensão Portal/metabolismo , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Estudos Prospectivos , Adulto Jovem , Fator de von Willebrand/genéticaRESUMO
Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders related to hematopoietic stem and progenitor cell dysfunction. Several studies have shown the role of the bone marrow microenvironment in regulating hematopoietic stem, and progenitor function and their individual abnormalities have been associated with disease pathogenesis. In this study, we simultaneously evaluated hematopoietic stem cells (HSC), hematopoietic stem progenitor cells (HSPCs) and different stromal elements in a cohort of patients with MDS-refractory cytopenia with multilineage dysplasia (RCMD). Karyotyping of these patients revealed variable chromosomal abnormalities in 73.33% of patients. Long-term HSC and lineage-negative CD34+CD38- cells were reduced while among the HPCs, there was an expansion of common myeloid progenitor and loss of granulocyte-monocyte progenitors. Interestingly, loss of HSCs was accompanied by aberrant frequencies of endothelial (ECs) (CD31+CD45-CD71-) and mesenchymal stem cells (MSCs) (CD31-CD45-71-) and its subsets associated with HSC niche. We further demonstrate down-regulation of HSC maintenance genes such as Cxcl12, VEGF in mesenchymal cells and a parallel upregulation in endothelial cells. Altogether we report for the first time quantitative and qualitative de novo changes in hematopoietic stem and its associated niche in a cohort of MDS-RCMD patients. These findings further reinforce the role of different components of the bone marrow microenvironment in MDS pathogenesis and emphasize the need for comprehensive simultaneous evaluation of all niche elements in such studies.
Assuntos
Células-Tronco Hematopoéticas/patologia , Síndromes Mielodisplásicas/patologia , Nicho de Células-Tronco , Medula Óssea/patologia , Linhagem da Célula , Aberrações Cromossômicas , Células Endoteliais/patologia , Humanos , Células-Tronco Mesenquimais/patologia , Células Progenitoras Mieloides/patologia , Células Estromais/patologiaRESUMO
Thioacetamide (TAA) administration is widely used for induction of liver cirrhosis in rats, where reactive oxygen radicals (ROS) and nitric oxide (NO) participate in development of liver damage. Cardiac dysfunction is an important complication of liver cirrhosis, but the role of ROS or NO in cardiac abnormalities during liver cirrhosis is not well understood. This was investigated in animals after TAA-induced liver cirrhosis and temporal changes in oxidative stress, NO and mitochondrial function in the heart evaluated. TAA induced elevation in cardiac levels of nitrate before development of frank liver cirrhosis, without gross histological alterations. This was accompanied by an early induction of P38 MAP kinase, which is influenced by ROS and plays an important signaling role for induction of iNOS. Increased nitrotyrosine, protein oxidation and lipid peroxidation in the heart and cardiac mitochondria, suggestive of oxidative stress, also preceded frank liver cirrhosis. However, compromised cardiac mitochondrial function with a decrease in respiratory control ratio and increased mitochondrial swelling was seen later, when cirrhosis was evident. In conclusion, TAA induces elevations in ROS and NO in the heart in parallel to early liver damage. This leads to later development of functional deficits in cardiac mitochondria after development of liver cirrhosis.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cardiopatias/metabolismo , Cirrose Hepática/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tioacetamida , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Peroxidação de Lipídeos , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Mitocôndrias Cardíacas/patologia , Dilatação Mitocondrial , Miócitos Cardíacos/patologia , Nitratos/metabolismo , Estresse Oxidativo , Ratos Wistar , Transdução de Sinais , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.
Assuntos
Loci Gênicos , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Longo não Codificante/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Acetilcisteína/farmacologia , Animais , Antígenos CD/metabolismo , Proteínas de Ligação ao Cálcio , Feto/metabolismo , Impressão Genômica , Células HEK293 , Humanos , Fígado/citologia , Fígado/embriologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/ultraestrutura , Mutação/genética , Biogênese de Organelas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirolimo/farmacologiaRESUMO
BACKGROUND: Primary colonic epithelial defects leading to inflammatory responses are considered central to the development of ulcerative colitis (UC). However, a systematic analysis of various colonic subcompartments in the pathogenesis of UC before inflammation remains elusive. Here, we explored changes in colonic subcompartments and their associated niche signals in patient mucosal biopsies and in an animal model of colitis. METHODS: Analysis of mucosal biopsies obtained from uninvolved and involved regions of patients with UC and Crohn's disease was performed and compared with normal subjects. Temporal analysis of colonic subcompartments was performed in mice administered with 5% dextran sodium sulphate. Phenotypic enumeration of the crypt subcompartment was complemented with flow cytometric analysis. Members of Notch and Wnt signaling pathways were analyzed by molecular, biochemical, and colocalization studies. RESULTS: Phenotypic enumeration of colonocytes' subcompartments from patients revealed significant alterations of the lower crypt, enriched in stem cell and progenitors, independent of inflammation. These changes, unique to UC, were confirmed by immunohistochemistry and molecular analysis. In parallel, a defect in proliferation and Muc2 synthesis was observed. Animal data before inflammation recapitulated human studies. Mechanistic studies revealed that changes in signaling through Wnt primarily affected colonic stem cells, whereas Notch affected progenitor function. CONCLUSIONS: Our results thus provide new insights into the development of inflammation and relapse in UC and suggest that the stem cell niche in the colon may influence pathogenesis of the disease.
Assuntos
Colite Ulcerativa/patologia , Doença de Crohn/patologia , Sulfato de Dextrana/administração & dosagem , Mucosa Intestinal/patologia , Mucina-2/metabolismo , Adolescente , Adulto , Idoso , Animais , Biópsia , Colite Ulcerativa/induzido quimicamente , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND AND AIM: Glutamine is an important energy source for the intestinal epithelium, and its supplementation protects intestinal epithelial cells by induction of glutathione. However, mechanisms of glutathione induction in cells at various stages of differentiation along the crypt to villus axis are not well understood. This study examined induction of glutathione in response to glutamine along the intestinal villus-crypt axis and evaluated regulatory mediators involved in the process. METHODS: Animals were administered 4% glutamine in feed for 7 days, following which enterocytes at various stages of differentiation were isolated and glutathione levels and signaling mediators involved in its regulation were studied. RESULTS: In control animals, glutathione levels were higher in the intestinal crypt than in the villus or middle region. This was accompanied by elevated expression of the modifier subunit of glutathione synthetase (GCLM) and the transcription factor Nrf2 when compared with cells from the villus and middle regions. These levels were further enhanced by glutamine throughout the intestine, although the effects were more dramatic in the crypt. In parallel to glutathione induction, glutamine supplementation also altered actin dynamics and proliferation in cells of the crypt. CONCLUSIONS: These results suggest that the variation of glutathione levels along the villus-crypt axis in the intestine is due to gradients in expression of mediators such as glutamate cysteine ligase modifier subunit and Nrf2. The protective effects of glutamine supplementation seem to be most pronounced in the crypt, where it upregulates proliferation, glutathione levels and alters actin dynamics.
Assuntos
Glutamina , Glutationa , Mucosa Intestinal , Animais , Feminino , Masculino , Citoesqueleto de Actina/metabolismo , Administração Oral , Diferenciação Celular , Separação Celular , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutamina/administração & dosagem , Glutamina/farmacologia , Glutationa/metabolismo , Glutationa Sintase/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Ratos Wistar , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
Multiple bone marrow stromal cell types have been identified as hematopoietic stem cell (HSC)-regulating niche cells. However, whether HSC progeny can serve directly as HSC niche cells has not previously been shown. Here we report a dichotomous role of megakaryocytes (MKs) in both maintaining HSC quiescence during homeostasis and promoting HSC regeneration after chemotherapeutic stress. We show that MKs are physically associated with HSCs in the bone marrow of mice and that MK ablation led to activation of quiescent HSCs and increased HSC proliferation. RNA sequencing (RNA-seq) analysis revealed that transforming growth factor ß1 (encoded by Tgfb1) is expressed at higher levels in MKs as compared to other stromal niche cells. MK ablation led to reduced levels of biologically active TGF-ß1 protein in the bone marrow and nuclear-localized phosphorylated SMAD2/3 (pSMAD2/3) in HSCs, suggesting that MKs maintain HSC quiescence through TGF-ß-SMAD signaling. Indeed, TGF-ß1 injection into mice in which MKs had been ablated restored HSC quiescence, and conditional deletion of Tgfb1 in MKs increased HSC activation and proliferation. These data demonstrate that TGF-ß1 is a dominant signal emanating from MKs that maintains HSC quiescence. However, under conditions of chemotherapeutic challenge, MK ablation resulted in a severe defect in HSC expansion. In response to stress, fibroblast growth factor 1 (FGF1) signaling from MKs transiently dominates over TGF-ß inhibitory signaling to stimulate HSC expansion. Overall, these observations demonstrate that MKs serve as HSC-derived niche cells to dynamically regulate HSC function.