RESUMO
Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
Assuntos
Toxinas Bacterianas/metabolismo , Enterococcus , Leucócitos Mononucleares , Fatores de Virulência/metabolismo , Animais , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidade , Cavalos , Camundongos , Testes de Sensibilidade Microbiana , SuínosRESUMO
Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/ß-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike ß-catenin gain-of-function models, which induce high Wnt/ß-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/ß-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing ß-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/ß-catenin activation, which is regulated by ZNRF3.
Assuntos
Córtex Suprarrenal/metabolismo , Homeostase/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/crescimento & desenvolvimento , Doenças do Córtex Suprarrenal/fisiopatologia , Animais , Proliferação de Células/genética , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Modelos Animais , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND & AIMS: RET tyrosine kinase is necessary for enteric nervous system development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, patients with HSCR often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility. METHODS: RetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET regulates gut motility in vivo, genetic, and pharmacologic approaches were used to disrupt RET in all RET-expressing cells, a subset of enteric neurons, or intestinal epithelial cells. RESULTS: Distinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the epithelium, rather than in enteric neurons, slowed GI motility selectively in male mice. RET kinase inhibition phenocopied this effect. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin or L-cells that release peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), both of which can alter motility. RET kinase inhibition exaggerated PYY and GLP-1 release in a nutrient-dependent manner without altering serotonin secretion in mice and human organoids. PYY receptor blockade rescued dysmotility in mice lacking epithelial RET. CONCLUSIONS: RET signaling normally limits nutrient-dependent peptide release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction.
Assuntos
Sistema Nervoso Entérico , Doença de Hirschsprung , Lactente , Humanos , Masculino , Camundongos , Animais , Peptídeo YY , Serotonina , Doença de Hirschsprung/genética , Células Enteroendócrinas , Intestino Delgado , Peptídeo 1 Semelhante ao Glucagon , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
BACKGROUND & AIMS: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). METHODS: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. RESULTS: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. CONCLUSIONS: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Células Caliciformes , Células-Tronco/fisiologia , Mucosa Intestinal/metabolismo , Diferenciação Celular/genética , Doenças Inflamatórias Intestinais/metabolismo , Colite/metabolismoRESUMO
OBJECTIVE: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.
Assuntos
Doença de Crohn , Animais , Humanos , Camundongos , Células CACO-2 , Doença de Crohn/tratamento farmacológico , Doença de Crohn/metabolismo , Mucosa Intestinal/metabolismo , Camundongos Knockout , Quinase de Cadeia Leve de Miosina/metabolismo , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo/metabolismo , Junções Íntimas/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In adult tissues such as adipose tissue, post-mitotic cells like adipocytes can be replaced by differentiation of a population of tissue-resident stem cells. Expression of mouse telomerase reverse transcriptase (mTert) is a hallmark of stem cell populations, and previous efforts to identify tissue-resident adult stem cells by measuring mTert expression have increased our understanding of stem cell biology significantly. Here, we used a doxycycline-inducible mouse model to perform longitudinal, live-animal lineage-tracing of mTert-expressing cells for more than 1 year. We identified a rare (<2%) population of stem cells in different fat depots that express putative preadipocyte markers. The adipose-derived mTert-positive cells are capable of self-renewal and possess adipogenic potential. Finally, we demonstrate that high-fat diet (HFD) can initiate differentiation of these cells in vivo. These data identify a population of adipose stem cells that contribute to the depot-specific response to HFD.
Assuntos
Telomerase , Adipogenia/genética , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular , Camundongos , Células-Tronco/metabolismo , Telomerase/genética , Telomerase/metabolismoRESUMO
Skeletal progenitor/stem cells (SSCs) play a critical role in postnatal bone growth and maintenance. Telomerase (Tert) activity prevents cellular senescence and is required for maintenance of stem cells in self-renewing tissues. Here we investigated the role of mTert-expressing cells in postnatal mouse long bone and found that mTert expression is enriched at the time of adolescent bone growth. mTert-GFP+ cells were identified in regions known to house SSCs, including the metaphyseal stroma, growth plate, and the bone marrow. We also show that mTert-expressing cells are a distinct SSC population with enriched colony-forming capacity and contribute to multiple mesenchymal lineages, in vitro. In contrast, in vivo lineage-tracing studies identified mTert+ cells as osteochondral progenitors and contribute to the bone-forming cell pool during endochondral bone growth with a subset persisting into adulthood. Taken together, our results show that mTert expression is temporally regulated and marks SSCs during a discrete phase of transitional growth between rapid bone growth and maintenance.
Assuntos
Células Epiteliais/metabolismo , Células-Tronco/metabolismo , Telomerase/metabolismo , Animais , Medula Óssea/metabolismo , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , CamundongosRESUMO
Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Colo/metabolismo , Epitélio/metabolismo , Receptores Frizzled/metabolismo , Animais , Antígenos/metabolismo , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Sítios de Ligação , Células CHO , Sistemas CRISPR-Cas , Linhagem Celular , Clostridioides difficile/patogenicidade , Cricetulus , Feminino , Receptores Frizzled/química , Receptores Frizzled/deficiência , Receptores Frizzled/genética , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Organoides/metabolismo , Domínios Proteicos , Proteoglicanas/metabolismo , Fatores de Virulência/metabolismo , Proteínas Wnt/metabolismoRESUMO
Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trios from the two families. Intestinal biopsy samples from affected individuals were used for histology and immunofluorescence and to generate enteroids ex vivo. Histopathologic evaluation demonstrated chronic inflammatory changes in the stomach, duodenum, and colon. Immunofluorescence demonstrated diminished staining for OLFM4, a marker for intestinal stem cells (ISCs). The enteroids generated from WNT2B-deficient intestinal epithelium could not be expanded and did not survive passage. Addition of CHIR-99021 (a GSK3A and GSK3B inhibitor and activator of canonical WNT/ß-CATENIN signaling) could not rescue WNT2B-deficient enteroids. Addition of supplemental recombinant murine WNT2B was able to perpetuate small enteroids for multiple passages but failed to expand their number. Enteroids showed a 10-fold increase in the expression of LEF1 mRNA and a 100-fold reduction in TLR4 expression, compared with controls by quantitative RT-PCR, indicating alterations in canonical WNT and microbial pattern-recognition signaling. In summary, individuals with homozygous nonsense mutations in WNT2B demonstrate severe intestinal dysregulation associated with decreased ISC number and function, likely explaining their diarrheal phenotype. WNT2B deficiency should be considered for individuals with neonatal-onset diarrhea.
Assuntos
Códon sem Sentido/genética , Diarreia/genética , Glicoproteínas/genética , Proteínas Wnt/genética , Criança , Pré-Escolar , Feminino , Homozigoto , Humanos , Lactente , Intestinos/patologia , Masculino , RNA Mensageiro/genética , Transdução de Sinais/genética , Células-Tronco/patologiaRESUMO
Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines.
Assuntos
Colite/metabolismo , Colo/imunologia , Citocinas/metabolismo , Animais , Autofagia , Comunicação Celular , Diferenciação Celular , Colo/metabolismo , Colo/patologia , Epitélio/imunologia , Epitélio/metabolismo , Microbioma Gastrointestinal , Homeostase , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Cinética , Camundongos , Análise Multivariada , Fosforilação , Análise de Componente Principal , Transdução de Sinais , Sirolimo/farmacologia , Biologia de Sistemas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
Studies from five independent laboratories conclude that bone marrow stem cells transdifferentiate into endometrial stroma, epithelium, and endothelium. We investigated the nature of bone marrow-derived cells in the mouse endometrium by reconstituting irradiated wild type recipients with bone marrow containing transgenic mTert-green fluorescent protein (GFP) or chicken ß-actin (Ch ß-actin)-GFP reporters. mTert-GFP is a telomerase marker identifying hematopoietic stem cells and subpopulations of epithelial, endothelial, and immune cells in the endometrium. Ch ß-actin-GFP is a ubiquitous reporter previously used to identify bone marrow-derived cells in the endometrium. Confocal fluorescence microscopy for GFP and markers of endometrial and immune cells were used to characterize bone marrow-derived cells in the endometrium of transplant recipients. No evidence of GFP+ bone marrow-derived stroma, epithelium, or endothelium was observed in the endometrium of mTert-GFP or Ch ß-actin-GFP recipients. All GFP+ cells detected in the endometrium were immune cells expressing the pan leukocyte marker CD45, including CD3+ T cells and F4/80+ macrophages. Further examination of the Ch ß-actin-GFP transplant model revealed that bone marrow-derived F4/80+ macrophages immunostained weakly for CD45. These macrophages were abundant in the stroma, infiltrated the epithelial and vascular compartments, and could easily be mistaken for bone marrow-derived endometrial cells. We conclude that it is unlikely that bone marrow cells are able to transdifferentiate into endometrial stroma, epithelium, and endothelium. This result has important therapeutic implications, as the expectation that bone marrow stem cells contribute directly to endometrial regeneration is shaping strategies designed to regenerate endometrium in Asherman's syndrome and to control aberrant endometrial growth in endometriosis. Stem Cells 2018;36:91-102.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Animais , Diferenciação Celular , Linhagem da Célula , Modelos Animais de Doenças , CamundongosRESUMO
OBJECTIVE: Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated. APPROACH AND RESULTS: In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase-/- Tregs into Rag2-/- ApoE-/- (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. CONCLUSIONS: Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.
Assuntos
Aterosclerose/enzimologia , Linfócitos T CD4-Positivos/enzimologia , Proliferação de Células , Ativação Linfocitária , Linfócitos T Reguladores/enzimologia , Telomerase/metabolismo , Transferência Adotiva , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Knockout , Camundongos Knockout para ApoE , Estresse Oxidativo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Telomerase/deficiência , Telomerase/genética , Homeostase do TelômeroRESUMO
The intestine's ability to recover from catastrophic injury requires quiescent intestinal stem cells (q-ISCs). While rapidly cycling (Lgr5+) crypt base columnar (CBC) ISCs normally maintain the intestine, they are highly sensitive to pathological injuries (irradiation, inflammation) and must be restored by q-ISCs to sustain intestinal homeostasis. Despite clear relevance to human health, virtually nothing is known regarding the factors that regulate q-ISCs. A comprehensive understanding of these mechanisms would likely lead to targeted new therapies with profound therapeutic implications for patients with gastrointestinal conditions. We briefly review the current state of the literature, highlighting homeostatic mechanisms important for q-ISC regulation, listing key questions in the field, and offer strategies to address them. Developmental Dynamics 245:718-726, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Homeostase/genética , Homeostase/fisiologia , Humanos , Modelos Biológicos , Transdução de Sinais , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
Long-lived and self-renewing adult stem cells (SCs) are essential for homeostasis in a wide range of tissues and can include both rapidly cycling and quiescent (q)SC populations. Rapidly cycling SCs function principally during normal tissue maintenance and are highly sensitive to stress, whereas qSCs exit from their quiescent state in response to homeostatic imbalance and regenerative pressure. The regulatory mechanisms underlying the quiescent state include factors essential for cell cycle control, stress response and survival pathways, developmental signalling pathways, and post-transcriptional modulation. Here, we review these regulatory mechanisms citing observations from the intestine and other self-renewing tissues.
Assuntos
Células-Tronco Adultas/fisiologia , Intestinos/citologia , Animais , Autofagia , Proteínas Inibidoras de Quinase Dependente de Ciclina/fisiologia , Proteínas de Ligação a DNA/fisiologia , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Quinases/fisiologiaRESUMO
Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca2+ levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage-dependent L- and T-type Ca2+ channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately -85 mV and has been considered electrically silent, low voltage-activated T-type Ca2+ channels are assumed to provide the primary Ca2+ signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain-of-function mutations in L-type CaV 1.3 channels in aldosterone-producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca2+ signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV 3.2 channels can generate a significant and recurring Ca2+ signal, and how CaV 1.3 channels may contribute to the Ca2+ signal that drives aldosterone production.
Assuntos
Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Canais de Cálcio/metabolismo , Zona Glomerulosa/metabolismo , Animais , Cálcio/metabolismo , HumanosRESUMO
STUDY HYPOTHESIS: The mouse endometrium harbours stem/progenitor cells that express the stem cell marker mouse telomerase reverse transcriptase (mTert). STUDY FINDING: We used a mouse carrying a transgenic reporter for mTert promoter activity to identify rare endometrial populations of epithelial and endothelial cells that express mTert. WHAT IS KNOWN ALREADY: Stem/progenitor cells are hypothesized to be responsible for the remarkable regenerative capacity of the endometrium, but the lack of convenient endometrial stem/progenitor markers in the mouse has hampered investigations into the identity of these cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A mouse containing a green fluorescent protein (GFP) reporter under the control of the telomerase reverse transcriptase promoter (mTert-GFP) was used to identify potential stem/progenitor cells in the endometrium. mTert promoter activity was determined using fluorescence microscopy and flow cytometry to identify GFP(+) cells. GFP(+) cells were examined for epithelial, stromal, endothelial, leucocyte and proliferation markers and bromodeoxyuridine retention to determine their identity. The endometrium of ovariectomized mice was compared to that of intact cycling mice to establish the role of ovarian hormones in maintaining mTert-expressing cells. MAIN RESULTS AND THE ROLE OF CHANCE: We found that mTert-GFP is expressed by rare luminal and glandular epithelial cells (0.3% of epithelial cells by flow cytometry), rare CD45(-) cells in the stromal compartment (0.028 ± 0.010% of stromal cells by microscopy) and many CD45(+) leucocytes. Ovariectomy resulted in significant decrease of mTert-GFP(+) epithelial cells (P = 0.029 for luminal epithelium; P = 0.034 for glandular epithelium) and a decrease in the percentage of mTert-GFP(+) CD45(+) leucocytes in the stromal compartment (P = 0.015). However, CD45(-) mTert-GFP(+) cells in the stromal compartment were maintained in ovariectomized mice. This population is enriched for cells bearing the endothelial marker CD31 (10.3% of CD90(-) CD45(-) and 97.8% CD90(+) CD45(-) by flow cytometry). CD45(-) mTert-GFP(+) cells also immunostained for the endothelial marker von Willebrand factor. These results suggest that the endometrial epithelium and vasculature are foci of stem/progenitor activity and provide a system to investigate molecular mechanisms involved in endometrial regeneration and repair. LIMITATIONS, REASONS FOR CAUTION: The stem/progenitor activity of endometrial mTert-GFP(+) cells needs to be experimentally verified. WIDER IMPLICATIONS OF THE FINDINGS: The identification and characterization of mTert-expressing progenitor cells in the mouse will facilitate the identification of equivalent populations in the human endometrium that are likely to be involved in endometrial function, fertility and disease. LARGE-SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was funded by National Health and Medical Research Council (NHMRC) of Australia grants (1085435, C.E.G., J.A.D.), 1021127 (C.E.G.), NHMRC Senior Research Fellowship (1042298, C.E.G.), the Victorian Infrastructure Support Program, U.S. National Institutes of Health grant R01 DK084056 (D.T.B.) and the Harvard Stem Cell Institute (D.T.B.). The authors have no conflicts of interest to declare.
Assuntos
Endométrio/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Células-Tronco/metabolismo , Telomerase/genética , Animais , Biomarcadores/metabolismo , Proliferação de Células , Endométrio/citologia , Células Endoteliais/citologia , Células Epiteliais/citologia , Feminino , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Ovariectomia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regiões Promotoras Genéticas , Células-Tronco/citologia , Telomerase/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5(+)) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5(+) cells. mTert-expressing cells distribute in a pattern along the crypt-villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert(+) cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.
Assuntos
Mucosa Intestinal/citologia , Células-Tronco Multipotentes/metabolismo , Telomerase/metabolismo , Animais , Linhagem da Célula/fisiologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Células-Tronco Multipotentes/citologia , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Klotho plays a critical role in the regulation of ion and fluid homeostasis. A previous study reported that haplo-insufficiency of Klotho in mice results in increased aldosterone synthase (CYP11B2) expression, elevated plasma aldosterone, and high blood pressure. This phenotype was presumed to be the result of diminished Klotho expression in zona glomerulosa (zG) cells of the adrenal cortex; however, systemic effects on adrenal aldosterone production could not be ruled out. To examine whether Klotho expressed in the zG is indeed a critical regulator of aldosterone synthesis, we generated a tamoxifen-inducible, zG-specific mouse model of Klotho deficiency by crossing Klotho-flox mice with Cyp11b2-CreERT mice (zG-Kl-KO). Tamoxifen-treated Cyp11b2-CreERT animals (zG-Cre) served as controls. Rosa26-mTmG reporter mice were used for Cre-dependent lineage-marking. Two weeks after tamoxifen induction, the specificity of the zG-Cre line was verified using immunofluorescence analysis to show that GFP expression was restricted to the zG. RNA in situ hybridization revealed a 65% downregulation of Klotho messenger RNA expression in the zG of zG-Kl-KO female mice at age 12 weeks compared to control mice. Despite this significant decrease, zG-Kl-KO mice exhibited no difference in plasma aldosterone levels. However, adrenal CYP11B2 expression and the CYP11B2 promotor regulatory transcription factors, NGFIB and Nurr1, were enhanced. Together with in vitro experiments, these results suggest that zG-derived Klotho modulates Cyp11b2 but does not evoke a systemic phenotype in young adult mice on a normal diet. Further studies are required to investigate the role of adrenal Klotho on aldosterone synthesis in aged animals.
Assuntos
Córtex Suprarrenal , Hiperaldosteronismo , Feminino , Camundongos , Animais , Zona Glomerulosa/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Aldosterona/metabolismo , Córtex Suprarrenal/metabolismo , Hiperaldosteronismo/genética , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: Sulfadoxine-pyrimethamine (SP) antimalarial therapy has been suggested to potentially increase the birth weight of infants in pregnant women in sub-Saharan Africa, independently of malarial infection. Here, we utilized female intestinal organoid-derived cells cultured within microfluidic Organ Chips to investigate whether SP could directly impact intestinal function and thereby improve the absorption of essential fats and nutrients crucial for fetal growth. METHODS: Using a human organ-on-a-chip model, we replicated the adult female intestine with patient organoid-derived duodenal epithelial cells interfaced with human intestinal endothelial cells. Nutrient-deficient (ND) medium was perfused to simulate malnutrition, resulting in the appearance of enteric dysfunction indicators such as villus blunting, reduced mucus production, impaired nutrient absorption, and increased inflammatory cytokine secretion. SP was administered to these chips in the presence or absence of human peripheral blood mononuclear cells (PBMCs). FINDINGS: Our findings revealed that SP treatment effectively reversed multiple intestinal absorptive abnormalities observed in malnourished female Intestine Chips, as validated by transcriptomic and proteomic analyses. SP also reduced the production of inflammatory cytokines and suppressed the recruitment of PBMCs in ND chips. INTERPRETATION: Our results indicate that SP could potentially increase birth weights by preventing enteric dysfunction and suppressing intestinal inflammation. This underscores the potential of SP as a targeted intervention to improve maternal absorption, subsequently contributing to healthier fetal growth. While SP treatment shows promise in addressing malabsorption issues that can influence infant birth weight, we did not model pregnancy in our chips, and thus its usefulness for treatment of malnourished pregnant women requires further investigation through clinical trials. FUNDING: The Bill and Melinda Gates Foundation, and the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the HDDC Organoid Core of the P30 DK034854.
Assuntos
Antimaláricos , Desnutrição , Complicações Parasitárias na Gravidez , Sulfadoxina , Adulto , Feminino , Humanos , Gravidez , Peso ao Nascer , Células Endoteliais , Leucócitos Mononucleares , Proteômica , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Antimaláricos/uso terapêutico , Combinação de Medicamentos , Intestinos , Desnutrição/complicações , Desnutrição/tratamento farmacológicoRESUMO
The sense of taste is essential for survival, as it allows animals to distinguish between foods that are nutritious from those that are toxic. However, innate responses to different tastants can be modulated or even reversed under pathological conditions. Here, we examined whether and how the internal status of an animal impacts taste valence by using Drosophila models of hyperproliferation in the gut. In all three models where we expressed proliferation-inducing transgenes in intestinal stem cells (ISCs), hyperproliferation of ISCs caused a tumor-like phenotype in the gut. While tumor-bearing flies had no deficiency in overall food intake, strikingly, they exhibited an increased gustatory preference for aristolochic acid (ARI), which is a bitter and normally aversive plant-derived chemical. ARI had anti-tumor effects in all three of our gut hyperproliferation models. For other aversive chemicals we tested that are bitter but do not have anti-tumor effects, gut tumors did not affect avoidance behaviors. We demonstrated that bitter-sensing gustatory receptor neurons (GRNs) in tumor-bearing flies respond normally to ARI. Therefore, the internal pathology of gut hyperproliferation affects neural circuits that determine taste valence postsynaptic to GRNs rather than altering taste identity by GRNs. Overall, our data suggest that increased consumption of ARI may represent an attempt at self-medication. Finally, although ARI's potential use as a chemotherapeutic agent is limited by its known toxicity in the liver and kidney, our findings suggest that tumor-bearing flies might be a useful animal model to screen for novel anti-tumor drugs.