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1.
Immunity ; 57(2): 319-332.e6, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38295798

RESUMO

Tuft cells in mucosal tissues are key regulators of type 2 immunity. Here, we examined the impact of the microbiota on tuft cell biology in the intestine. Succinate induction of tuft cells and type 2 innate lymphoid cells was elevated with loss of gut microbiota. Colonization with butyrate-producing bacteria or treatment with butyrate suppressed this effect and reduced intestinal histone deacetylase activity. Epithelial-intrinsic deletion of the epigenetic-modifying enzyme histone deacetylase 3 (HDAC3) inhibited tuft cell expansion in vivo and impaired type 2 immune responses during helminth infection. Butyrate restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids blocked tuft cell expansion. Collectively, these data define a HDAC3 mechanism in stem cells for tuft cell differentiation that is dampened by a commensal metabolite, revealing a pathway whereby the microbiota calibrate intestinal type 2 immunity.


Assuntos
Mucosa Intestinal , Microbiota , Adulto , Camundongos , Humanos , Animais , Células em Tufo , Butiratos/farmacologia , Butiratos/metabolismo , Imunidade Inata , Linfócitos/metabolismo , Intestinos , Histona Desacetilases/metabolismo , Diferenciação Celular
2.
Am J Physiol Gastrointest Liver Physiol ; 325(6): G493-G500, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37697924

RESUMO

Deep crypt secretory (DCS) cells are a population of epithelial cells located at the colonic crypt base that share some similarities to Paneth and goblet cells. They were initially defined as c-Kit expressing cells, though subsequent work showed that they are more specifically marked by Reg4 in the murine colon. The best-understood function of DCS cells at present is supporting the stem cell niche by generating Notch and EGF ligands. However, as these cells also express immunoregulatory (e.g., Ccl6) and host defense (e.g., Retnlb) genes, it is likely they have additional functions in maintaining colonic health outside of maintenance of the stem niche. Recent advances in single-cell transcriptomic profiling hint at additional epithelial and immune roles that may exist for these cells and have aided in elucidating their developmental lineage. This review highlights the emerging evidence supporting a crucial role for DCS cells in intestinal physiology, the current understanding of how these cells are regulated, and their potential role(s) in colonic disease.


Assuntos
Mucosa Intestinal , Celulas de Paneth , Camundongos , Animais , Intestinos , Colo , Células Caliciformes , Diferenciação Celular/fisiologia
3.
Am J Physiol Gastrointest Liver Physiol ; 320(6): G990-G1001, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33826403

RESUMO

Proinflammatory macrophages are essential drivers of colitis and express the growth factor receptor ErbB4. This study tested the role of ErbB4 and its specific ligand, NRG4, in regulating macrophage function. We show that endogenous NRG4-ErbB4 signaling limits macrophage production of proinflammatory cytokines in vitro and limits colitis severity in vivo and thus is a potential target for therapeutic intervention.


Assuntos
Inflamação/metabolismo , Macrófagos/metabolismo , Neurregulinas/metabolismo , Receptor ErbB-4/metabolismo , Transdução de Sinais/fisiologia , Animais , Colite/metabolismo , Colo/metabolismo , Citocinas/metabolismo , Inflamação/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Ativação de Macrófagos/fisiologia , Camundongos , Camundongos Knockout
5.
PLoS Pathog ; 11(2): e1004663, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25658601

RESUMO

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylori that was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation.


Assuntos
Proliferação de Células , Células Epiteliais/imunologia , Mucosa Gástrica/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Receptores de Hialuronatos/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Fundo Gástrico/imunologia , Fundo Gástrico/microbiologia , Mucosa Gástrica/microbiologia , Deleção de Genes , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Humanos , Camundongos , Receptores Proteína Tirosina Quinases/imunologia
6.
PLoS Pathog ; 10(7): e1004275, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25033386

RESUMO

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 106 H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10(6)) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10(6)) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.


Assuntos
Mucosa Gástrica , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Úlcera Gástrica/imunologia , Úlcera Gástrica/microbiologia , Ácido Acético/efeitos adversos , Ácido Acético/farmacologia , Animais , Mucosa Gástrica/imunologia , Mucosa Gástrica/lesões , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Infecções por Helicobacter/patologia , Indicadores e Reagentes/efeitos adversos , Indicadores e Reagentes/farmacologia , Camundongos , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia
7.
J Physiol ; 593(8): 1809-27, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25605613

RESUMO

KEY POINTS: An in vitro approach to study gastric development is primary mouse-derived epithelium cultured as three-dimensional spheroids known as organoids. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Organoids maintained in co-culture with immortalized stomach mesenchymal cells express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. We report the use of these models for studies of epithelial cell biology and cell damage and repair. ABSTRACT: Studies of gastric function and disease have been limited by the lack of extended primary cultures of the epithelium. An in vitro approach to study gastric development is primary mouse-derived antral epithelium cultured as three-dimensional spheroids known as organoids. There have been no reports on the use of organoids for gastric function. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Both models were generated from single glands dissociated from whole fundic tissue and grown in basement membrane matrix (Matrigel) and organoid growth medium. Model 1 enriches for a stem cell-like niche via simple passage of the organoids. Maintained in Matrigel and growth medium, proliferating organoids expressed high levels of stem cell markers CD44 and Lgr5. Model 2 is a system of gastric organoids co-cultured with immortalized stomach mesenchymal cells (ISMCs). Organoids maintained in co-culture with ISMCs express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. Thus, we report the use of these models for studies of epithelial cell biology and cell damage and repair.


Assuntos
Técnicas de Cocultura/métodos , Células Epiteliais/citologia , Mucosa Gástrica/citologia , Organoides/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Camundongos
8.
Helicobacter ; 20(1): 19-28, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25495001

RESUMO

BACKGROUND: Helicobacter pylori (H. pylori) infection leads to acute induction of Sonic Hedgehog (Shh) in the stomach that is associated with the initiation of gastritis. The mechanism by which H. pylori induces Shh is unknown. Shh is a target gene of transcription factor Nuclear Factor-κB (NFκB). We hypothesize that NFκB mediates H. pylori-induced Shh. MATERIALS AND METHODS: To visualize Shh ligand expression in response to H. pylori infection in vivo, we used a mouse model that expresses Shh fused to green fluorescent protein (Shh::GFP mice) in place of wild-type Shh. In vitro, changes in Shh expression were measured in response to H. pylori infection using 3-dimensional epithelial cell cultures grown from whole dissociated gastric glands (organoids). Organoids were generated from stomachs collected from the fundic region of control and mice expressing a parietal cell-specific deletion of Shh (PC-Shh(KO) mice). RESULTS: Within 2 days of infection, H. pylori induced Shh expression within parietal cells of Shh::GFP mice. Organoids expressed all major gastric cell markers, including parietal cell marker H(+) ,K(+) -ATPase and Shh. H. pylori infection of gastric organoids induced Shh expression; a response that was blocked by inhibiting NFκB signaling and correlated with IκB degradation. H. pylori infection of PC-Shh(KO) mouse-derived organoids did not result in the induction of Shh expression. CONCLUSION: Gastric organoids allow for the study of the interaction between H. pylori and the differentiated gastric epithelium independent of the host immune response. H. pylori induces Shh expression from the parietal cells, a response mediated via activation of NFκB signaling.


Assuntos
Técnicas Citológicas/métodos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Proteínas Hedgehog/biossíntese , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno , NF-kappa B/metabolismo , Animais , Fusão Gênica Artificial , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/análise , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Camundongos , Camundongos Knockout , Organoides , Proteínas Recombinantes de Fusão/análise
9.
Dig Dis Sci ; 59(3): 569-82, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24202649

RESUMO

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) promote gastric cancer in response to gastritis. In culture, BM-MSCs are prone to mutation with continued passage but it is unknown whether a similar process occurs in vivo in response to gastritis. AIM: The purpose of this study was to identify the role of chronic gastritis in the transformation of BM-MSCs leading to an activated cancer-promoting phenotype. METHODS: Age matched C57BL/6 (BL/6) and gastrin deficient (GKO) mice were used for isolation of stomach, serum and mesenchymal stem cells (MSCs) at 3 and 6 months of age. MSC activation was assessed by growth curve analysis, fluorescence-activated cell sorting and xenograft assays. To allow for the isolation of bone marrow-derived stromal cells and assay in response to chronic gastritis, IRG/Vav-1(Cre) mice that expressed both enhanced green fluorescent protein-expressing hematopoietic cells and red fluorescent protein-expressing stromal cells were generated. In a parabiosis experiment, IRG/Vav-1(Cre) mice were paired to either an uninfected Vav-1(Cre) littermate or a BL/6 mouse inoculated with Helicobacter pylori. RESULTS: GKO mice displayed severe atrophic gastritis accompanied by elevated gastric tissue and circulating transforming growth factor beta (TGFß) by 3 months of age. Compared to BM-MSCs isolated from uninflamed BL/6 mice, BM-MSCs isolated from GKO mice displayed an increased proliferative rate and elevated phosphorylated-Smad3 suggesting active TGFß signaling. In xenograft assays, mice injected with BM-MSCs from 6-month-old GKO animals displayed tumor growth. RFP+ stromal cells were rapidly recruited to the gastric mucosa of H. pylori parabionts and exhibited changes in gene expression. CONCLUSIONS: Gastritis promotes the in vivo activation of BM-MSCs to a phenotype reminiscent of a cancer-promoting cell.


Assuntos
Transformação Celular Neoplásica , Mucosa Gástrica/patologia , Gastrite Atrófica/patologia , Células-Tronco Mesenquimais/patologia , Fenótipo , Animais , Biomarcadores/metabolismo , Proliferação de Células , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Gastrinas/deficiência , Gastrite Atrófica/metabolismo , Gastrite Atrófica/microbiologia , Proteínas Hedgehog/metabolismo , Infecções por Helicobacter/patologia , Helicobacter pylori , Immunoblotting , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parabiose , Reação em Cadeia da Polimerase em Tempo Real , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo
10.
Gastroenterology ; 142(5): 1150-1159.e6, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22285806

RESUMO

BACKGROUND & AIMS: Macrophages mediate the epithelial response to Helicobacter pylori and are involved in the development of gastritis. Sonic Hedgehog (Shh) regulates gastric epithelial differentiation and function, but little is known about its immunoregulatory role in the stomach. We investigated whether gastric Shh acts as a macrophage chemoattractant during the innate immune response to H pylori infection. METHODS: Mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and control mice were infected with H pylori. Levels of gastric Shh, cytokines, and chemokines were assayed by quantitative reverse-transcriptase polymerase chain reaction or by a Luminex-based multiplex assay 2, 7, or 180 days after infection. Circulating concentrations of Shh were measured by enzyme-linked immunosorbent assay. Bone marrow chimera experiments were performed with mice that have myeloid cell-specific deletion of the Hedgehog signal transduction protein Smoothened (LysMCre/Smo(KO)). Macrophage recruitment was measured in gastric tissue and peripheral blood by fluorescence-activated cell sorting analysis. RESULTS: Control mice infected with H pylori for 6 months developed an inflammatory response characterized by infiltration of CD4(+) T cells and increased levels of interferon gamma and interleukin 1ß in the stomach. PC-Shh(KO) mice did not develop gastritis, even after 6 months of infection with H pylori. Control mice had increased concentrations of Shh, accompanied by the recruitment of CD11b(+)F4/80(+)Ly6C(high) macrophages 2 days after infection. Control mice that received bone marrow transplants from control mice had an influx of macrophages to the gastric mucosa in response to H pylori infection; this was not observed in H pylori-infected control mice that received bone marrow transplants from LysMCre/Smo(KO) mice. CONCLUSIONS: H pylori induces release of Shh from the stomach; Shh acts as a macrophage chemoattractant during initiation of gastritis.


Assuntos
Fatores Quimiotáticos/fisiologia , Proteínas Hedgehog/fisiologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Macrófagos/fisiologia , Estômago/imunologia , Animais , Gastrite/etiologia , Proteínas Hedgehog/sangue , Infecções por Helicobacter/complicações , Interleucina-12/fisiologia , Interleucina-1beta/fisiologia , Camundongos , Transdução de Sinais
11.
Inflamm Bowel Dis ; 29(11): 1778-1792, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37265326

RESUMO

BACKGROUND: Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. METHODS: NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. RESULTS: During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. CONCLUSIONS: NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.


Neuregulin-4 (NRG4) is a growth factor that protects the epithelial cells lining the colon from injury and restrains innate (non-specific) immune responses. Here we show that NRG4's role in inflammation is context-specific, and mice that lack NRG4 have impaired adaptive immunity in a model of chronic immune-mediated colitis.


Assuntos
Colite , Mucosa Intestinal , Camundongos , Animais , Lipocalina-2/metabolismo , Mucosa Intestinal/patologia , Colite/patologia , Imunidade Adaptativa , Citocinas/metabolismo , Colo/patologia , Sulfato de Dextrana , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Camundongos Knockout
12.
Cell Mol Gastroenterol Hepatol ; 15(4): 971-984, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36414210

RESUMO

BACKGROUND & AIMS: Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal interleukin (IL)33/IL13 signaling loop. Here, we tested the hypothesis that IL13, induced by epithelial Sprouty2 down-regulation, promotes DCS cell differentiation and function. METHODS: Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single-cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL13 and IL13 receptor; bulk RNA sequencing and quantitative polymerase chain reaction were used to quantify changes in expression of identified HDPs. Cytokine-treated colonoids were assessed for DCS cells. A requirement for an IL33/IL13 signaling loop in the regulation of DCS cells was assessed in vivo using IL13 null mice. RESULTS: Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single-cell transcriptomics showed the HDP Retnlb/Resistin Like Beta (RELMß) is highly enriched in DCS cells. Retnlb/RELMß expression was increased in Spry2ΔIE colons. IL13, but not IL33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL33-mediated expansion of the DCS cell population in vivo was dependent on IL13, which was expressed predominantly by type II innate lymphoid cells in the colonic mucosa. CONCLUSIONS: Sprouty2 limits colonic DCS cell differentiation through suppression of IL13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMß. Loss of epithelial Sprouty2 activates type II innate lymphoid cells to release IL13, promoting expansion of the DCS cell population and increased colonic RELMß levels.


Assuntos
Imunidade Inata , Interleucina-13 , Animais , Camundongos , Diferenciação Celular , Epitélio , Linfócitos , Camundongos Knockout , Proteínas Associadas a Pancreatite , Proteínas
13.
J Physiol ; 590(6): 1495-509, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22289908

RESUMO

Maternal adiponectin levels are inversely correlated to birth weight, suggesting that maternal adiponectin limits fetal growth. We hypothesized that full-length adiponectin (fADN) infusion in pregnant mice down-regulates placental amino acid transporters and decreases fetal growth. Starting at embryonic day (E) 14.5, fADN (0.62 ± 0.02 µg (g body weight)(−1) day(−1), n = 7) or vehicle (control, n = 9) were infused in pregnant C57/BL6 mice by mini-osmotic pump. At E18.5, dams were killed and placental homogenates and trophoblast plasma membrane (TPM) vesicles were prepared. Infusion of fADN elevated maternal serum fADN by 4-fold and decreased fetal weights by 18%. Adiponectin receptor 2, but not adiponectin receptor 1, was expressed in TPM. fADN infusion decreased TPM System A (­56%, P < 0.001) and System L amino acid transporter activity (­50%, P < 0.03). TPM protein expression of SNAT1, 2 and 4 (System A amino acid transporter isoforms) and LAT1 and LAT2, but not CD98, (System L amino acid transporter isoforms) was down-regulated by fADN infusion. To identify possible mechanisms underlying these changes we determined the phosphorylation of proteins in signalling pathways known to regulate placental amino acid transporters. fADN decreased phosphorylation of insulin receptor substrate-1 (Tyr-608), Akt (Thr-308 and Ser-473), S6 kinase 1 (Thr-389), eukaryotic initiation factor 4E binding protein 1 (Thr-37/46 and Thr-70) and ribosomal protein S6 (Ser-235/236) and increased the phosphorylation of peroxisome proliferator-activated receptor α (PPARα) (Ser-21) in the placenta. These data suggest that maternal adiponectin decreases fetal growth by down-regulation of placental amino acid transporters, which limits fetal nutrient availability. This effect may be mediated by inhibition of insulin/IGF-I and mTOR signalling pathways, which are positive regulators of placental amino acid transporters. We have identified a novel physiological mechanism by which the endocrine functions of maternal adipose tissue influence fetal growth.


Assuntos
Adiponectina/farmacologia , Sistemas de Transporte de Aminoácidos/metabolismo , Desenvolvimento Fetal/efeitos dos fármacos , Placenta/efeitos dos fármacos , Adiponectina/sangue , Animais , Peso Corporal/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Insulina/sangue , Camundongos , Camundongos Endogâmicos C57BL , PPAR alfa/metabolismo , Placenta/metabolismo , Gravidez , Receptores de Adiponectina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Trofoblastos/metabolismo
14.
Gastroenterology ; 138(2): 550-61, 561.e1-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19909751

RESUMO

BACKGROUND & AIMS: Sonic Hedgehog (Shh) is expressed in the adult stomach, but its role as a gastric morphogen is unclear. We sought to identify mechanisms by which Shh might regulate gastric epithelial cell function and differentiation. METHODS: Mice with a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) were created. Gastric morphology and function were studied in control and HKCre/Shh(KO) mice between 1 and 8 months of age. RESULTS: In contrast to control mice, HKCre/Shh(KO) mice developed gastric hypochlorhydria, hypergastrinemia, and a phenotype that resembled foveolar hyperplasia. The fundic mucosa of HKCre/Shh(KO) mice had an expanded surface pit cell lineage that was documented by increased incorporation of bromodeoxyuridine and was attributed to the hypergastrinemia. Compared with controls, numbers of total mucous neck and zymogen cells were significantly decreased in stomachs of HKCre/Shh(KO) mice. In addition, zymogen and neck cell markers were coexpressed in the same cell populations, indicating disrupted differentiation of the zymogen cell lineage from the mucous neck cells in the stomachs of HKCre/Shh(KO) mice. Laser capture microdissection of the surface epithelium, followed by quantitative reverse-transcription polymerase chain reaction, revealed a significant increase in expression of Indian Hedgehog, glioma-associated oncogene homolog 1, Wnt, and cyclin D1. Laser capture microdissection analysis also showed a significant increase in Snail with a concomitant decrease in E-cadherin. CONCLUSIONS: In the stomachs of adult mice, loss of Shh from parietal cells results in hypochlorhydria and hypergastrinemia. Hypergastrinemia might subsequently induce increased Hedgehog and Wnt signaling in the surface pit epithelium, resulting in hyperproliferation.


Assuntos
Proliferação de Células , Mucosa Gástrica/patologia , Gastrite/metabolismo , Proteínas Hedgehog/metabolismo , Células Parietais Gástricas/metabolismo , Acloridria/metabolismo , Acloridria/fisiopatologia , Animais , Atrofia , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Epitélio/metabolismo , Epitélio/patologia , Mucosa Gástrica/metabolismo , Gastrite/fisiopatologia , Proteínas Hedgehog/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células Parietais Gástricas/patologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
15.
Nat Commun ; 12(1): 836, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547321

RESUMO

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.


Assuntos
Colite/genética , Glicogênio Sintase Quinase 3 beta/genética , Homeostase/genética , Interleucina-33/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Feminino , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Células HT29 , Homeostase/efeitos dos fármacos , Humanos , Interleucina-33/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Dodecilsulfato de Sódio/administração & dosagem
16.
Am J Physiol Gastrointest Liver Physiol ; 299(6): G1252-65, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20847300

RESUMO

In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5(Ski)) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5(Ski) cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/ß-catenin and ZO-1/occludin protein complexes was observed in HKCre/Shh(KO) mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1.


Assuntos
Caderinas/metabolismo , Citoesqueleto/fisiologia , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Junções Íntimas/fisiologia , Actinas/metabolismo , Animais , Anticorpos Monoclonais , Caderinas/genética , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas Hedgehog/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico/fisiologia , RNA Interferente Pequeno , Alcaloides de Veratrum/farmacologia , Proteína da Zônula de Oclusão-1
17.
Cell Death Dis ; 8(2): e2622, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230865

RESUMO

Efficient clearance of pro-inflammatory macrophages from tissues after resolution of a challenge is critical to prevent prolonged inflammation. Defects in clearance can contribute to conditions such as inflammatory bowel disease, and thus may be therapeutically targetable. However, the signaling pathways that induce termination of pro-inflammatory macrophages are incompletely defined. We tested whether the ErbB4 receptor tyrosine kinase, previously not known to have role in macrophage biology, is involved in this process. In vitro, pro-inflammatory activation of cultured murine and human macrophages induced ErbB4 expression; in contrast, other ErbB family members were not induced in pro-inflammatory cells, and other innate immune lineages (dendritic cells, neutrophils) did not express detectable ErbB4 levels. Treatment of activated pro-inflammatory macrophages with the ErbB4 ligand neuregulin-4 (NRG4) induced apoptosis. ErbB4 localized to the mitochondria in these cells. Apoptosis was accompanied by loss of mitochondrial membrane potential, and was dependent upon the proteases that generate the cleaved ErbB4 intracellular domain fragment, suggesting a requirement for this fragment and mitochondrial pathway apoptosis. In vivo, ErbB4 was highly expressed on pro-inflammatory macrophages but not neutrophils during experimental DSS colitis in C57Bl/6 mice. Active inflammation in this model suppressed NRG4 expression, which may allow for macrophage persistence and ongoing inflammation. Consistent with this notion, NRG4 levels rebounded during the recovery phase, and administration of exogenous NRG4 during colitis reduced colonic macrophage numbers and ameliorated inflammation. These data define a novel role for ErbB4 in macrophage apoptosis, and outline a mechanism of feedback inhibition that may promote resolution of colitis.


Assuntos
Apoptose/fisiologia , Colo/metabolismo , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Macrófagos/metabolismo , Receptor ErbB-4/metabolismo , Animais , Células Cultivadas , Colite/metabolismo , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurregulinas/metabolismo , Neutrófilos/metabolismo , Transdução de Sinais/fisiologia
18.
Cell Death Differ ; 24(5): 855-865, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28304405

RESUMO

Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1+ cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Celulas de Paneth/metabolismo , Fosfatidilinositol 3-Quinases/genética , Receptor ErbB-3/genética , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Comunicação Celular , Contagem de Células , Diferenciação Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Células HT29 , Humanos , Íleo/citologia , Íleo/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Celulas de Paneth/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-3/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia
19.
Methods Mol Biol ; 1422: 23-31, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27246019

RESUMO

Three-dimensional primary epithelial-derived gastric organoids have recently been established as an important tool to study gastric development, physiology, and disease. Specifically, mouse-derived fundic gastric organoids (mFGOs) co-cultured with Immortalized Stomach Mesenchymal Cells (ISMCs) reflect expression patterns of mature fundic cell types seen in vivo, thus allowing for long-term in vitro studies of gastric epithelial cell physiology, regeneration, and bacterial-host interactions. Here, we describe the development and culture of mFGOs, co-cultured with ISMCs.


Assuntos
Técnicas de Cocultura/métodos , Mucosa Gástrica/citologia , Células-Tronco Mesenquimais/citologia , Organoides/citologia , Animais , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Organoides/metabolismo
20.
Sci Rep ; 5: 17185, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26597788

RESUMO

Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration.


Assuntos
Células-Tronco/fisiologia , Papilas Gustativas/citologia , Animais , Ciclo Celular , Proliferação de Células , Rastreamento de Células , Receptores de Hialuronatos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organoides/citologia , Receptores Acoplados a Proteínas G/metabolismo , Técnicas de Cultura de Tecidos
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