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1.
J Virol ; 96(7): e0023522, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35311549

RESUMO

Here, we report the appearance of natural killer B (NKB) cells within the colon during simian immunodeficiency virus (SIV) infection of susceptible monkeys. Using RNA sequencing (RNAseq) and flow cytometry, we show that NKB cells are unique cells with features and functions of both NK and B cells. NKB cells express receptors and ligands found on B cells that are important for (i) antigen presentation; (ii) activities associated with class switching, affinity maturation, and B-cell memory formation in secondary lymphoid follicles; and (iii) antigen recognition. The predominant immunoglobulins (Igs) expressed on NKB cells are IgA, although NKB cells can express surface IgM and IgG. There is dominant lambda expression over the kappa light chain characteristic of mucosal B cells. In addition to B-cell aspects, NKB cells express NK cell activation receptors and Fas ligand. We show in this study that NKB cells express perforin and granzymes and lyse cells in a lytic assay. In addition to NK cell cytolytic function, NKB cells also produce the inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and interleukin-18 (IL-18). Finally, we noted the increased capacity of NKB cells to proliferate compared to NK cells and CD8+ T cells from the SIV-infected colon. The increased proliferation and inflammatory cytokine production may be related to the relatively high expression levels of IL-15 receptor beta, IL-7 receptor, IL-18 receptor, and 41BB relative to the same receptors on CD8 and NK cells. The properties of NKB cells may point to their role in the enhanced inflammation observed in the SIV-infected gut. IMPORTANCE There is low-level but significant mucosal inflammation in the gastrointestinal tract secondary to human immunodeficiency virus (HIV) infection that has long-term consequences for the infected host. This inflammation most likely originates from the immune response that appears as a consequence of HIV. Here, we show in an animal model of HIV that the chronically SIV-infected gut contains cytotoxic natural killer B cells that produce inflammatory cytokines and proliferate during infection.


Assuntos
Células Matadoras Naturais , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD8-Positivos/imunologia , Colo/citologia , Colo/imunologia , Colo/virologia , Citocinas/metabolismo , Inflamação/patologia , Células Matadoras Naturais/imunologia , Macaca mulatta , Receptores de Células Matadoras Naturais/metabolismo , Vírus da Imunodeficiência Símia/imunologia
2.
Bioengineered ; 13(3): 6490-6499, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35220895

RESUMO

Ulcerative colitis (UC) is a type of chronic disease of inflammation, and matrine has anti-inflammatory activity. However, it is unclear that whether matrine can alleviate UC. This study aimed to evaluate the effect of matrine on DSS-induced intestinal epithelial cell injury. Cell viability was performed by MTT assay. Then cell apoptosis was analyzed using the TUNEL assay and flow cytometry. The levels of interleukin (IL)-2, IL-6, TNF-α, and IL-1ß were evaluated using qRT-PCR. Myeloperoxidase (MPO) activity was detected using ELISA assay. Nitric oxide (NO) production was detected by the Griess reagent. Bax, cleaved caspase-3, Bcl-2, JAK2, p-JAK2, STAT3, p-STAT3, STAT5, p-STAT5 levels were measured by Western blot. Bax (6A7) was asses using immunoprecipitation and immunofluorescence assays. The results illustrated that cell viability was inhibited as the concentration of DSS increased. Matrine did not affect cell viability at the concentration of 0-2 mg/ml but inhibited cell viability in a time-independent manner. Matrine suppressed the levels of pro-inflammatory factors, MPO activity, NO production, and apoptosis of DSS-stimulated cells. Furthermore, we found that matrine inhibited the levels of p-JAK2/JAK2 and p-STAT3/STAT3 but did not affect p-STAT5/STAT5. AG490 treatment further enhanced the effect of matrine on the apoptosis and pro-inflammatory factor levels in DSS-induced cells. In summary, matrine protected NCM460 cell against injury by inactivating the JAK2/STAT3 pathway. These data suggested for the first time that matrine may effective in treating UC.


Assuntos
Alcaloides , Apoptose/efeitos dos fármacos , Colo , Mucosa Intestinal , Substâncias Protetoras , Quinolizinas , Alcaloides/química , Alcaloides/farmacologia , Linhagem Celular , Colite Ulcerativa , Colo/citologia , Colo/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Quinolizinas/química , Quinolizinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Acta Physiol (Oxf) ; 234(3): e13774, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34985202

RESUMO

AIM: The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. METHODS: Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. RESULTS: pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. CONCLUSIONS: The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.


Assuntos
Colo , Trocadores de Sódio-Hidrogênio , Animais , Linhagem da Célula , Colo/citologia , Concentração de Íons de Hidrogênio , Camundongos , Microvilosidades/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Transportadores de Sulfato/metabolismo
4.
PLoS Biol ; 20(1): e3001527, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35089911

RESUMO

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.


Assuntos
DNA/genética , Desoxirribonuclease I/metabolismo , Loci Gênicos , Organoides/metabolismo , Reparo de DNA por Recombinação , Coloração e Rotulagem/métodos , Alelos , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Colo/citologia , Colo/metabolismo , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Desoxirribonuclease I/genética , Eletroporação/métodos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Técnicas de Introdução de Genes , Vetores Genéticos , Genoma Humano , Heterozigoto , Humanos , Organoides/citologia
5.
J Gastroenterol Hepatol ; 37(1): 134-143, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34477242

RESUMO

BACKGROUND AND AIM: Efficient intestinal wound healing is essential for good prognoses of ulcerative colitis (UC). Although bile acids and the transmembrane G-protein-coupled receptor (TGR) 5 have been reported to affect wound healing in intestinal epithelial cells, the detailed underlying mechanisms are unclear. Here, we investigated the role of TGR5 in wound healing in the context of colonic epithelial cells in the presence of bile acids. METHODS: The expression of TGR5 in the colonic epithelium of both a dextran sulfate sodium (DSS)-induced colitis mouse model (recovery phase), and UC patients in clinical remission, was evaluated. Young adult mouse colonic epithelial (YAMC) cells were then used to evaluate wound healing after treatment with deoxycholic acid (DCA); TGR5 was silenced in YAMC cells via shRNA-transfection, and a wound-healing assay in the presence of DCA was performed. Furthermore, we investigated the role of the activation of AKT in the context of wound healing. RESULTS: The expression of TGR5 was decreased in the colonic epithelium of both mice with DSS-induced colitis and UC patients. Additionally, DCA significantly delayed wound healing in YAMC cells but not in TGR5 silenced ones. Of note, the DCA-induced activation of AKT signaling in YAMC cells was inhibited by TGR5 silencing, and AKT inhibitors prevented the wound healing delay induced by DCA. CONCLUSIONS: Overall, we show that DCA delays wound healing in the context of colonic epithelial cells through AKT activation. These results may support the development of new therapeutic approaches for epithelial regeneration in UC.


Assuntos
Colo , Ácido Desoxicólico , Células Epiteliais , Cicatrização , Animais , Ácidos e Sais Biliares , Colite Ulcerativa/tratamento farmacológico , Colo/citologia , Colo/metabolismo , Ácido Desoxicólico/farmacologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Cicatrização/efeitos dos fármacos
6.
EMBO Rep ; 23(3): e53246, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34939731

RESUMO

Regulatory T lymphocyte (Treg) homing reactions mediated by G protein-coupled receptor (GPCR)-ligand interactions play a central role in maintaining intestinal immune homeostasis by restraining inappropriate immune responses in the gastrointestinal tract. However, the origin of Treg homing to the colon remains mysterious. Here, we report that the C10ORF99 peptide (also known as CPR15L and AP57), a cognate ligand of GPR15 that controls Treg homing to the colon, originates from a duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. Evolutionary analysis and experimental data indicate that the GPR15-C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes and their expression patterns are positively correlated with herbivore diet in the colon. With the first herbivorous diet in early amniotes, a new biological process (herbivorous diet short-chain fatty acid-C10ORF99/GPR15-induced Treg homing colon immune homeostasis) emerged, and we propose an evolutionary model whereby GPR15-C10ORF99 functional pairing has initiated the first colonic Treg homing reaction in amniotes. Our findings also highlight that GPCR-ligand pairing leads to physiological adaptation during vertebrate evolution.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Colo/citologia , Proteínas de Ligação a DNA , Receptores Acoplados a Proteínas G , Linfócitos T Reguladores , Animais , Colo/imunologia , Ligantes , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Linfócitos T Reguladores/citologia
7.
Development ; 149(1)2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34910127

RESUMO

Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.


Assuntos
Actinas/metabolismo , Colite Ulcerativa/metabolismo , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteína Wnt3A/metabolismo , Cicatrização , Actinas/genética , Animais , Células Cultivadas , Colo/citologia , Colo/metabolismo , Colo/fisiologia , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Proteína Wnt3A/genética
8.
FASEB J ; 35(12): e21992, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34719821

RESUMO

The colonic epithelial barrier is vital to preserve gut and host health by maintaining the immune homeostasis between host and microbes. The mechanisms underlying beneficial or harmful host-microbe interactions are poorly understood and impossible to study in vivo given the limited accessibility and ethical constraints. Moreover, existing in vitro models lack the required cellular complexity for the routine, yet profound, analysis of the intricate interplay between different types of host and microbial cells. We developed and characterized a broadly applicable, easy-to-handle in vitro triple coculture model that combines chemically-induced macrophage-like, goblet and epithelial cells covered by a mucus layer, which can be coincubated with complex human-derived gut microbiota samples for 16 h. Comparison with a standard epithelial monolayer model revealed that triple cocultures produce thicker mucus layers, morphologically organize in a network and upon exposure to human-derived gut microbiota samples, respond via pro-inflammatory cytokine production. Both model systems, however, were not suffering from cytotoxic stress or different microbial loads, indicating that the obtained endpoints were caused by the imposed conditions. Addition of the probiotic Lactobacillus rhamnosus GG to assess its immunomodulating capacity in the triple coculture slightly suppressed pro-inflammatory cytokine responses, based on transcriptomic microarray analyses. TNF conditioning of the models prior to microbial exposure did not cause shifts in cytokines, suggesting a strong epithelial barrier in which TNF did not reach the basolateral side. To conclude, the triple coculture model is tolerable towards manipulations and allows to address mechanistic host-microbe research questions in a stable in vitro environment.


Assuntos
Técnicas de Cocultura/métodos , Colo/imunologia , Células Epiteliais/imunologia , Microbioma Gastrointestinal , Interações entre Hospedeiro e Microrganismos , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Colo/citologia , Colo/metabolismo , Colo/microbiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Lactobacillus rhamnosus/fisiologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Transcriptoma
9.
STAR Protoc ; 2(4): 100872, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34746855

RESUMO

We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).


Assuntos
Colo/citologia , Ensaio Cometa/métodos , Dano ao DNA/genética , Mucosa Intestinal/citologia , Animais , Técnicas de Cultura de Células , Camundongos
10.
Physiol Rep ; 9(21): e15099, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34755491

RESUMO

Our previous study indicated that streptozotocin (STZ)-induced diabetes leads to colonic platelet-derived growth factor receptor-α-positive (PDGFRα+ ) cell proliferation accompanied by slow colonic transit in mice; however, the mechanism of this effect is unclear. The present study used western blotting, immunohistochemistry, and quantitative PCR to investigate whether proteinase-activated receptor 2 (PAR2) mediates PDGFRα+ cell proliferation. Our results showed that PDGFRα, PAR2, and Ki-67 coexpression was increased in the diabetic colonic muscle layer. PDGFRα and PAR2 mRNA and protein expression levels were also markedly enhanced in the diabetic colonic muscle layer. Mice treated with 2-furoyl-LIGRLO-amide (2-F-L-a), a PAR2 agonist, exhibited significant colon elongation and increased smooth muscle weight. In the 2-F-L-a-treated mice, PDGFRα, PAR2, and Ki-67 coexpression was increased and PDGFRα and PAR2 mRNA and protein expression was significantly enhanced in the colonic smooth muscle layer. 2-F-L-a also increased proliferation and PDGFRα expression in NIH/3T3 cells cultured in high glucose, while LY294002, a PI3K antagonist, decreased cell proliferation and PDGFRα expression. PI3K and Akt protein and mRNA expression and p-Akt protein expression in diabetic and 2-F-L-a-treated mice were markedly reduced in colonic smooth muscle. 2-F-L-a also reduced PI3K, Akt, and p-Akt protein expression in NIH/3T3 cells, while the PI3K antagonist LY294002 increased this expression. The results indicate that PAR2 is involved in the proliferation of PDGFRα+ cells through the PI3K/Akt signaling pathway in the colon of STZ-induced diabetic mice, which may contribute to the slow transit and constipation that are associated with diabetes.


Assuntos
Proliferação de Células , Colo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Receptor PAR-2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Células Cultivadas , Colo/citologia , Colo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Células NIH 3T3 , Oligopeptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais
11.
Int J Mol Sci ; 22(20)2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34681598

RESUMO

Butyrate is considered the primary energy source of colonocytes and has received wide attention due to its unique health benefits. Insight into the mechanistic effects of butyrate on cellular and metabolic function relies mainly on research in in-vitro-cultured cells. However, cells in culture differ from those in vivo in terms of metabolic phenotype and nutrient availability. For translation, it is therefore important to understand the impact of different nutrients on the effects of butyrate. We investigated the metabolic consequences of butyrate exposure under various culturing conditions, with a focus on the interaction between butyrate and glucose. To investigate whether the effects of butyrate were different between cells with high and low mitochondrial capacity, we cultured HT29 cells under either low- (0.5 mM) or high- (25 mM) glucose conditions. Low-glucose culturing increased the mitochondrial capacity of HT29 cells compared to high-glucose (25 mM) cultured HT29 cells. Long-term exposure to butyrate did not alter mitochondrial bioenergetics, but it decreased glycolytic function, regardless of glucose availability. In addition, both high- and low-glucose-grown HT29 cells showed increased lipid droplet accumulation following long-term butyrate exposure. Acute exposure of cultured cells (HT29 and Caco-2) to butyrate increased their oxygen consumption rate (OCR). A simultaneous decrease in extracellular acidification rate (ECAR) was observed. Furthermore, in the absence of glucose, OCR did not increase in response to butyrate. These results lead us to believe that butyrate itself was not responsible for the observed increase in OCR, but, instead, butyrate stimulated pyruvate flux into mitochondria. Indeed, blocking of the mitochondrial pyruvate carrier prevented a butyrate-induced increase in oxygen consumption. Taken together, our results indicate that butyrate itself is not oxidized in cultured cells but instead alters pyruvate flux and induces lipid accumulation.


Assuntos
Butiratos/farmacologia , Gotículas Lipídicas/metabolismo , Mitocôndrias/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Linhagem Celular Tumoral , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos
12.
Front Immunol ; 12: 727952, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34566985

RESUMO

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.


Assuntos
Separação Celular , Colo/citologia , Citometria de Fluxo , Íleo/citologia , Mucosa Intestinal/citologia , Jejuno/citologia , Fagócitos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fagócitos/imunologia , Fenótipo
13.
Front Immunol ; 12: 727626, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484237

RESUMO

Multiplexed imaging is a recently developed and powerful single-cell biology research tool. However, it presents new sources of technical noise that are distinct from other types of single-cell data, necessitating new practices for single-cell multiplexed imaging processing and analysis, particularly regarding cell-type identification. Here we created single-cell multiplexed imaging datasets by performing CODEX on four sections of the human colon (ascending, transverse, descending, and sigmoid) using a panel of 47 oligonucleotide-barcoded antibodies. After cell segmentation, we implemented five different normalization techniques crossed with four unsupervised clustering algorithms, resulting in 20 unique cell-type annotations for the same dataset. We generated two standard annotations: hand-gated cell types and cell types produced by over-clustering with spatial verification. We then compared these annotations at four levels of cell-type granularity. First, increasing cell-type granularity led to decreased labeling accuracy; therefore, subtle phenotype annotations should be avoided at the clustering step. Second, accuracy in cell-type identification varied more with normalization choice than with clustering algorithm. Third, unsupervised clustering better accounted for segmentation noise during cell-type annotation than hand-gating. Fourth, Z-score normalization was generally effective in mitigating the effects of noise from single-cell multiplexed imaging. Variation in cell-type identification will lead to significant differential spatial results such as cellular neighborhood analysis; consequently, we also make recommendations for accurately assigning cell-type labels to CODEX multiplexed imaging.


Assuntos
Diagnóstico por Imagem/métodos , Análise de Célula Única/métodos , Algoritmos , Análise por Conglomerados , Colo/citologia , Colo/diagnóstico por imagem , Humanos
14.
Science ; 373(6556): 813-818, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385401

RESUMO

A Western-style, high-fat diet promotes cardiovascular disease, in part because it is rich in choline, which is converted to trimethylamine (TMA) by the gut microbiota. However, whether diet-induced changes in intestinal physiology can alter the metabolic capacity of the microbiota remains unknown. Using a mouse model of diet-induced obesity, we show that chronic exposure to a high-fat diet escalates Escherichia coli choline catabolism by altering intestinal epithelial physiology. A high-fat diet impaired the bioenergetics of mitochondria in the colonic epithelium to increase the luminal bioavailability of oxygen and nitrate, thereby intensifying respiration-dependent choline catabolism of E. coli In turn, E. coli choline catabolism increased levels of circulating trimethlamine N-oxide, which is a potentially harmful metabolite generated by gut microbiota.


Assuntos
Colo/fisiologia , Dieta Hiperlipídica , Escherichia coli/metabolismo , Mucosa Intestinal/fisiologia , Metilaminas/metabolismo , Animais , Hipóxia Celular , Colina/administração & dosagem , Colina/metabolismo , Colo/citologia , Metabolismo Energético , Células Epiteliais/fisiologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fezes/microbiologia , Microbioma Gastrointestinal , Inflamação , Mucosa Intestinal/metabolismo , Masculino , Metilaminas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nitratos/metabolismo , Obesidade , Consumo de Oxigênio
15.
Cell Commun Signal ; 19(1): 85, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380509

RESUMO

BACKGROUND: Vitamin D receptor (VDR) plays a vital protective role in oral and colonic epithelial cells. Albeit we know that VDR expression is reduced in the mucosal epithelial layers of autoimmune diseases, the mechanism by which VDR is decreased remains elusive. METHODS: VDR and zinc finger protein 36 (ZFP36) levels in human samples and cell lines were detected by real-time PCR, western blot and immunostaining. Luciferase report assay was used to test cis-elements in VDR gene promoter, real-time PCR was applied to measure mRNA decay and western blot was performed to evaluate protein degradation. RNA affinity chromatography assay was used to test protein-mRNA interaction. Co-immunoprecipitation was used to detect protein-protein interaction. The role of ZFP36 in AU-rich elements (AREs) in the 3' untranslated region (UTR) of VDR mRNA was also measured by luciferase report assay. RESULTS: We identify ZFP36 can bind with the AREs in the 3'UTR of VDR mRNA, leading to mRNA degradation in oral and colonic epithelial cells under inflammatory circumstance. Either ZFP36 protein or AREs of VDR mRNA mutation abolishes this protein-mRNA binding process. After the key amino acid's mutation, ZFP36 fails to decrease VDR mRNA expression. We also find that VDR physically binds with Y box-binding protein 1 (YBX-1) to block YBX-1's nuclear translocation and ameliorate cell death in the presence of inflammation. CONCLUSION: These findings provide insights into the cause of VDR decrease in oral and colonic epithelial cells under inflammatory condition and explain how VDR maintains cell viability in these cells. Video abstract.


Assuntos
Inflamação/genética , RNA Mensageiro/genética , Receptores de Calcitriol/genética , Tristetraprolina/genética , Morte Celular/genética , Linhagem Celular , Colo/citologia , Colo/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Boca/citologia , Boca/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética
16.
Sci Rep ; 11(1): 15889, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354183

RESUMO

Enteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6-8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.


Assuntos
Colo/citologia , Pseudo-Obstrução Intestinal/terapia , Células-Tronco Neurais/citologia , Animais , Técnicas de Cultura de Células/métodos , Colo/fisiologia , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/fisiologia , Feminino , Pseudo-Obstrução Intestinal/fisiopatologia , Intestino Delgado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Crista Neural/citologia , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Organoides/citologia , Organoides/metabolismo , Transplante de Células-Tronco/métodos
17.
Am J Physiol Cell Physiol ; 321(3): C471-C488, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288721

RESUMO

Intestinal NaCl, HCO3-, and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pHi), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi control.


Assuntos
Colo/metabolismo , Enterócitos/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Colo/citologia , Colo/efeitos dos fármacos , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Guanidinas/farmacologia , Células HT29 , Homeostase/genética , Humanos , Concentração de Íons de Hidrogênio , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia
18.
Nutrients ; 13(6)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072741

RESUMO

The colonic epithelium is never exposed to a single factor, therefore studies on the effect of combinations of factors naturally and persistently present in the intestines are of special importance for understanding the phenomena occurring at this place. The aim of the study was to investigate the combined effect of 1 mM phytate and 1 mM butyrate (PA1B1) on cell lines derived from cancer (HCT116 and HT-29) and healthy (NCM460D) human colonic epithelium. Colorimetric and flow cytometry methods were used to determine the proliferation rate, cell cycle, and apoptosis. Selected markers of proliferation, inflammatory, and survival pathways were investigated at the mRNA and/or protein level. The combination of phytate and butyrate disturbed the cell cycle and triggered apoptosis and/or death in both studied cancer colonocytes to a higher extent compared to healthy colonocytes. Moreover, in healthy colonocytes, phytate activated the survival pathway without stimulation of inflammatory response. This may indicate that the response of healthy colonocytes to phytate protects colonic epithelium from the loss of integrity and tightness that would occur if inflammation developed. Based on the obtained results we postulate that studies on both cancer and/or healthy colonocytes should be carried out in the presence of butyrate as the permanent component of colonic contents. This should be of special importance when anti-proliferative/pro-apoptotic activity or inflammatory status of colonocytes is to be investigated.


Assuntos
Apoptose/efeitos dos fármacos , Butiratos/farmacologia , Colo/citologia , Neoplasias do Colo , Ácido Fítico/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dieta , Células HCT116 , Células HT29 , Humanos , Mucosa Intestinal/citologia
19.
Regul Toxicol Pharmacol ; 124: 104967, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34062205

RESUMO

Aloe-emodin, one of the molecules belonging to the group of hydroxyanthracene derivatives, was recently described as genotoxic in vivo. Indeed, the EFSA judged that aloe-emodin, together with other similar molecules (emodin and danthron) and extracts from the leaf of Aloe species containing hydroxyanthracene derivatives, could represent a risk factor for colorectal cancer mediated by a genotoxic effect. Given the marked uncertainty regarding the conclusions in the opinion of the EFSA ANS Panel and conflicts in the epidemiological data on which the opinion is based, a new in vivo study (in vivo alkaline comet assay in mice - OECD 489) was conducted to test the potential genotoxicity of aloe-emodin at doses of 250, 500, 1000 and 2000 mg/kg bw/day on preparations of single cells from the kidney and colon of treated male mice. Following treatment with the test item, no clinical signs were observed in animals in any treatment group. Slight body-weight loss was randomly observed in all groups treated with the test item and was more evident in the groups dosed at 1000 and 2000 mg/kg bw/day. Under these experimental conditions, aloe-emodin showed no genotoxic activity. Possible oxidative damage to colon tissues could not be excluded based on the results obtained after repair enzyme treatment.


Assuntos
Antraquinonas/toxicidade , Dano ao DNA/efeitos dos fármacos , Administração Oral , Animais , Antraquinonas/administração & dosagem , Colo/citologia , Colo/efeitos dos fármacos , Colo/patologia , Ensaio Cometa/métodos , Relação Dose-Resposta a Droga , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Camundongos
20.
JCI Insight ; 6(14)2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34138755

RESUMO

Cancer cells reprogram cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to break down dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here, we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis-associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer-derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth were not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models, and it provides therapeutic targets for colon cancer.


Assuntos
Neoplasias Associadas a Colite/imunologia , Mitocôndrias/metabolismo , Mitofagia/imunologia , Nutrientes/deficiência , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colite/induzido quimicamente , Colite/complicações , Colite/imunologia , Colite/patologia , Neoplasias Associadas a Colite/tratamento farmacológico , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/patologia , Colo/citologia , Colo/imunologia , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Metabolômica , Camundongos , Camundongos Transgênicos , Mitocôndrias/imunologia , Mitofagia/efeitos dos fármacos
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