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1.
Sci Rep ; 11(1): 13777, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215818

RESUMO

Necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP) are two of the most common emergencies of the gastrointestinal tract in preterm infants with very low birth weight (VLBW, birth weight < 1500 g). Identification of risk factors among these children is crucial for earlier diagnosis and prompt intervention. In this study, we investigated a relationship between ABO blood groups and the risk for surgical NEC/FIP. We genotyped the ABO locus (rs8176746 and rs8176719) in VLBW infants enrolled in a prospective, population-based cohort study of the German Neonatal Network (GNN). Of the 10,257 VLBW infants, 441 (4.3%) had surgical NEC/FIP. In univariate analyses, the blood group AB was more prevalent in VLBW infants with surgical NEC/FIP compared to non-AB blood groups (OR 1.51, 95% CI 1.07-2.13, p = 0.017; absolute risk difference 2.01%, 95% CI 0.06-3.96%). The association between blood group AB and surgical NEC/FIP was observed in a multivariable logistic regression model (OR of 1.58, 95% CI 1.10-2.26, p = 0.013) as well. In summary, our study suggests that the risk of surgical NEC and FIP is higher in patients with blood group AB and lower in those having non-AB blood groups.


Assuntos
Sistema ABO de Grupos Sanguíneos/sangue , Enterocolite Necrosante/epidemiologia , Doenças do Recém-Nascido/epidemiologia , Doenças do Prematuro/epidemiologia , Perfuração Intestinal/epidemiologia , Pré-Escolar , Enterocolite Necrosante/sangue , Enterocolite Necrosante/patologia , Enterocolite Necrosante/cirurgia , Feminino , Doenças Fetais/sangue , Doenças Fetais/patologia , Doenças Fetais/cirurgia , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/sangue , Doenças do Recém-Nascido/patologia , Doenças do Recém-Nascido/cirurgia , Recém-Nascido Prematuro/sangue , Doenças do Prematuro/patologia , Doenças do Prematuro/cirurgia , Recém-Nascido de muito Baixo Peso , Perfuração Intestinal/sangue , Perfuração Intestinal/patologia , Perfuração Intestinal/cirurgia , Masculino , Fatores de Risco
2.
Mol Med Rep ; 24(3)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34296298

RESUMO

The miR­34a/SIRT1 signaling axis is an important signaling axis in tumors and diseases. Notably, low SIRT1 expression in the intestinal tissues of patients with necrotizing enterocolitis (NEC) has been reported. However, whether miR­34a/SIRT1 signaling as a target to protect the intestines during the NEC process is unclear and remains to be elucidated. Blood samples were collected from 30 patients with NEC, and an NEC rat model was used. The miR­34a and SIRT1 gene and protein expression levels were assayed by qPCR and Western blotting method. The inflammatory cytokine levels and oxidative stress levels were detected using the ELISA method. The results demonstrated that birth weight, albumin and glucose concentrations were significantly decreased in the NEC patient group compared with the control group, but the C­reactive protein (CRP) and procalcitonin (PCT) concentrations were significantly increased. The miR­34a expression level was notably increased in the NEC group, but the SIRT1 expression level was markedly decreased. Notably, the miR­34a was significantly correlated with NEC severity and the concentrations of CRP, PCT, IL­6, TNF­α, IL­1ß, IL­8, MCP­1, VCAM1 and malondialdehyde (MDA), but was significantly negatively correlated with SIRT1 gene expression and the concentration of IL­10. Intestinal villi damage in NEC rats was decreased with miR­34a inhibition and SIRT1 activation treatment by decreasing the levels of inflammatory cytokines, including IL­6, TNF­α, IL­1ß and IL­8, and oxidative stress proteins, including MCP­1, VCAM1, and MDA, as well as increasing the level of the anti­inflammatory cytokine IL­10. In addition, the results indicated that miR­34a inhibition and SIRT1 activation strongly protected the intestine and decreased the damage caused by NEC, not only by decreasing the protein levels of SIRT1, TNF­α, IL­1ß, IL­6 and IL­8, but also by increasing the IL­10 protein levels. The miR­34a inhibition and SIRT1 activation may decrease the damage caused by NEC by decreasing proinflammatory cytokines and oxidative stress proteins and by increasing the anti­inflammatory cytokine pathway. Based on the aforementioned analysis, the miR­34a and SIRT1 proteins may be potential novel therapeutic targets in NEC.


Assuntos
Enterocolite Necrosante/metabolismo , Inflamação/metabolismo , MicroRNAs/metabolismo , Sirtuína 1/metabolismo , Animais , Animais Recém-Nascidos , Correlação de Dados , Citocinas/sangue , Citocinas/genética , Modelos Animais de Doenças , Regulação para Baixo/genética , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Feminino , Humanos , Recém-Nascido , Masculino , MicroRNAs/antagonistas & inibidores , Estresse Oxidativo/genética , Ratos Sprague-Dawley , Transdução de Sinais/genética , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética
3.
J Exp Med ; 218(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34269788

RESUMO

Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/Mϕ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/Mϕ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/Mϕ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.


Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Enterocolite Necrosante/patologia , Mucosa Gástrica/patologia , Monócitos/patologia , Receptores de Superfície Celular , Receptores de IgG , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Vasos Sanguíneos/patologia , Estudos de Casos e Controles , Quimiotaxia , Enterocolite Necrosante/cirurgia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Lactente , Recém-Nascido , Intestino Delgado/irrigação sanguínea , Intestino Delgado/patologia , Monócitos/imunologia , Neutropenia/etiologia , Neutropenia/patologia , Neutrófilos/patologia , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Análise de Sequência de RNA , Análise de Célula Única
4.
PLoS One ; 16(6): e0246412, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34111125

RESUMO

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.


Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Estresse Fisiológico , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Enterocolite Necrosante/fisiopatologia , Fórmulas Infantis
5.
Methods Mol Biol ; 2321: 101-110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34048010

RESUMO

Necrotizing enterocolitis (NEC) is an acute inflammatory disease that unforeseeably develops in very low birth weight premature infants. NEC is characterized by impairment of the intestinal barrier resulting in intestinal necrosis and multisystem organ failure. Animal models of NEC have contributed significantly to a better understanding of the underlying molecular mechanisms of the disease and facilitated the exploration of potential new therapeutic strategies. Here, we provide a detailed protocol that recapitulates some of the main histological and transcriptional features of human NEC in newborn mice.


Assuntos
Enterocolite Necrosante/patologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , Recém-Nascido , Doenças do Recém-Nascido/patologia , Inflamação/patologia , Mucosa Intestinal/patologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nascimento Prematuro/patologia , Transcrição Genética/fisiologia
7.
Sci Rep ; 11(1): 7200, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785826

RESUMO

The intestinal stroma provides an important microenvironment for immune cell activation. The perturbation of this tightly regulated process can lead to excessive inflammation. We know that upregulated Toll-like receptor 4 (TLR4) in the intestinal epithelium plays a key role in the inflammatory condition of preterm infants, such as necrotizing enterocolitis (NEC). However, the surrounding stromal contribution to excessive inflammation in the pre-term setting awaits careful dissection. Ex vivo co-culture of embryonic day 14.5 (E14.5) or adult murine intestinal stromal cells with exogenous monocytes was undertaken. We also performed mRNAseq analysis of embryonic and adult stromal cells treated with vehicle control or lipopolysaccharide (LPS), followed by pathway and network analyses of differentially regulated transcripts. Cell characteristics were compared using flow cytometry and pHrodo red phagocytic stain, candidate gene analysis was performed via siRNA knockdown and gene expression measured by qPCR and ELISA. Embryonic stromal cells promote the differentiation of co-cultured monocytes to CD11bhighCD11chigh mononuclear phagocytes, that in turn express decreased levels of CD103. Global mRNAseq analysis of stromal cells following LPS stimulation identified TLR signaling components as the most differentially expressed transcripts in the immature compared to adult setting. We show that CD14 expressed by CD11b+CD45+ embryonic stromal cells is a key inducer of TLR mediated inflammatory cytokine production and phagocytic activity of monocyte derived cells. We utilise transcriptomic analyses and functional ex vivo modelling to improve our understanding of unique molecular cues provided by the immature intestinal stroma.


Assuntos
Enterocolite Necrosante/patologia , Inflamação/patologia , Intestinos/patologia , Monócitos/patologia , Células Estromais/patologia , Animais , Células Cultivadas , Técnicas de Cocultura , Enterocolite Necrosante/genética , Redes Reguladoras de Genes , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Células Estromais/metabolismo , Transcriptoma
8.
J Neuroinflammation ; 18(1): 66, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33676524

RESUMO

BACKGROUND: Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal disease in premature neonates with high mortality and morbidity, while the underlining mechanism of intestinal injury and profound neurological dysfunction remains unclear. Here, we aimed to investigate the involvement of NLPR3 inflammasome activation in NEC-related enterocolitis and neuroinflammation, especially long-term cognitive impairment, meanwhile, explore the protective effect of NLRP3 inhibitor MCC950 on NEC in mice. METHODS: NLRP3 inflammasome activation in the intestine and brain was assessed in the NEC mouse model, and NLRP3 inhibitor MCC950 was administrated during the development of NEC. Survival rate, histopathological injury of the intestine and brain, and expression of mature IL-1ß and other pro-inflammatory cytokines were analyzed. Long-term cognitive impairment was evaluated by behavioral test. RESULTS: The expression of NLRP3 and mature IL-1ß in the intestine and brain was greatly upregulated in NEC mice compared to the controls. MCC950 treatment efficiently improved NEC survival rate, reduced intestinal and brain inflammation, and ameliorated the severity of pathological damage in both organs. Additionally, in vivo blockage of NLRP3 inflammasome with MCC950 in early life of NEC pups potently protected against NEC-associated long-term cognitive impairment. CONCLUSIONS: Our findings suggest that NLRP3 inflammasome activation participates in NEC-induced intestinal and brain injury, and early intervention with NLRP3 inhibitor may provide beneficial therapeutic effect on NEC infants.


Assuntos
Disfunção Cognitiva/imunologia , Enterocolite Necrosante/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Furanos/farmacologia , Indenos/farmacologia , Inflamassomos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sulfonamidas/farmacologia
9.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669331

RESUMO

Chorioamnionitis, an important cause of preterm birth, is linked to necrotizing enterocolitis (NEC). NEC is characterized by a disrupted mucus barrier, goblet cell loss, and endoplasmic reticulum (ER) stress of the intestinal epithelium. These findings prompted us to investigate the mechanisms underlying goblet cell alterations over time in an ovine chorioamnionitis model. Fetal lambs were intra-amniotically (IA) exposed to lipopolysaccharides (LPS) for 5, 12, or 24 h, or 2, 4, 8, or 15 d before premature delivery at 125 d gestational age (GA). Gut inflammation, the number, distribution, and differentiation of goblet cells, ER stress, and apoptosis were measured. We found a biphasic reduction in goblet cell numbers 24 h-2 d after, and 15 d after IA LPS exposure. The second decrease of goblet cell numbers was preceded by intestinal inflammation, apoptosis, and crypt ER stress, and increased SAM-pointed domain-containing ETS transcription factor (SPDEF)-positive cell counts. Our combined findings indicated that ER stress drives apoptosis of maturating goblet cells during chorioamnionitis, ultimately reducing goblet cell numbers. As similar changes have been described in patients suffering from NEC, these findings are considered to be clinically important for understanding the predecessors of NEC, and targeting ER stress in this context is interesting for future therapeutics.


Assuntos
Corioamnionite/patologia , Corioamnionite/veterinária , Enterocolite Necrosante/patologia , Enterocolite Necrosante/reabilitação , Enterocolite Necrosante/veterinária , Feto/patologia , Células Caliciformes/patologia , Animais , Animais Recém-Nascidos , Apoptose , Contagem de Células , Diferenciação Celular , Corioamnionite/induzido quimicamente , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Enterocolite Necrosante/induzido quimicamente , Feminino , Idade Gestacional , Humanos , Lipopolissacarídeos/efeitos adversos , Gravidez , Nascimento Prematuro , Ovinos
10.
Am J Physiol Gastrointest Liver Physiol ; 320(4): G658-G674, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33566727

RESUMO

Necrotizing enterocolitis (NEC), a life-threatening intestinal disease, is becoming a larger proportionate cause of morbidity and mortality in premature infants. To date, therapeutic options remain elusive. Based on recent cell therapy studies, we investigated the effect of a human placental-derived stem cell (hPSC) therapy on intestinal damage in an experimental NEC rat pup model. NEC was induced in newborn Sprague-Dawley rat pups for 4 days via formula feeding, hypoxia, and LPS. NEC pups received intraperitoneal (ip) injections of either saline or hPSC (NEC-hPSC) at 32 and 56 h into NEC induction. At 4 days, intestinal macroscopic and histological damage, epithelial cell composition, and inflammatory marker expression of the ileum were assessed. Breastfed (BF) littermates were used as controls. NEC pups developed significant bowel dilation and fragility in the ileum. Further, NEC induced loss of normal villi-crypt morphology, disruption of epithelial proliferation and apoptosis, and loss of critical progenitor/stem cell and Paneth cell populations in the crypt. hPSC treatment improved macroscopic intestinal health with reduced ileal dilation and fragility. Histologically, hPSC administration had a significant reparative effect on the villi-crypt morphology and epithelium. In addition to a trend of decreased inflammatory marker expression, hPSC-NEC pups had increased epithelial proliferation and decreased apoptosis when compared with NEC littermates. Further, the intestinal stem cell and crypt niche that include Paneth cells, SOX9+ cells, and LGR5+ stem cells were restored with hPSC therapy. Together, these data demonstrate hPSC can promote epithelial healing of NEC intestinal damage.NEW & NOTEWORTHY These studies demonstrate a human placental-derived stem cell (hPSC) therapeutic strategy for necrotizing enterocolitis (NEC). In an experimental model of NEC, hPSC administration improved macroscopic intestinal health, ameliorated epithelial morphology, and supported the intestinal stem cell niche. Our data suggest that hPSC are a potential therapeutic approach to attenuate established intestinal NEC damage. Further, we show hPSC are a novel research tool that can be utilized to elucidate critical neonatal repair mechanisms to overcome NEC.


Assuntos
Apoptose , Proliferação de Células , Enterocolite Necrosante/cirurgia , Íleo/patologia , Mucosa Intestinal/patologia , Celulas de Paneth/patologia , Placenta/transplante , Transplante de Células-Tronco , Animais , Animais Recém-Nascidos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/genética , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Feminino , Humanos , Íleo/metabolismo , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Celulas de Paneth/metabolismo , Placenta/citologia , Gravidez , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição SOX9 , Nicho de Células-Tronco , Cicatrização
11.
Eur J Immunol ; 51(5): 1110-1125, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547649

RESUMO

Intestinal trefoil factor 3 (TFF3) plays an important role in repairing the intestinal mucosa. However, the detailed mechanism regarding immune regulation by TFF3 is not well defined. Here, we reported that treatment of mouse BM cells and human peripheral blood mononuclear cells from healthy volunteers with TFF3 activated polymorphnuclear myeloid-derived suppressor cells (PMN-MDSCs) in vitro. We also found that prostaglandin E2 is a major TFF3-mediated MDSC target, and that NF-κB/COX2 signaling was involved in this process. Moreover, TFF3 treatment or transfer of TFF3-derived PMN-MDSCs (TFF3-MDSCs) to experimental necrotizing enterocolitis (NEC) mice caused PMN-MDSC accumulation in the lamina propria (LP), which was associated with decreased intestinal inflammation, permeability, bacterial loading, and prolonged survival. Interestingly, no NEC severity remission was observed in Rag1 KO mice that were given TFF3-MDSCs, but coinjection with CD4+ T cells significantly relieved NEC inflammation. Overall, TFF3 mediates the NF-κB/COX2 pathway to regulate PMN-MDSC activation and attenuates NEC in a T-cell-dependent manner, which suggests a novel mechanism in preventing NEC occurrence.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/metabolismo , Células Supressoras Mieloides/metabolismo , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Transdução de Sinais , Fator Trefoil-3/genética , Animais , Animais Recém-Nascidos , Dinoprostona/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Enterocolite Necrosante/patologia , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/imunologia , Neutrófilos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator Trefoil-3/metabolismo
12.
Nat Commun ; 12(1): 1042, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589625

RESUMO

Necrotizing enterocolitis (NEC) is a disease of premature infants characterized by acute intestinal necrosis. Current dogma suggests that NEC develops in response to post-natal dietary and bacterial factors, and so a potential role for in utero factors in NEC remains unexplored. We now show that during pregnancy, administration of a diet rich in the aryl hydrocarbon receptor (AHR) ligand indole-3-carbinole (I3C), or of breast milk, activates AHR and prevents NEC in newborn mice by reducing Toll-like receptor 4 (TLR4) signaling in the newborn gut. Protection from NEC requires activation of AHR in the intestinal epithelium which is reduced in mouse and human NEC, and is independent of leukocyte activation. Finally, we identify an AHR ligand ("A18") that limits TLR4 signaling in mouse and human intestine, and prevents NEC in mice when administered during pregnancy. In summary, AHR signaling is critical in NEC development, and maternally-delivered, AHR-based therapies may alleviate NEC.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Enterocolite Necrosante/genética , Indóis/administração & dosagem , Leite Humano/fisiologia , Receptores de Hidrocarboneto Arílico/genética , Receptor 4 Toll-Like/genética , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/imunologia , Dieta/métodos , Modelos Animais de Doenças , Enterocolite Necrosante/imunologia , Enterocolite Necrosante/patologia , Enterocolite Necrosante/prevenção & controle , Feminino , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Ligantes , Exposição Materna , Camundongos , Gravidez , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/imunologia , Transdução de Sinais , Suínos , Receptor 4 Toll-Like/imunologia
13.
Inflamm Res ; 70(3): 343-358, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33564933

RESUMO

BACKGROUND: Activation of intestinal macrophages is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC), yet its precise mechanisms remain unclear. OBJECTIVE: The purpose of this study is to investigate the role of macrophages and TNF-α via an inflammatory MicroRNA in NEC. MATERIALS AND METHODS: Immunofluorescence (IF) staining of CD68, iNOS, and Arg-1 was employed to identify phenotypes of macrophage in the intestines of NEC infants and NEC mice. Expression of TNF-α, c-kit, and miR-222 was evaluated by qRT-PCR, Western blot, and immunochemical staining from the tissue samples. RESULTS: Large number of M1 macrophage infiltration was found in the NEC intestines. Expression of CD68, iNOS, and TNF-α were significantly increased, while c-kit was decreased distinctly in the NEC group. In the early phase of NEC mouse model, inhibition of M1 macrophages reduced the incidence of NEC and intestinal inflammation. We found that TNF-α upregulated the expression of miRNA-222 and inhibited the expression of c-kit. Conversely, such decrease of c-kit expression could be reversed by miR-222 antagonists. Furtherly, dual-luciferase assay confirmed that c-kit can be inhibited by miR-222 directly. CONCLUSION: Macrophages activation in NEC intestine results in an increased inflammatory response and TNF-α production, accompanied with miR-222 upregulation and c-kit suppression. Modulations of M1 macrophages, TNF-α or miR-222 may be potential therapeutic targets for NEC treatment.


Assuntos
Enterocolite Necrosante/imunologia , Macrófagos/imunologia , MicroRNAs , Proteínas Proto-Oncogênicas c-kit/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Enterocolite Necrosante/patologia , Feminino , Humanos , Recém-Nascido , Intestino Delgado/imunologia , Intestino Delgado/patologia , Ativação de Macrófagos , Masculino , Camundongos Endogâmicos C57BL , Regulação para Cima
14.
Pediatr Surg Int ; 37(3): 325-332, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33547933

RESUMO

PURPOSE: Necrotizing enterocolitis (NEC) is a severe neonatal gastrointestinal disease that can cause damage to remote organs. Previous studies have shown that inflammatory and oxidative injury occur in the liver during NEC. Mitochondrial DNA (mtDNA) plays an important role in hepatic injuries of many other diseases. We aimed to investigate the mechanism of mitochondrial dysfunction in hepatic oxidative injury during NEC. METHODS: NEC was induced in C57BL/6 mice (approval: 44032) by hypoxia, gavage feeding with hyperosmolar formula, and lipopolysaccharide administration from postnatal days 5 to 9 (n = 15). Two additional groups with hypoxia only (n = 10) and hypoxia and hyperosmolar formula (n = 13) were also examined. Breastfed pups were used as control (n = 15). Liver was harvested on postnatal day 9. Gene expressions of mtDNA markers cytochrome c oxidase subunit 3 (COX3), cytochrome b (CYTB) and NADH-ubiquinone oxidoreductase chain 1 (ND1) were measured by real-time qPCR. Mitochondrial morphology marker HSP60 and oxidative stress marker NRF2 were detected by immunofluorescence staining and compared between NEC and control. Data were presented as mean ± SD and compared using Student's t test; p < 0.05 was considered significant. RESULTS: Gene expression of mtDNA markers (COX3, CYTB, and ND1) were significantly decreased in the liver of NEC mice relative to control, hypoxia alone, and hypoxia with hyperosmolar formula. Immunofluorescence showed depletion of HSP60 indicating decreased mitochondria in NEC liver relative to control. Furthermore, a higher protein expression of NRF2 was observed indicating higher oxidative stress in NEC liver relative to control. CONCLUSIONS: Intestinal injury in experimental NEC leads to a systemic inflammatory response affecting the liver. Hepatic oxidative injury in NEC is characterized by decreased mitochondria and mtDNA depletion. This study provides insight into the mechanism of liver injury in NEC.


Assuntos
Enterocolite Necrosante/patologia , Fígado/metabolismo , Mitocôndrias/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Modelos Animais de Doenças , Nutrição Enteral/efeitos adversos , Expressão Gênica , Humanos , Hipóxia , Recém-Nascido , Doenças do Recém-Nascido , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo
15.
Mol Immunol ; 131: 23-32, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33465592

RESUMO

Necrotizing enterocolitis (NEC) is a devastating neonatal gastrointestinal emergency. Fucosylated glycans on intestinal epithelial cells (IECs) play a central role in the maintenance of intestinal homeostasis. Nevertheless, its association with necrotizing enterocolitis is not clear. We examined paraffin-embedded intestinal specimens from participants and found that the NEC patients showed lower intestinal epithelial fucosylation levels than the control patients. In the mouse model of NEC, the percentage of fucosylated epithelial cells (F-ECs) and ILC3s was decreased. Also, the expression levels of IL-22 and Fut2 were reduced. Moreover, the critical role of epithelial fucosylation in NEC was further confirmed by administering the anti-IL-22 antibody, which caused an increase in histological damage, body weight loss, intestinal permeability and proinflammatory cytokine release correlated with a reduction of F-ECs. Overall, intestinal fucosylation deficiency led to increased susceptibility and severity of NEC. Further studies are needed to determine whether modification of intestinal fucosylation affects the development of NEC.


Assuntos
Enterocolite Necrosante/patologia , Células Epiteliais/patologia , Mucosa Intestinal/patologia , Intestinos/patologia , Animais , Pré-Escolar , Citocinas/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/metabolismo , Células Epiteliais/metabolismo , Feminino , Fucosiltransferases/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucinas/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Camundongos
16.
Bosn J Basic Med Sci ; 21(1): 33-38, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32651971

RESUMO

Tumor necrosis factor-alpha (TNF-α) has an important role in hypoxia/reoxygenation (H/R)-induced intestinal damage. It was shown that blocking TNF-α with infliximab has beneficial effects on experimental necrotizing enterocolitis and hypoxic intestinal injury. However, there is no data about the effect of adalimumab on H/R-induced intestinal damage. Therefore, we aimed to determine potential dose-dependent benefits of adalimumab in such damage in neonatal rats. Wistar albino rat pups were assigned to one of the four groups: control group, hypoxia group, low-dose adalimumab (5 mg/kg/day) treated group (LDAT), and high-dose adalimumab (50 mg/kg/day) treated group (HDAT). On the fourth day of the experiment, all rats except for the control group were exposed to H/R followed by euthanasia. Malondialdehyde (MDA), myeloperoxidase (MPO), TNF-α, total antioxidant capacity (TAC), and total oxidant capacity (TOC) were measured in intestinal tissue. TAC and TOC values were used to calculate the oxidative stress index (OSI). Histopathological injury scores (HIS) were also evaluated in the tissue samples. MDA levels were significantly lower in the LDAT and HDAT groups (p < 0.001). TNF-α levels were significantly lower in the LDAT group (p < 0.001). OSI was significantly higher in the H/R group than in the control and LDAT groups (p < 0.001). Mean HIS values in the LDAT group were significantly lower than those in the H/R and HDAT groups (p < 0.001). This experimental study showed that low-dose adalimumab appears to have a beneficial effect on intestinal injury induced with H/R in neonatal rats.


Assuntos
Adalimumab/farmacologia , Enterocolite Necrosante/tratamento farmacológico , Hipóxia/patologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Adalimumab/administração & dosagem , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Malondialdeído/metabolismo , Estresse Oxidativo , Peroxidase/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo
17.
FASEB J ; 35(1): e21265, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33373067

RESUMO

Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases affecting premature infants. It has been shown that NEC is associated with disrupted intestinal barrier and dysregulated endoplasmic reticulum (ER)-stress response. It has also been shown that stem cells derived from amniotic fluid (AFSC) rescued intestinal injury in experimental NEC. Herein, we hypothesized that the beneficial effects of AFSC in the injured intestine are due to the restoration of intestinal barrier function. We evaluated intestinal barrier function using an ex vivo intestinal organoid model of NEC. We found that AFSC restored the expression and localization of tight junction proteins in intestinal organoids, and subsequently decreased epithelial permeability. AFSC rescued tight junction expression by inducing a protective ER stress response that prevents epithelial cell apoptosis in injured intestinal organoids. Finally, we validated these results in our experimental mouse model of NEC and confirmed that AFSC induced sustained ER stress and prevented intestinal apoptosis. This response led to the restoration of tight junction expression and localization, which subsequently reduced intestinal permeability in NEC pups. These findings confirm that intestinal barrier function is disrupted during NEC intestinal injury, and further demonstrate the disruption can be reversed by the administration of AFSC through the activation of the ER stress pathway. This study provides insight into the pathogenesis of NEC and highlights potential therapeutic targets for the treatment of NEC.


Assuntos
Líquido Amniótico/metabolismo , Estresse do Retículo Endoplasmático , Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco/metabolismo , Junções Íntimas/metabolismo , Animais , Apoptose , Enterocolite Necrosante/patologia , Mucosa Intestinal/patologia , Camundongos , Organoides/metabolismo , Organoides/patologia , Permeabilidade , Ratos , Células-Tronco/patologia , Junções Íntimas/patologia
19.
Clin Epigenetics ; 12(1): 190, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33308304

RESUMO

BACKGROUND: Necrotizing enterocolitis (NEC) remains one of the overall leading causes of death in premature infants, and the pathogenesis is unpredictable and not well characterized. The aim of our study was to determine the molecular phenotype of NEC via transcriptomic and epithelial cell-specific epigenomic analysis, with a specific focus on DNA methylation. METHODS: Using laser capture microdissection, epithelial cell-specific methylation signatures were characterized by whole-genome bisulfite sequencing of ileal and colonic samples at the time of surgery for NEC and after NEC had healed at reanastomosis (n = 40). RNA sequencing was also performed to determine the transcriptomic profile of these samples, and a comparison was made to the methylome data. RESULTS: We found that surgical NEC has a considerable impact on the epigenome by broadly increasing DNA methylation levels, although these effects are less pronounced in genomic regions associated with the regulation of gene expression. Furthermore, NEC-related DNA methylation signatures were influenced by tissue of origin, with significant differences being noted between colon and ileum. We also identified numerous transcriptional changes in NEC and clear associations between gene expression and DNA methylation. CONCLUSIONS: We have defined the intestinal epigenomic and transcriptomic signatures during surgical NEC, which will advance our understanding of disease pathogenesis and may enable the development of novel precision medicine approaches for NEC prediction, diagnosis and phenotyping.


Assuntos
Enterocolite Necrosante/genética , Enterocolite Necrosante/cirurgia , Células Epiteliais/metabolismo , Microdissecção e Captura a Laser/métodos , Animais , Estudos de Casos e Controles , Colo/patologia , Colo/cirurgia , Ilhas de CpG/genética , Metilação de DNA , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/patologia , Epigenômica/métodos , Células Epiteliais/patologia , Estudo de Associação Genômica Ampla/métodos , Humanos , Íleo/patologia , Íleo/cirurgia , Recém-Nascido , Intestinos/patologia , Microdissecção e Captura a Laser/efeitos adversos , Modelos Animais , Análise de Sequência de RNA/métodos , Transcriptoma/genética
20.
Nat Commun ; 11(1): 5794, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188181

RESUMO

Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/TH17 polarization. In murine NEC, pro-inflammatory type 3 NKp46-RORγt+Tbet+ innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46+RORγt+ ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.


Assuntos
Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/imunologia , Imunidade Adaptativa , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Enterocolite Necrosante/sangue , Enterocolite Necrosante/patologia , Homeostase , Humanos , Imunidade Inata , Recém-Nascido , Mediadores da Inflamação/metabolismo , Interleucina-1 , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Toll-Like/metabolismo
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