RESUMEN
Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.
Asunto(s)
Colesterol , Estrés del Retículo Endoplásmico , Receptores sigma , Colesterol/metabolismo , Receptores sigma/metabolismo , Receptores sigma/genética , Humanos , Animales , Ratones , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ligandos , Línea Celular Tumoral , Muerte Celular/efectos de los fármacos , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Humanos , Animales , Ratones , Femenino , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Apoptosis , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Línea Celular TumoralRESUMEN
OBJECTIVE: Neonatal Opioid Withdrawal Syndrome (NOWS) has been associated with the development of necrotizing enterocolitis (NEC) in term and late-preterm neonates. In this study, we used stool gene expression to determine if an increase in baseline inflammation in the intestine of infants with NOWS is associated with these findings. STUDY DESIGN: Stool samples were prospectively collected between days 1-3 and days 4-9 after delivery for opioid-exposed ( n = 9) or non-exposed neonates (n = 8). Stool gene expression for TLR4 and HMGB1 was determined via real-time PCR. RESULTS: TLR4 expression was higher in the stool of the non-exposed group in both time periods, between days 1-3 (P < 0.0001) and days 4-9 (P < 0.05) after delivery. No significant difference in HMGB1 expression was found at either time point (P > 0.05). CONCLUSION: These findings point to an important interplay between opioid exposure and/or NOWS and the inflammatory milieu of the neonatal intestine.
Asunto(s)
Analgésicos Opioides , Enterocolitis Necrotizante , Proteína HMGB1 , Síndrome de Abstinencia Neonatal , Receptor Toll-Like 4 , Humanos , Receptor Toll-Like 4/metabolismo , Recién Nacido , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Femenino , Masculino , Enterocolitis Necrotizante/metabolismo , Enterocolitis Necrotizante/genética , Analgésicos Opioides/efectos adversos , Estudios Prospectivos , Heces/química , Mucosa Intestinal/metabolismo , Estudios de Casos y Controles , Intestinos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field's understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.
Asunto(s)
Enterocolitis Necrotizante , Enfermedades del Recién Nacido , Microbiota , Animales , Lactante , Ratones , Humanos , Recién Nacido , Disbiosis , Células Endoteliales , MicrofluídicaRESUMEN
Introduction: Necrotizing enterocolitis (NEC) is a potentially fatal intestinal disease primarily affecting preterm infants. Early diagnosis of neonates with NEC is crucial to improving outcomes; however, traditional diagnostic tools remain inadequate. Biomarkers represent an opportunity to improve the speed and accuracy of diagnosis, but they are not routinely used in clinical practice. Methods: In this study, we utilized an aptamer-based proteomic discovery assay to identify new serum biomarkers of NEC. We compared levels of serum proteins in neonates with and without NEC and identified ten differentially expressed serum proteins between these groups. Results: We detected two proteins, C-C motif chemokine ligand 16 (CCL16) and immunoglobulin heavy constant alpha 1 and 2 heterodimer (IGHA1 IGHA2), that were significantly increased during NEC and eight that were significantly decreased. Generation of receiver operating characteristic (ROC) curves revealed that alpha-fetoprotein (AUC = 0.926), glucagon (AUC = 0.860), and IGHA1 IGHA2 (AUC = 0.826) were the proteins that best differentiated patients with and without NEC. Discussion: These findings indicate that further investigation into these serum proteins as a biomarker for NEC is warranted. In the future, laboratory tests incorporating these differentially expressed proteins may improve the ability of clinicians to diagnose infants with NEC rapidly and accurately.
RESUMEN
Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Antígenos de Neoplasias , Neoplasias Pancreáticas/terapia , Linfocitos T CD8-positivos , Carcinoma Ductal Pancreático/terapia , Inmunoterapia , Neoplasias PancreáticasRESUMEN
Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease.
Asunto(s)
Células Endoteliales , Enterocolitis Necrotizante , Lactante , Recién Nacido , Humanos , Recien Nacido Prematuro , Mucosa Intestinal , Dispositivos Laboratorio en un ChipRESUMEN
OBJECTIVE: Necrotizing enterocolitis (NEC) is the most common and lethal gastrointestinal disease affecting preterm infants. NEC develops suddenly and is characterized by gut barrier destruction, an inflammatory response, intestinal necrosis and multi-system organ failure. There is currently no method for early NEC detection, and the pathogenesis of NEC remains unclear. DESIGN: To further understand the molecular mechanisms that support NEC, we used solution phase hybridization and next-generation DNA sequencing of bisulfite converted DNA to perform targeted genome-wide analysis of DNA methylation at high read depth. RESULTS: We found that ileal samples from surgical NEC infants (n = 5) exist in a broadly hypermethylated state relative to their non-NEC counterparts (n = 9). These trends were not uniform, with hypermethylation being most consistently observed outside CpG islands and promoters. We further identified several biologically interesting gene promoters that displayed differential methylation in NEC and a number of biological pathways that appear dysregulated in NEC. We also found that DNA methylation patterns identified in ileal NEC tissue were correlated with those found and published previously in stool samples from NEC-affected infants. CONCLUSION: We confirmed that surgical NEC is associated with broad DNA hypermethylation in the ileum, and this may be detectable in stool samples of affected individuals. Thus, an epigenomic liquid biopsy of stool may have significant potential as a biomarker with respect to the diagnostic/predictive detection of NEC. Our findings, along with recent similar observations in colon, suggest that epigenomic dysregulation is a significant feature of surgical NEC. These findings motivate future studies which will involve the longitudinal screening of samples obtained prior to the onset of NEC. Our long-term goal is the development of novel screening, diagnostic and phenotyping methods for NEC.
Asunto(s)
Enterocolitis Necrotizante , Enfermedades del Recién Nacido , Islas de CpG , Metilación de ADN , Enterocolitis Necrotizante/diagnóstico , Enterocolitis Necrotizante/genética , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Intestino Delgado/patologíaRESUMEN
Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.
Asunto(s)
Antígenos de Neoplasias/genética , Muerte Celular , Células Epiteliales/fisiología , Serpinas/genética , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Serpinas/metabolismoRESUMEN
Recapitulating human NEC using animal models has been insightful in dissecting the signaling pathways, immune-mediated mechanisms, genetic signatures, and the intestinal architecture of NEC. This protocol describes an in vivo murine NEC model, using hypoxia and formula containing lipopolysaccharide and enteric bacteria derived from an infant with NEC. With this mouse model, we aim to further dissect NEC pathogenesis and develop new therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Mihi et al. (2021).
Asunto(s)
Modelos Animales de Enfermedad , Enterocolitis Necrotizante , Animales , Animales Recién Nacidos , Heces/química , Hipoxia/fisiopatología , Íleon/patología , Íleon/fisiopatología , Lipopolisacáridos/efectos adversos , RatonesRESUMEN
The application of metabolomics in neonatology offers an approach to investigate the complex relationship between nutrition and infant health. Characterization of the metabolome of human milk enables an investigation into nutrients that affect the neonatal metabolism and identification of dietary interventions for infants at risk of diseases such as necrotizing enterocolitis (NEC). In this study, we aimed to identify differences in the metabolome of breast milk of 48 mothers with preterm infants with NEC and non-NEC healthy controls. A minimum significant difference was observed in the human milk metabolome between the mothers of infants with NEC and mothers of healthy control infants. However, significant differences in the metabolome related to fatty acid metabolism, oligosaccharides, amino sugars, amino acids, vitamins and oxidative stress-related metabolites were observed when comparing milk from mothers with control infants of ≤1.0 kg birth weight and >1.5 kg birth weight. Understanding the functional biological features of mothers' milk that may modulate infant health is important in the future of tailored nutrition and care of the preterm newborn.
Asunto(s)
Recien Nacido Prematuro/metabolismo , Metabolómica , Leche Humana/metabolismo , Madres , Aminoácidos/análisis , Amino Azúcares/análisis , Peso al Nacer , Metabolismo Energético , Ácidos Grasos/análisis , Femenino , Glucólisis , Humanos , Recién Nacido , Masculino , Análisis Multivariante , Oligosacáridos/análisis , Estrés Oxidativo , Análisis de Componente PrincipalRESUMEN
Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal that Il22 expression in neonatal murine intestine is negligible until weaning, and both human and murine neonates lack IL-22 production during NEC. Mice deficient in IL-22 or lacking the IL-22 receptor in the intestine display a similar susceptibility to NEC, consistent with the lack of endogenous IL-22 during development. Strikingly, treatment with recombinant IL-22 during NEC substantially reduces inflammation and enhances epithelial regeneration. These findings may provide a new therapeutic strategy to attenuate NEC.
Asunto(s)
Enterocolitis Necrotizante/inmunología , Interleucinas/genética , Mucosa Intestinal/inmunología , Proteínas Recombinantes/farmacología , Regeneración/inmunología , Animales , Animales Recién Nacidos , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Quimiocina CXCL2/genética , Quimiocina CXCL2/inmunología , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/tratamiento farmacológico , Enterocolitis Necrotizante/microbiología , Enterocolitis Necrotizante/patología , Microbioma Gastrointestinal/inmunología , Regulación del Desarrollo de la Expresión Génica , Humanos , Recién Nacido , Enfermedades del Recién Nacido/inmunología , Enfermedades del Recién Nacido/microbiología , Enfermedades del Recién Nacido/patología , Recien Nacido Prematuro , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucinas/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Regeneración/genética , Transducción de Señal , Destete , Interleucina-22RESUMEN
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.
Asunto(s)
Enterocolitis Necrotizante/patología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Humanos , Recién Nacido , Enfermedades del Recién Nacido/patología , Inflamación/patología , Mucosa Intestinal/patología , Intestinos/patología , Ratones , Ratones Endogámicos C57BL , Nacimiento Prematuro/patología , Transcripción Genética/fisiologíaRESUMEN
Necrotizing enterocolitis (NEC) causes significant morbidity and mortality in premature infants; therefore, the identification of therapeutic and preventative strategies against NEC remains a high priority. The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) is well known to contribute to the regulation of intestinal microbial communities and amelioration of intestinal inflammation. However, the role of AhR signaling in NEC is unclear. Experimental NEC was induced in 4-d-old wild-type mice or mice lacking AhR expression in the intestinal epithelial cells or AhR expression in CD11c+ cells (AhRΔCD11c) by subjecting animals to twice daily hypoxic stress and gavage feeding with formula supplemented with LPS and enteric bacteria. During NEC, compared with wild-type mice treated with vehicle, littermates treated with an AhR proligand, indole-3-carbinol, had reduced expression of Il1b and Marco, a scavenger receptor that mediates dendritic cell activation and the recognition and clearance of bacterial pathogens by macrophages. Furthermore, indole-3-carbinol treatment led to the downregulation of genes involved in cytokine and chemokine, as revealed by pathway enrichment analysis. AhR expression in the intestinal epithelial cells and their cre-negative mouse littermates were similarly susceptible to experimental NEC, whereas AhRΔCD11c mice with NEC exhibited heightened inflammatory responses compared with their cre-negative mouse littermates. In seeking to determine the mechanisms involved in this increased inflammatory response, we identified the Tim-4- monocyte-dependent subset of macrophages as increased in AhRΔCD11c mice compared with their cre-negative littermates. Taken together, these findings demonstrate the potential for AhR ligands as a novel immunotherapeutic approach to the management of this devastating disease.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Enterocolitis Necrotizante/tratamiento farmacológico , Indoles/farmacología , Mucosa Intestinal/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/metabolismo , Enterocolitis Necrotizante/microbiología , Enterocolitis Necrotizante/patología , Humanos , Indoles/uso terapéutico , Interleucina-1beta/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Aim: Neonatal necrotizing enterocolitis (NEC) is a deadly and unpredictable gastrointestinal disease, for which no biomarker exists. We aimed to describe the methylation patterns in stool and colon from infants with NEC. Methods: We performed a high-resolution genome-wide epigenomic analysis using solution-phase hybridization and next-generation sequencing of bisulfite-converted DNA. Results: Our data reveal significant genomic hypermethylation in NEC tissues compared with non-NEC controls. These changes were more pronounced in regions outside CpG islands and gene regulatory elements, suggesting that NEC-specific hypermethylation is not a nonspecific global phenomenon. Conclusions: This study provides evidence of a methylomic signature associated with NEC that is detectable noninvasively and provides a new opportunity for the development of a novel diagnostic method for NEC.
Asunto(s)
Biomarcadores , Metilación de ADN , Susceptibilidad a Enfermedades , Enterocolitis Necrotizante/etiología , Islas de CpG , Enterocolitis Necrotizante/diagnóstico , Enterocolitis Necrotizante/metabolismo , Epigénesis Genética , Epigenómica/métodos , Heces , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Recién Nacido , Masculino , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
Type 17 cytokines have been strongly implicated in mucosal immunity, in part by regulating the production of antimicrobial peptides. Using a mouse model of Citrobacter rodentium infection, which causes colitis, we found that intestinal IL-17RA and IL-17RC were partially required for control of infection in the colon and IL-17 regulates the production of luminal hydrogen peroxide as well as expression of Tnsf13 Reduced Tnfsf13 expression was associated with a profound defect in generating C. rodentium-specific IgA+ Ab-secreting cells. Taken together, intestinal IL-17R signaling plays key roles in controlling invading pathogens, in part by regulating luminal hydrogen peroxide as well as regulating the generation of pathogen-specific IgA+ Ab-secreting cells.
Asunto(s)
Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Inmunoglobulina A Secretora/inmunología , Mucosa Intestinal/inmunología , Oxidorreductasas/inmunología , Receptores de Interleucina-17/inmunología , Transducción de Señal/inmunología , Animales , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/genética , Humanos , Peróxido de Hidrógeno/inmunología , Inmunoglobulina A Secretora/genética , Ratones , Ratones Noqueados , Oxidorreductasas/genética , Receptores de Interleucina-17/genética , Transducción de Señal/genéticaRESUMEN
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.
Asunto(s)
Enterocolitis Necrotizante/genética , Enterocolitis Necrotizante/cirugía , Células Epiteliales/metabolismo , Captura por Microdisección con Láser/métodos , Animales , Estudios de Casos y Controles , Colon/patología , Colon/cirugía , Islas de CpG/genética , Metilación de ADN , Enterocolitis Necrotizante/etiología , Enterocolitis Necrotizante/patología , Epigenómica/métodos , Células Epiteliales/patología , Estudio de Asociación del Genoma Completo/métodos , Humanos , Íleon/patología , Íleon/cirugía , Recién Nacido , Intestinos/patología , Captura por Microdisección con Láser/efectos adversos , Modelos Animales , Análisis de Secuencia de ARN/métodos , Transcriptoma/genéticaRESUMEN
BACKGROUND: Preterm infants are susceptible to unique pathology due to their immaturity. Mouse models are commonly used to study immature intestinal disease, including necrotizing enterocolitis (NEC). Current NEC models are performed at a variety of ages, but data directly comparing intestinal developmental stage equivalency between mice and humans are lacking. METHODS: Small intestines were harvested from C57BL/6 mice at 3-4 days intervals from birth to P28 (n = 8 at each age). Preterm human small intestine samples representing 17-23 weeks of completed gestation were obtained from the University of Pittsburgh Health Sciences Tissue Bank, and at term gestation during reanastamoses after resection for NEC (n = 4-7 at each age). Quantification of intestinal epithelial cell types and messenger RNA for marker genes were evaluated on both species. RESULTS: Overall, murine and human developmental trends over time are markedly similar. Murine intestine prior to P10 is most similar to human fetal intestine prior to viability. Murine intestine at P14 is most similar to human intestine at 22-23 weeks completed gestation, and P28 murine intestine is most similar to human term intestine. CONCLUSION: Use of C57BL/6J mice to model the human immature intestine is reasonable, but the age of mouse chosen is a critical factor in model development.
Asunto(s)
Epitelio/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Intestinos/crecimiento & desarrollo , Animales , Enterocolitis Necrotizante/metabolismo , Epitelio/patología , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Homeostasis , Humanos , Enfermedades Intestinales/metabolismo , Intestinos/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
Importance: Necrotizing enterocolitis (NEC) in preterm infants is an often-fatal gastrointestinal tract emergency. A robust NEC biomarker that is not confounded by sepsis could improve bedside management, lead to lower morbidity and mortality, and permit patient selection in randomized clinical trials of possible therapeutic approaches. Objective: To evaluate whether aberrant intestinal alkaline phosphatase (IAP) biochemistry in infant stool is a molecular biomarker for NEC and not associated with sepsis. Design, Setting, and Participants: This multicenter diagnostic study enrolled 136 premature infants (gestational age, <37 weeks) in 2 hospitals in Louisiana and 1 hospital in Missouri. Data were collected and analyzed from May 2015 to November 2018. Exposures: Infant stool samples were collected between 24 and 40 or more weeks postconceptual age. Enrolled infants underwent abdominal radiography at physician and hospital site discretion. Main Outcomes and Measures: Enzyme activity and relative abundance of IAP were measured using fluorometric detection and immunoassays, respectively. After measurements were performed, biochemical data were evaluated against clinical entries from infants' hospital stay. Results: Of 136 infants, 68 (50.0%) were male infants, median (interquartile range [IQR]) birth weight was 1050 (790-1350) g, and median (IQR) gestational age was 28.4 (26.0-30.9) weeks. A total of 25 infants (18.4%) were diagnosed with severe NEC, 19 (14.0%) were suspected of having NEC, and 92 (66.9%) did not have NEC; 26 patients (19.1%) were diagnosed with late-onset sepsis, and 14 (10.3%) had other non-gastrointestinal tract infections. For severe NEC, suspected NEC, and no NEC samples, median (IQR) fecal IAP content, relative to the amount of IAP in human small intestinal lysate, was 99.0% (51.0%-187.8%) (95% CI, 54.0%-163.0%), 123.0% (31.0%-224.0%) (95% CI, 31.0%-224.0%), and 4.8% (2.4%-9.8%) (95% CI, 3.4%-5.9%), respectively. For severe NEC, suspected NEC, and no NEC samples, median (IQR) enzyme activity was 183 (56-507) µmol/min/g (95% CI, 63-478 µmol/min/g) of stool protein, 355 (172-608) µmol/min/g (95% CI, 172-608 µmol/min/g) of stool protein, and 613 (210-1465) µmol/min/g (95% CI, 386-723 µmol/min/g) of stool protein, respectively. Mean (SE) area under the receiver operating characteristic curve values for IAP content measurements were 0.97 (0.02) (95% CI, 0.93-1.00; P < .001) at time of severe NEC, 0.97 (0.02) (95% CI, 0.93-1.00; P < .001) at time of suspected NEC, 0.52 (0.07) (95% CI, 0.38-0.66; P = .75) at time of sepsis, and 0.58 (0.08) (95% CI, 0.42-0.75; P = .06) at time of other non-gastrointestinal tract infections. Mean (SE) area under the receiver operating characteristic curve values for IAP activity were 0.76 (0.06) (95% CI, 0.64-0.86; P < .001), 0.62 (0.07) (95% CI, 0.48-0.77; P = .13), 0.52 (0.07) (95% CI, 0.39-0.67; P = .68), and 0.57 (0.08) (95% CI, 0.39-0.69; P = .66), respectively. Conclusions and Relevance: In this diagnostic study, high amounts of IAP protein in stool and low IAP enzyme activity were associated with diagnosis of NEC and may serve as useful biomarkers for NEC. Our findings indicated that IAP biochemistry was uniquely able to distinguish NEC from sepsis.