Blood transcriptomic markers of necrotizing enterocolitis in preterm pigs.

BACKGROUND: Necrotizing enterocolitis (NEC), a severe gut disorder in preterm infants, is difficult to predict due to poor specificity and sensitivity of clinical signs and biomarkers. Using preterm piglets as a model, we hypothesized that early development of NEC affects blood gene expression, potentially related to early systemic immune responses. METHODS: A retrospective analysis of clinical, tissue, and blood data was performed on 129 formula-fed piglets with NEC diagnosis at necropsy on day 5. Subgroups of NEC (n = 20) and control piglets (CON, n = 19) were analyzed for whole-blood transcriptome. RESULTS: Preterm piglets had variable NEC lesions, especially in the colon region, without severe clinical signs (e.g. normal growth, activity, hematology, digestion, few piglets with bloody stools). Transcriptome analysis showed 344 differentially expressed genes (DEGs) between NEC and CON piglets. Validation experiment showed that AOAH, ARG2, FKBP5, PAK2, and STAT3 were among the genes affected by severe lesions on day 5, when analyzed in whole blood and in dried blood spots (DBS). CONCLUSION: Whole-blood gene expressions may be affected in preterm pigs before clinical signs of NEC get severe. Blood gene expression analysis, potentially using DBS samples, is a novel tool to help identify new early biomarkers of NEC. IMPACT: Preterm pig model was used to investigate if blood transcriptomics could be used to identify new early blood biomarkers of NEC progression. Whole-blood transcriptome revealed upregulation of target genes in NEC cases when clinical symptoms are subtle, and mainly colon regions were affected. Differential NEC-associated gene expressions could be detected also in dried blood spots, potentially allowing easy collection of small blood volumes in infants.

Subclinical necrotizing enterocolitis-induced systemic immune suppression in neonatal preterm pigs.

Preterm infants are at high risks of sepsis and necrotizing enterocolitis (NEC). Some develop sepsis shortly after suspected or confirmed NEC, implying that NEC may predispose to sepsis but the underlying mechanisms are unknown. Using NEC-sensitive preterm pigs as models, we investigated the immune status in animals following development of subclinical NEC-like lesions with variable severities. Caesarean-delivered preterm pigs were reared until day 5 or day 9. Blood was analyzed for T-cell subsets, neutrophil phagocytosis, transcriptomics, and immune responses to in vitro LPS challenge. Gut tissues were used for histology and cytokine analyses. Pigs with/without macroscopic NEC lesions were scored as healthy, mild, or severe NEC. Overall NEC incidence was similar on day 5 and day 9 (61%-62%) but with lower severity on day 9, implying gradual mucosal repair following the early phase of NEC. Pigs with NEC showed decreased goblet cell density and increased MPO+ and CD3+ cell infiltration in the distal small intestine or colon. Mild or severe NEC lesions had limited effects on circulating parameters on day 5. On day 9, pigs with NEC lesions (especially severe lesions) showed systemic immune suppression, as indicated by elevated Treg frequency, impaired neutrophil phagocytosis, low expression of genes related to innate immunity and Th1 polarization, and diminished LPS-induced immune responses. In conclusion, we shows evidence for NEC-induced systemic immune suppression, even with mild and subclinical NEC lesions. The results help to explain that preterm infants suffering from NEC may show high sensitivity to later secondary infections and sepsis.NEW & NOTEWORTHY Necrotizing enterocolitis (NEC) and sepsis are common diseases in preterm infants. Many develop sepsis following an episode of suspected NEC, suggesting NEC as a predisposing factor for sepsis but mechanisms are unclear. Using preterm pigs as a model, now we show that subclinical NEC lesions, independent of clinical confounding factors, induces systemic immune suppression. The results may help to explain the increased risks of infection and sepsis in preterm infants with previous NEC diagnosis.

Prenatal inflammation suppresses blood Th1 polarization and gene clusters related to cellular energy metabolism in preterm newborns.

Chorioamnionitis (CA, fetal membrane inflammation) predisposes to preterm birth and is associated with increased neonatal infection risk, but the separate effects of prematurity, CA, and postnatal adaptations on this risk are unclear. Using pigs as models for infants, we examined the systemic immune-metabolic status in cesarean-delivered preterm pigs, with and without CA induced by intra-amniotic (IA) LPS exposure. At birth, cord blood of preterm pigs showed neutropenia and low expressions of innate and adaptive immune genes, relative to term pigs. IA LPS induced CA and fetal systemic innate immune activation via complement and neutrophil-related pathways. These were mainly modulated via cellular regulations rather than granulopoiesis, as validated by the in vitro LPS stimulation of cord blood. After birth, IA LPS-exposed preterm pigs did not follow normal immune-metabolic ontogenies found in fetuses or newborns without prenatal insults, but showed consistently high levels of Treg, impaired Th1 polarization, and reduced expressions of multiple genes related to cellular oxidative phosphorylation and ribosomal activities. In conclusion, our results provide cellular and molecular evidence for CA-induced distinct neonatal immune-metabolic status with increased disease tolerance strategy, suggesting mechanisms for the clinical observation of elevated sepsis risks in immune-compromised preterm infants born with CA.

Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation.

Prenatal inflammation may predispose to preterm birth and postnatal inflammatory disorders such as necrotizing enterocolitis (NEC). Bioactive milk ingredients may help to support gut maturation in such neonates, but mother's milk is often insufficient after preterm birth. We hypothesized that supplementation with bioactive ingredients from bovine milk [osteopontin (OPN), caseinoglycomacropeptide (CGMP), colostrum (COL)] supports gut, immunity, and NEC resistance in neonates born preterm after gram-negative infection before birth. Using preterm pigs as a model for preterm infants, fetal pigs were given intraamniotic injections of lipopolysaccharide (LPS; 1 mg/fetus) and delivered 3 days later (90% gestation). For 5 days, groups of LPS-exposed pigs were fed formula (FOR), bovine colostrum (COL), or formula enriched with OPN or CGMP. LPS induced intraamniotic inflammation and postnatal systemic inflammation but limited effects on postnatal gut parameters and NEC. Relative to FOR, COL feeding to LPS-exposed pigs showed less diarrhea, NEC severity, reduced gut IL-1ß and IL-8 levels, greater gut goblet cell density and digestive enzyme activities, and blood helper T-cell fraction. CGMP improved neonatal arousal and gut lactase activities and reduced LPS-induced IL-8 secretion in intestinal epithelial cells (IECs) in vitro. Finally, OPN tended to reduce diarrhea and stimulated IEC proliferation in vitro. No effects on villus morphology, circulating cytokines, or colonic microbiota were observed among groups. In conclusion, bioactive milk ingredients exerted only modest effects on gut and systemic immune parameters in preterm pigs exposed to prenatal inflammation. Short-term, prenatal exposure to inflammation may render the gut less sensitive to immune-modulatory milk effects. NEW & NOTEWORTHY Prenatal inflammation is a risk factor for preterm birth and postnatal complications including infections. However, from clinical studies, it is difficult to separate the effects of only prenatal inflammation from preterm birth. Using cesarean-delivered preterm pigs with prenatal inflammation, we documented some beneficial gut effects of bioactive milk diets relative to formula, but prenatal inflammation appeared to decrease the sensitivity of enteral feeding. Special treatments and diets may be required for this neonatal population.

Prenatal Intra-Amniotic Endotoxin Induces Fetal Gut and Lung Immune Responses and Postnatal Systemic Inflammation in Preterm Pigs.

Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS-induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1ß levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses.

Neonatal gut and immune maturation is determined more by postnatal age than by postconceptional age in moderately preterm pigs.

Preterm infants have immature organ functions that predispose them to gut and immune disorders. Developmental delays at preterm birth may affect various organs differently at term-corrected age. We hypothesized that gut and immune maturation in moderately preterm neonates depends more on birth and postnatal factors than on advancing postconceptional age (PCA). Using preterm pigs as models, we investigated how gut and immune parameters develop until term-corrected age and how these differ from those in term counterparts. Preterm ( n = 43, 106 days of gestation) and term pigs ( n = 41, 116 days of gestation) were delivered by caesarean section and euthanized at birth ( day 1) or postnatal day 11 (term-corrected age for preterm pigs) using identical rearing conditions. Relative to term pigs, preterm pigs had lower blood oxygenation, glucose, and cortisol levels, lower gut lactase activity, villus height, and goblet cell density, and lower blood neutrophil, helper T, and cytotoxic T cell numbers at birth. Despite slower growth in preterm pigs, most intestinal and immune parameters increased markedly after birth in both groups. However, some parameters remained negatively affected by preterm birth until postnatal day 11 (goblet cells, gut permeability, and cytotoxic T cells). The colon microbiota showed limited differences between preterm and term pigs at this time. At the same PCA, preterm 11-day-old pigs had higher blood leukocyte numbers and gut enzyme activities but lower villus height and blood cytotoxic T cell numbers relative to newborn term pigs. Birth and postnatal factors, not advancing PCA, are key determinants of gut and immune maturation in moderately preterm neonates. NEW & NOTEWORTHY Postnatally, preterm infants are often considered to reach a physiological maturation similar to that in term infants when they reach term-corrected postconceptional age (PCA). Using preterm pigs as models, we show that PCA may be a poor measure of gut and immune maturation because environmental triggers (regardless of PCA at birth) are critical. Possibly, PCA is only relevant to evaluate physiological maturation of organs that develop relatively independent of the external environment (e.g., the brain).

Drug Repositioning of Inflammatory Bowel Disease Based on Co-Target Gene Expression Signature of Glucocorticoid Receptor and TET2.

The glucocorticoid receptor (GR) and ten-eleven translocation 2 (TET2), respectively, play a crucial role in regulating immunity and inflammation, and GR interacts with TET2. However, their synergetic roles in inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), remain unclear. This study aimed to investigate the co-target gene signatures of GR and TET2 in IBD and provide potential therapeutic interventions for IBD. By integrating public data, we identified 179 GR- and TET2-targeted differentially expressed genes (DEGs) in CD and 401 in UC. These genes were found to be closely associated with immunometabolism, inflammatory responses, and cell stress pathways. In vitro inflammatory cellular models were constructed using LPS-treated HT29 and HCT116 cells, respectively. Drug repositioning based on the co-target gene signatures of GR and TET2 derived from transcriptomic data of UC, CD, and the in vitro model was performed using the Connectivity Map (CMap). BMS-536924 emerged as a top therapeutic candidate, and its validation experiment within the in vitro inflammatory model confirmed its efficacy in mitigating the LPS-induced inflammatory response. This study sheds light on the pathogenesis of IBD from a new perspective and may accelerate the development of novel therapeutic agents for inflammatory diseases including IBD.

Coordinated transcriptional and post-transcriptional epigenetic regulation during skeletal muscle development and growth in pigs.

BACKGROUND: N6-methyladenosine (m6A) and DNA 5-methylcytosine (5mC) methylation plays crucial roles in diverse biological processes, including skeletal muscle development and growth. Recent studies unveiled a potential link between these two systems, implicating the potential mechanism of coordinated transcriptional and post-transcriptional regulation in porcine prenatal myogenesis and postnatal skeletal muscle growth. METHODS: Immunofluorescence and co-IP assays were carried out between the 5mC writers and m6A writers to investigate the molecular basis underneath. Large-scale in-house transcriptomic data were compiled for applying weighted correlation network analysis (WGCNA) to identify the co-expression patterns of m6A and 5mC regulators and their potential role in pig myogenesis. Whole-genome bisulfite sequencing (WGBS) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were performed on the skeletal muscle samples from Landrace pigs at four postnatal growth stages (days 30, 60, 120 and 180). RESULTS: Significantly correlated expression between 5mC writers and m6A writers and co-occurrence of 5mC and m6A modification were revealed from public datasets of C2C12 myoblasts. The protein-protein interactions between the DNA methylase and the m6A methylase were observed in mouse myoblast cells. Further, by analyzing transcriptome data comprising 81 pig skeletal muscle samples across 27 developmental stages, we identified a 5mC/m6A epigenetic module eigengene and decoded its potential functions in pre- or post-transcriptional regulation in postnatal skeletal muscle development and growth of pigs. Following integrative multi-omics analyses on the WGBS methylome data and MeRIP-seq data for both m6A and gene expression profiles revealed a genome/transcriptome-wide correlated dynamics and co-occurrence of 5mC and m6A modifications as a consequence of 5mC/m6A crosstalk in the postnatal myogenesis progress of pigs. Last, we identified a group of myogenesis-related genes collaboratively regulated by both 5mC and m6A modifications in postnatal skeletal muscle growth in pigs. CONCLUSIONS: Our study discloses a potential epigenetic mechanism in skeletal muscle development and provides a novel direction for animal breeding and drug development of related human muscle-related diseases.

Impaired Neonatal Immunity and Infection Resistance Following Fetal Growth Restriction in Preterm Pigs.

Background: Infants born preterm or small for gestational age (SGA, due to fetal growth restriction) both show an increased risk of neonatal infection. However, it remains unclear how the co-occurrence of preterm birth and SGA may affect neonatal immunity and infection risk. We hypothesized that fetal growth restricted (FGR) preterm newborns possess impaired immune competence and increased susceptibility to systemic infection and sepsis, relative to corresponding normal birth weight (NBW) newborns. Methods: Using preterm pigs as a model for preterm infants, gene expression in lipopolysaccharide (LPS) stimulated cord blood was compared between NBW and FGR (lowest 25% birth weight percentile) preterm pigs. Next, clinical responses to a systemic Staphylococcus epidermidis (SE) challenge were investigated in newborn FGR and NBW preterm pigs. Finally, occurrence of spontaneous infections were investigated in 9 d-old FGR and NBW preterm pigs, with or without neonatal antibiotics treatment. Results: At birth, preterm FGR piglets showed diminished ex vivo cord blood responses to LPS for genes related to both innate and adaptive immunity, and also more severe septic responses following SE infection (e.g., higher blood lactate, decreased blood pH, neutrophil and platelet counts, relative to NBW pigs). After 9 d, FGR pigs had higher incidence and severity of spontaneous infections (e.g., higher bacterial densities in the bone marrow), increased regulatory T cell numbers, reduced neutrophil phagocytosis capacity, and impaired ex vivo blood gene responses to LPS, especially when receiving neonatal antibiotics. Conclusion: FGR at preterm birth is associated with poor immune competence, impaired infection resistance, and greater sepsis susceptibility in the immediate postnatal period. Our results may explain the increased morbidity and mortality of SGA preterm infants and highlight the need for clinical vigilance for this highly sensitive subgroup of preterm neonates.

Bovine Colostrum Before or After Formula Feeding Improves Systemic Immune Protection and Gut Function in Newborn Preterm Pigs.

Objectives: Maternal milk is often absent or in limited supply just after preterm birth. Many preterm infants are therefore fed infant formula as their first enteral feed despite an increased risk of feeding intolerance, necrotizing enterocolitis (NEC), and infection. Using preterm pigs as a model for preterm infants, we hypothesized that bovine colostrum given before or after formula feeding would alleviate formula-induced detrimental effects during the first days after preterm birth. Methods: A total of 74 preterm pigs received gradually increasing volumes of formula (F) or bovine colostrum (C) until day 5, when they were euthanized or transitioned to either C or F for another 4 days, resulting in six groups: C or F until day 5 (C5, F5, n = 11 each), C or F until day 9 (CC, FF n = 12-13 each), C followed by F (CF, n = 14), and F followed by C (FC, n = 13). Results: Systemically, colostrum feeding stimulated circulating neutrophil recruitment on day 5 (C5 vs. F5, P < 0.05). Relative to initial formula feeding, initial colostrum feeding promoted the development of systemic immune protection as indicated by a decreased T-helper cell population and an increased regulatory T-cell population (CC + CF vs. FC + FF, P < 0.01). In the gut, colostrum feeding improved intestinal parameters such as villus heights, enzymes, hexose absorption, colonic goblet cell density, and decreased the incidence of severe NEC (27 vs. 64%), diarrhea (16 vs. 49%), and gut permeability on day 5, coupled with lowered expression of LBP, MYD88, IL8, HIF1A, and CASP3 (C5 vs. F5, all P < 0.05). On day 9, the incidence of severe NEC was similarly low across groups (15-21%), but diarrhea resistance and intestinal parameters were further improved by colostrum feeding, relative to exclusive formula feeding (CC, CF, or FC vs. FF, respectively, all P < 0.05). The expression of MYD88 and CASP3 remained downregulated by exclusive colostrum feeding (CC vs. FF, P < 0.01) and colostrum before or after formula feeding down regulated HIF1A and CASP3 expression marginally. Conclusion: Colostrum feeding ameliorated detrimental effects of formula feeding on systemic immunity and gut health in preterm newborns, especially when given immediately after birth.