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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 post-conceptional 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 d of gestation) and term pigs (n=41, 116 d of gestation) were delivered by caesarean section, and euthanized at birth (d 1) or postnatal d 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 d 11 (goblet cells, gut permeability, cytotoxic-T cells). The colon microbiota showed limited differences between preterm and term pigs at this time. At the same PCA, preterm 11 d-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.

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-1β 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.

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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.

Conclusion: Colostrum feeding ameliorated detrimental effects of formula feeding on systemic immunity and gut health in preterm newborns, especially when given immediately after birth.

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.

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 sub-clinical NEC-like lesions with variable severities. Caesarean-delivered preterm pigs were reared until day 5 or 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 days 5 and 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 sub-clinical NEC lesions. The results help to explain that preterm infants suffering from NEC may show high sensitivity to later secondary infections and sepsis.