Patricia W. Denning

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic–eukaryotic cross‐talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)‐dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox‐mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.

Therapeutic Use of Prebiotics, Probiotics, and Postbiotics to Prevent Necrotizing Enterocolitis: What is the Current Evidence?

Necrotizing enterocolitis (NEC) is a leading cause of neonatal morbidity and mortality, and preventive therapies that are both effective and safe are urgently needed. Current evidence from therapeutic trials suggests that probiotics are effective in decreasing NEC in preterm infants, and probiotics are currently the most promising therapy for this devastating disease. However, concerns regarding safety and optimal dosing have limited the widespread adoption of routine clinical use of probiotics in preterm infants. This article summarizes the current evidence regarding the use of probiotics, prebiotics, and postbiotics in the preterm infant, including their therapeutic role in preventing NEC.

Intestinal Microbiota and Its Relationship with Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in infants born prematurely. After birth, the neonatal gut must acquire a healthy complement of commensal bacteria. Disruption or delay of this critical process, leading to deficient or abnormal microbial colonization of the gut, has been implicated as key risk factor in the pathogenesis of NEC. Conversely, a beneficial complement of commensal intestinal microbiota may protect the immature gut from inflammation and injury. Interventions aimed at providing or restoring a healthy complement of commensal bacteria, such as probiotic therapy, are currently the most promising treatment to prevent NEC. Shifting the balance of intestinal microbiota from a pathogenic to protective complement of bacteria can protect the gut from inflammation and subsequent injury that leads to NEC. Herein, we review the relationship of intestinal microbiota and NEC in preterm infants.

Human Breast Milk and the Gastrointestinal Innate Immune System

The gastrointestinal (GI) tract is a large potential portal for multiple infectious agents to enter the human body. The GI system performs multiple functions as part of the neonates innate immune system, providing critical defense during a vulnerable period. Multiple mechanisms and actions are enhanced by the presence of human breast milk. Bioactive factors found in human milk work together to create and maintain an optimal and healthy environment, allowing the intestines to deliver ideal nutrition to the host and afford protection by a variety of mechanisms.

The role of intestinal epithelial barrier function in the development of NEC

The intestinal epithelial barrier plays an important role in maintaining host health. Breakdown of intestinal barrier function is known to play a role in many diseases such as infectious enteritis, idiopathic inflammatory bowel disease, and neonatal inflammatory bowel diseases. Recently, increasing research has demonstrated the importance of understanding how intestinal epithelial barrier function develops in the premature neonate in order to develop strategies to promote its maturation. Optimizing intestinal barrier function is thought to be key to preventing neonatal inflammatory bowel diseases such as necrotizing enterocolitis. In this review, we will first summarize the key components of the intestinal epithelial barrier, what is known about its development, and how this may explain NEC pathogenesis. Finally, we will review what therapeutic strategies may be used to promote optimal development of neonatal intestinal barrier function in order to reduce the incidence and severity of NEC.

Lactobacillus rhamnosus (LGG) Regulates IL-10 Signaling in the Developing Murine Colon through Upregulation of the IL-10R2 Receptor Subunit

The intestinal microflora is critical for normal development, with aberrant colonization increasing the risk for necrotizing enterocolitis (NEC). In contrast, probiotic bacteria have been shown to decrease its incidence. Multiple pro- and anti-inflammatory cytokines have been identified as markers of intestinal inflammation, both in human patients with NEC and in models of immature intestine. Specifically, IL-10 signaling attenuates intestinal responses to gut dysbiosis, and disruption of this pathway exacerbates inflammation in murine models of NEC. However, the effects of probiotics on IL-10 and its signaling pathway, remain poorly defined. Real-time PCR profiling revealed developmental regulation of MIP-2, TNF-α, IL-12, IL-10 and the IL-10R2 subunit of the IL-10 receptor in immature murine colon, while the expression of IL-6 and IL-18 was independent of postnatal age. Enteral administration of the probiotic Lactobacillus rhamnosus GG (LGG) down-regulated the expression of TNF-α and MIP-2 and yet failed to alter IL-10 mRNA and protein expression. LGG did however induce mRNA expression of the IL-10R2 subunit of the IL-10 receptor. IL-10 receptor activation has been associated with signal transducer and activator of transcription (STAT) 3-dependent induction of members of the suppressors of cytokine signaling (SOCS) family. In 2 week-old mice, LGG also induced STAT3 phosphorylation, increased colonic expression of SOCS-3, and attenuated colonic production of MIP-2 and TNF-α. These LGG-dependent changes in phosphoSTAT3, SOCS3, MIP-2 and TNF-α were all inhibited by antibody-mediated blockade of the IL-10 receptor. Thus LGG decreased baseline proinflammatory cytokine expression in the developing colon through upregulation of IL-10 receptor-mediated signaling, most likely due to the combined induction of phospho-STAT3 and SOCS3. Furthermore, LGG-dependent increases in IL-10R2 were associated with reductions in TNF-α, MIP-2 and disease severity in a murine model of intestinal injury in the immature colon.

Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis.

Background Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17. Objective We sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ−/− mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls. Results In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A. Conclusion Complimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention.

Commensal and probiotic bacteria may prevent NEC by maturing intestinal host defenses

Necrotizing enterocolitis (NEC) is a devastating disease of prematurity with significant morbidity and mortality. Immaturity of intestinal host defenses predisposes the premature infant gut to injury. An abnormal bacterial colonization pattern with a deficiency of commensal bacteria may lead to a further breakdown of these host defense mechanisms, predisposing the infant to NEC. The presence of probiotic and commensal bacteria within the gut has been shown to mature the intestinal defense system through a variety of mechanisms. We have shown that commensal and probiotic bacteria can promote intestinal host defenses by reducing apoptotic signaling, blocking inflammatory signaling, and maturing barrier function in immature intestinal epithelia. Future studies aimed at elucidating the mechanisms by which probiotic and commensal bacteria exert their effects will be critical to developing effective preventive therapies for NEC.

Pathogenesis of NEC: Role of the innate and adaptive immune response

Necrotizing enterocolitis (NEC) is a devastating disease in premature infants with high case fatality and significant morbidity among survivors. Immaturity of intestinal host defenses predisposes the premature infant gut to injury. An abnormal bacterial colonization pattern with a deficiency of commensal bacteria may lead to a further breakdown of these host defense mechanisms, predisposing the infant to NEC. Here, we review the role of the innate and adaptive immune system in the pathophysiology of NEC.

Routine Supplementation of Lactobacillus rhamnosus GG and Risk of Necrotizing Enterocolitis in Very Low Birth Weight Infants

Objective To evaluate if routine supplementation of Lactobacillus rhamnosus GG ATCC 53103 (LGG) is associated with a decreased risk of necrotizing enterocolitis in very low birth weight (VLBW) infants. Study design Retrospective observational cohort study of VLBW (<1500 g) infants at a single center from 2008 to 2016. LGG supplementation with Culturelle at a dose of 2.5 to 5 × 109 CFU/day began in 2014. We used multivariable logistic regression to evaluate the association between LGG supplementation and necrotizing enterocolitis (modified Bell stage IIA or greater), after adjusting for potential confounders. We also compared changes in necrotizing enterocolitis incidence before and after implementation of LGG using a statistical process control chart. Results We evaluated 640 VLBW infants with a median gestational age of 28.7 weeks (IQR 26.3–30.6); 78 (12%) developed necrotizing enterocolitis. The median age at first dose of LGG was 6 days (IQR 3–10), and duration of supplementation was 32 days (IQR 18–45). The incidence of necrotizing enterocolitis in the epoch before LGG implementation was 10.2% compared with 16.8% after implementation. In multivariable analysis, LGG supplementation was associated with a higher risk of necrotizing enterocolitis (aOR 2.10, 95 % CI 1.25–3.54, P = .005). We found no special cause variation in necrotizing enterocolitis after implementation of LGG supplementation. There were no episodes of Lactobacillus sepsis during 5558 infant days of LGG supplementation. Conclusions In this study, routine LGG supplementation was not associated with a decreased risk of necrotizing enterocolitis. Our findings do not support the use of the most common probiotic preparation currently supplemented to VLBW infants in the US.