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

Abstract

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.

1. Introduction

The extremely preterm neonate faces many challenges following early birth. Their fragile gastrointestinal (GI) systems are in a critical state of development and are forced to adapt quickly to extrauterine life. This complex organ provides a large interface with the external environment. When the GI system functions properly, it not only facilitates optimal nutrition, but also plays a unique role in host defense. A breakdown in this essential function poses a significant threat to the health of the premature infant. More specifically, immaturity of intestinal host defenses may predispose infants to necrotizing enterocolitis (NEC). Despite advances in the medical care of premature infants, NEC rates remain unchanged [1-3], and it continues to be one of the most devastating and unpredictable diseases of prematurity. Unfortunate patients who contract this disease are at high risk for adverse neurodevelopmental outcomes [4,5]. The etiology of NEC has not been fully elucidated, but it is likely multifactorial, involving immaturity of intestinal host defenses and abnormal bacterial colonization [6-9].

Colonization of the initially sterile intestinal ecosystem occurs postnatally as dietary and environmental changes occur. Complete colonization resembling that of an adult is reached by two years of age and, remarkably, contains up to 1 × 10¹⁴ colony-forming units (CFUs) [10]. Appropriate colonization with commensal bacteria is important for intestinal function and development [11-13] and may play a central role in the postnatal maturation of intestinal host defenses [14]. Neonates who are born prematurely have an abnormal intestinal microbial composition [13,15,16], which may predispose them to a failure of postnatal evolution of critical innate defenses and lead to NEC. As further evidence for the importance of commensal bacteria, preterm infants with altered intestinal flora due to prolonged antibiotic therapy are more likely to develop NEC [17,18]. Other factors that influence proper colonization include maternal exposure to antibiotics, mode of delivery, human breast milk feedings, and the hospital environment. Preterm infants are more likely to be delivered by cesarean section and experience delayed enteral feeding, which make them less likely to acquire commensal flora perinatally from passage through the birth canal or from human milk feedings. This may lead to decreased colonization of beneficial probiotic bacteria, including species of Bifidobacterium, Lactobacillus and Bacteroides [19,20]. The hospital environment, with its preponderance of pathogenic organisms [21], also negatively affects the intestinal colonization of beneficial commensal bacteria.

Studies have shown commensal bacteria regulate many intestinal defenses including barrier function, mucin and IgA secretion, inflammation, and homeostatic processes such as proliferation and apoptosis [22-26]. We believe that immature intestinal host defenses play a critical role in the pathogenesis of neonatal intestinal inflammatory diseases, including NEC, and commensal bacteria (or their products) can promote the maturation of these host defenses. Understanding this process is crucial to developing potential therapeutic (prebiotic, probiotic or postbiotic) interventions to better treat or prevent these devastating diseases. Specific areas of host defense that can be promoted by commensal and probiotic bacteria include intestinal epithelial cell proliferation and apoptosis, innate immune regulation, and epithelial barrier function.

2. Murine model of postnatal intestinal development

Epidemiologic studies indicate that incidence and postnatal age of NEC onset is inversely proportional to gestational age [9,27-30]. Thus, infants born earlier not only have a higher NEC incidence but also develop NEC at a later postnatal age, with a developmental window of susceptibility at 30-32 weeks [9,27-30]. Susceptibility to NEC is likely due to developmental immaturity in intestinal host defenses such as exaggerated apoptotic and inflammatory responses [7,31] and decreased barrier function. In order to better understand developmental differences in intestinal host defenses that may play a role in NEC, our laboratory has successfully modeled premature intestinal epithelia in 0-3 week old preweaned mice. Rodents are altricial species and thus their intestines are functionally immature at birth [32]. Premature human intestines and preweaned murine intestines are similar in that mucosal immunity and GALT are maturing postnatally [14,33]. The neonatal murine intestinal epithelial architecture and barrier function are relatively immature at birth compared to the neonatal human intestine and continues to mature over the first three weeks of life [14,34]. Rodent intestines in the 2nd week of life are thought to represent the maturity of early 3rd trimester human intestines, and thus, we and others have successfully used murine intestines in the 2nd week of life to model premature human intestines [14,32,35-48].

Using this murine model of postnatal intestinal development, we have characterized the ontogeny of key intestinal host defenses. Specifically, we have shown that intestinal epithelial apoptotic [49,50] and proinflammatory [51] responses peak at 2 weeks in the murine gut at a time when intestinal epithelial barrier function remains immature [52]. This developmental period of dysregulated intestinal host defenses may resemble the developmental period of peak susceptibility to NEC seen in premature infants. Thus the 2-week-old murine intestine may be an ideal model for the preterm human intestine. Further, we have successfully employed this novel murine model of postnatal intestinal development to investigate the mechanisms by which commensal and probiotic bacteria promote regulation and maturation of intestinal host defenses.

3. Commensal and probiotic bacteria regulate apoptotic responses in immature intestinal epithelia

Apoptosis, when regulated correctly, is generally regarded as a protective process for the host and results in a balance between cell proliferation and cell death. It allows damaged cells to be removed in response to injurious stimuli (microbial, hypoxic, or chemical) [23] without further impairment of the surrounding tissue, but problems may arise when apoptotic activity is excessive or aberrant. There is histopathologic evidence in humans that apoptosis plays a role in the early events of the development of NEC [53,54], and, in animal models of NEC, epithelial apoptosis precedes gross bowel necrosis [55]. We have shown that immature intestinal epithelial cells exhibit exaggerated responses to apoptotic stimuli [49-51]. Commensal bacteria have been shown to reduce the incidence of NEC [56]. Therefore, we investigated whether commensal Escherichia coli could regulate apoptotic signaling in the developing gut.

Commensal strains of E. coli, which are obtained from the maternal GI tract, populate the intestines of term newborns very early in life [11,12,57]. Mirpuri et al. showed that a previously known, anti-inflammatory [22,58] commensal strain of E. coli isolated from healthy human colon reduces epithelial apoptosis in a murine model of developing intestine [50]. Ontogeny studies demonstrated that immature murine intestinal epithelia were most susceptible to inducible apoptosis at 2 weeks of postnatal age. However, when we fed neonatal mice with E. coli prior to inducing apoptosis, we found that intestinal epithelia in both the small intestine and colon showed a 50% reduction in induced apoptosis. E. coli mitigated apoptotic responses via IFN-αA-dependent upregulation of the antiapoptotic protein, GBP-1.

We have shown that probiotic bacteria can also regulate intestinal epithelial apoptotic responses. Probiotics are defined as ‘living micro-organisms, which upon ingestion in sufficient numbers, exert health benefits beyond basic nutrition’ [59]. These beneficial bacteria can foster a normal intestinal colonization pattern in addition to directly supporting optimal functioning of intestinal epithelial cells. Experimental NEC in animal models has shown a reduction in both the severity [60] and incidence [61,62] of NEC after receiving probiotics. There has been much enthusiasm surrounding similar results in human trials, with a reduced incidence of NEC following probiotic administration [56,63]. However, this excitement has been attenuated by the concern over their safety and little knowledge about the optimal species, dosing and duration of treatment to use. There have been reports of sepsis following probiotic therapy in immunocompromised children [64-66], which has raised the question of possible harm for similarly immunocompromised extremely preterm infants [67]. Specific bacteria that have been studied in clinical trials include species of Lactobacillus and Bifidobacterium, and Streptococcus thermophilus [68-75]. Lactobacillus rhamnosus GG (LGG) is of particular interest as it has been shown to be effective in preventing cytokine-induced apoptosis in adult intestinal epithelial cells [26,76] and is one of the most effective commensal species in mitigating inflammatory responses [22,58]. Our laboratory has recently reported that LGG can reduce apoptotic signaling in immature intestinal epithelia via upregulation of genes involved in cellular proliferation and migration and mitogen activated protein kinase (MAPK) pathways known to be important for growth, differentiation, and cytoprotection [49].

4. Probiotic bacteria regulate inflammatory and anti-inflammatory signaling in immature intestinal epithelia

In addition to regulating apopotic signaling, probiotic bacteria can regulate inflammatory signaling. We have recently shown that the probiotic LGG can regulate inflammation both by blocking inflammatory signaling via NF-κB [51] and by upregulating anti-inflammatory signaling via IL-10 [42]. Inflammation defends against harmful pathogens by alerting and directing cells of the innate immune system to a site of injury. The vast majority of the time this initial response works appropriately and guards the host from further damage, but uncontrolled inflammation may lead to acute or chronic inflammatory diseases. With a lack of commensal bacteria that have the ability to control inflammation [77], the premature intestinal environment is predisposed to exaggerated inflammatory responses, possibly leading to NEC [8,78]. In animal NEC models, the expression of known pro-inflammatory cytokines TNF-α [79-82], MIP-2 [83], and IL-6 [84,85] have been shown to be increased. Activation of the IL-10 pathway negatively regulates the expression of these cytokines [86-92], and there has been speculation that IL-10-dependent suppression of inflammatory mediators may be a final common pathway protecting the developing intestine from uncontrolled inflammation and NEC [42]. Indeed, IL-10 deficient mice develop spontaneous colitis [93] and are more susceptible to NEC [94]. Also, mutations in genes encoding IL-10 receptor subunit proteins were found in multiple infants who had severe, early onset enterocolitis [95].

Using our murine model of premature intestines, we recently reported that intestinal epithelial inflammatory responses peak at 2 weeks in the neonatal mouse. Further, we demonstrated that probiotic bacteria (LGG) could block intestinal epithelial inflammatory signaling via NF-κB by inducing homeostatic ROS signaling. Probiotic and commensal bacteria can induce intestinal epithelial ROS production which in turn can regulate homeostatic processes via inactive oxidation of key regulatory enzymes. We demonstrated that in immature intestinal epithelia, LGG can induce physiologic ROS expression which in turn prevents NF-κB activation through oxidative inactivation of its regulatory enzyme, Ubc12. Physiologic ROS signaling is distinct from oxidative stress (defined as an imbalance in the oxidation–reduction pathways leading to excessive ROS generation) and has been implicated in many disease processes [96-99]. Diseases specific to prematurity such as retinopathy of prematurity, chronic lung disease, intraventricular hemorrhage and NEC have all been linked to oxidative stress, but attempts at administering antioxidants to premature infants have led to disappointing results [100-102]. This is likely because physiologic ROS signaling regulates many homeostatic processes [99], and thus, the global suppression of that signaling may lead to undesirable effects. Expounding on the mechanisms by which commensal bacteria or probiotics induce innate immune responses via intestinal epithelial ROS regulation could be key to developing targeted preventive therapies for intestinal inflammatory diseases in neonates and children.

We also recently discovered that LGG can regulate anti-inflammatory pathways in the developing murine gut. Following the administration of LGG, the colonic expression of MIP-2 and TNF-α were decreased and the colonic expression of the IL-10R2 receptor subunit was induced [42]. We also demonstrated that the reduction in pro-inflammatory cytokine expression was dependent on the IL-10 receptor. These results suggest that the anti-inflammatory effects of LGG are mediated through induction of the IL-10R2 receptor subunit. Future studies in this area will focus on cell-specific sources of IL-10 and IL-10 receptors.

5. Commensal and probiotic bacteria improve tight junction-dependent epithelial barrier function in the developing intestine

Proper functioning of the epithelial barrier is imperative for intestinal health, and the breakdown of this critical defense has been implicated in the pathogenesis of multiple intestinal inflammatory diseases, including idiopathic inflammatory bowel disease (IBD) [103,104], infectious enteritis, and NEC [7,105-107]. Early postnatal maturation of the epithelium leads to more selective permeability in both premature neonates [108,109] and neonatal animal models [110-112]. As an essential component of barrier function, tight junctions (TJs) regulate paracellular permeability and maintain separation of tissue compartments by sealing the intercellular space [113,114]. Studies have implicated abnormal TJ protein expression in the development of an impaired barrier function in both IBD [115] and rodent models of NEC [116,117]. The presence of commensal bacteria has been shown to maintain and improve barrier function through interactions with toll-like receptors (TLRs) [118] and by the up-regulation of beneficial genes [23]. The abnormal microbial colonization patterns and lack of normal commensal bacteria in premature neonates can further compromise their intestinal barrier, which may lead to intestinal inflammation and injury secondary to the systemic entry of toxin from the gut lumen [108,119-121].

Using our murine model of intestinal development, we characterized intestinal barrier function and TJ expression in the immature gut [52]. We demonstrated that intestinal barrier function (as measured by gut permeability) matures between 2 and 3 weeks of life. Claudin 3 was the most upregulated TJ protein during this developmental period. Further, we determined that both commensal and probiotic bacteria were able to induce maturation of both intestinal barrier function and Claudin 3 expression, likely via toll-like receptor signaling. Finally, we were able to demonstrate for the first time that both live and heat-killed probiotic bacteria (LGG) could accelerate maturation of intestinal barrier function and Claudin 3 expression in vivo. These studies provide evidence for and a potential mechanism by which postbiotics may be used to mature key host defenses in order to prevent NEC in the premature gut.

6. Summary

Commensal bacteria foster a healthy intestinal microbiome, and these resident bacteria are vital in protecting the host from devastating intestinal diseases, including NEC. Infants born prematurely have low bacterial diversity due to their immaturity, hospital environment, and necessary medical care. Premature intestines also have immature host defenses which may lead to increased apoptotic signaling, increased inflammatory signaling, and reduced gut barrier function. Prebiotic, probiotic, or postbiotic therapy may be able to restore essential strains of commensal flora necessary for maturation of these intestinal host defenses in the preterm gut. Lactobacillus species specifically appear to be a commensal that is lacking in preterm infants and is also more susceptible to eradication in response to antibiotic treatment and stress [11]. We have shown that Lactobacillus species can augment host intestinal defenses by promoting cytoprotective gene expression and anti-inflammatory signaling, blocking inflammatory signaling, and improving gut barrier function (Fig. 1). Further exploration of the mechanisms by which probiotics and commensal bacteria exert their effects will hopefully result in effective therapies for the premature neonate that will decrease the incidence and severity of NEC.

Fig. 1.

Overview of mechanisms by which probiotics exert their effects, leading to a decreased incidence of NEC.

Abbreviations

IFN

interferon

NF-κB

nuclear factor kappa B

IL

interleukin

TNF-α

tumor necrosis factor alpha

MIP

macrophage inflammatory protein

ROS

reactive oxygen species

LGG

Lactobacillus rhamnosus GG