Maternal helminth infection protects offspring from high-fat-diet-induced obesity through altered microbiota and SCFAs.

Helminth-induced Th2 immunity and gut microbiota have been recently shown to be highly effective in modulating metabolic syndromes in animal models. This study aimed to determine whether maternal immunity and microbial factors affect the induction and development of obesity in offspring. Here, Heligomosomoides polygyrus (Hp)-infected or control female C57BL/6J mice mated with normal males and their offspring were fed a high-fat diet (HFD) for 9 weeks after weaning. Our results showed that Hp-induced maternal outcomes during gestation and lactation significantly impacted offspring metabolic phenotypes. This was evidenced by results showing that offspring from helminth-infected mothers on an HFD (Hp-offspring + HFD) gained significantly less body weight than those from uninfected mothers (Cont-offspring + HFD). Hp-offspring + HFD exhibited no Th2 phenotype but displayed a pattern of gut microbiota composition similar to that of Hp-infected mothers. Cross-fostering experiments confirmed that the helminth-induced maternal attenuation of offspring obesity was mediated through both prenatal and postnatal effects. Our results further showed that helminth-infected dams and their offspring had a markedly altered gut microbiome composition, with increased production of short-chain fatty acids (SCFAs). Intriguingly, Hp-infected mothers and Hp-offspring + HFD showed increased SCFA receptor (GPR) expression in adipose and colonic tissues compared to noninfected mothers and Cont-offspring + HFD, respectively. Moreover, SCFA supplementation to the pups of uninfected control mothers during lactation protected against HFD-induced weight gain, which corresponded with changes in gut bacterial colonization. Collectively, our findings provide new insights into the complex interaction of maternal immune status and gut microbiome, Hp infection, and the immunity and gut microbiome in obese-prone offspring in infant life.

The potential role of adherence factors in probiotic function in the gastrointestinal tract of adults and pediatrics: a narrative review of experimental and human studies.

Numerous studies point to the important role of probiotic bacteria in gastrointestinal health. Probiotics act through mechanisms affecting enteric pathogens, epithelial barrier function, immune signaling, and conditioning of indigenous microbiota. Once administered, probiotics reach the gastrointestinal tract and interact with the host through bacterial surface molecules, here called adhesion factors, which are either strain- or specie-specific. Probiotic adhesion, through structural adhesion factors, is a mechanism that facilitates persistence within the gastrointestinal tract and triggers the initial host responses. Thus, an understanding of specific probiotic adhesion mechanisms could predict how specific probiotic strains elicit benefits and the potential of adherence factors as a proxy to predict probiotic function. This review summarizes the present understanding of probiotic adherence in the gastrointestinal tract. It highlights the bacterial adhesion structure types, their molecular communication with the host and the consequent impact on intestinal diseases in both adult and pediatric populations. Finally, we discuss knockout/isolation studies as direct evidence for adhesion factors conferring anti-inflammatory and pathogen inhibition properties to a probiotic.What is known: Probiotics can be used to treat clinical conditions.Probiotics improve dysbiosis and symptoms.Clinical trials may not confirm in vitro and animal studies.What is new: Adhesion structures may be important for probiotic function.Need to systematically determine physical characteristics of probiotics before selecting for clinical trials.Probiotics may be genetically engineered to add to clinical efficacy.

Maternal Influences and Intervention Strategies on the Development of Food Allergy in Offspring.

Food allergies and other immune-mediated diseases have become serious health concerns amongst infants and children in developed and developing countries. The absence of available cures limits disease management to allergen avoidance and symptomatic treatments. Research has suggested that the presence of maternal food allergies may expose the offspring to genetic predisposition, making them more susceptible to allergen sensitization. The following review has focused on epidemiologic studies regarding maternal influences of proneness to develop food allergy in offspring. The search strategy was "food allergy OR maternal effects OR offspring OR prevention". A systematically search from PubMed/MEDLINE, Science Direct and Google Scholar was conducted. Specifically, it discussed the effects of maternal immunity, microbiota, breastfeeding, genotype and allergy exposure on the development of food allergy in offspring. In addition, several commonly utilized prenatal and postpartum strategies to reduce food allergy proneness were presented, including early diagnosis of high-risk infants and various dietary interventions.

The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis.

An excessive intestinal inflammatory response may have a role in the pathogenesis of necrotizing enterocolitis (NEC) in very preterm infants. Indole-3-lactic acid (ILA) of breastmilk tryptophan was identified as the anti-inflammatory metabolite involved in probiotic conditioned media from Bifidobacteria longum subsp infantis. This study aimed to explore the molecular endocytic pathways involved in the protective ILA effect against inflammation. H4 cells, Caco-2 cells, C57BL/6 pup and adult mice were used to compare the anti-inflammatory mechanisms between immature and mature enterocytes in vitro and in vivo. The results show that ILA has pleiotropic protective effects on immature enterocytes including anti-inflammatory, anti-viral, and developmental regulatory potentials in a region-dependent and an age-dependent manner. Quantitative transcriptomic analysis revealed a new mechanistic model in which STAT1 pathways play an important role in IL-1ß-induced inflammation and ILA has a regulatory effect on STAT1 pathways. These studies were validated by real-time RT-qPCR and STAT1 inhibitor experiments. Different protective reactions of ILA between immature and mature enterocytes indicated that ILA's effects are developmentally regulated. These findings may be helpful in preventing NEC for premature infants.

Short-chain fatty acid butyrate, a breast milk metabolite, enhances immature intestinal barrier function genes in response to inflammation in vitro and in vivo.

Infants born under 1,500 g have an increased incidence of necrotizing enterocolitis in the ileum and the colon, which is a life-threatening intestinal necrosis. This is in part due to excessive inflammation in the immature intestine to colonizing bacteria because of an immature innate immune response. Breastmilk complex carbohydrates create metabolites of colonizing bacteria in the form of short-chain fatty acids (SCFAs). We studied the effect of breastmilk metabolites, SCFAs, on immature intestine with regard to anti-inflammatory effects. This showed that acetate, propionate, and butyrate were all anti-inflammatory to an IL-1ß inflammatory stimulus. In this study, to further define the mechanism of anti-inflammation, we created transcription profiles of RNA from immature human enterocytes after exposure to butyrate with and without an IL-1ß inflammatory stimulus. We demonstrated that butyrate stimulates an increase in tight-junction and mucus genes and if we inhibit these genes, the anti-inflammatory effect is partially lost. SCFAs, products of microbial metabolism of complex carbohydrates of breastmilk oligosaccharides, have been found with this study to induce an anti-IL-1ß response that is associated with an upregulation of tight junctions and mucus genes in epithelial cells (H4 cells). These studies suggest that breastmilk in conjunction with probiotics can reduce excessive inflammation with metabolites that are anti-inflammatory and stimulate an increase in the mucosal barrier.NEW & NOTEWORTHY This study extends previous observations to define the anti-inflammatory properties of butyrate, a short-chain fatty acid produced by the metabolism of breastmilk oligosaccharides by colonizing bacteria. Using transcription profiling of immature enterocyte genes, after exposure to butyrate and an IL-1ß stimulus, we showed that tight-junction genes and mucus genes were increased, which contributed to the anti-inflammatory effect.

Helminth-Induced and Th2-Dependent Alterations of the Gut Microbiota Attenuate Obesity Caused by High-Fat Diet.

BACKGROUND & AIMS: Epidemiological and animal studies have indicated an inverse correlation between the rising prevalence of obesity and metabolic syndrome and exposure to helminths. Whether helminth-induced immune response contributes to microbiota remodeling in obesity remains unknown. The aim of this study is to explore the immune-regulatory role of helminth in the prevention of HFD-induced obesity through remodeling gut microbiome. METHODS: C57BL/6J WT and STAT6-/- mice were infected with Heligmosomoides polygyrus and followed by high fat diet (HFD) feeding for 6 weeks. The host immune response, body weight, and fecal microbiota composition were analyzed. We used adoptive transfer of M2 macrophages and microbiota transplantation approaches to determine the impact of these factors on HFD-obesity. We also examined stool microbiota composition and short chain fatty acids (SCFAs) concentration and determined the expression of SCFA-relevant receptors in the recipient mice. RESULTS: Helminth infection of STAT6-/- (Th2-deficient) mice and adoptive transfer of helminth-induced alternatively activated (M2) macrophages demonstrated that the helminth-associated Th2 immune response plays an important role in the protection against obesity and induces changes in microbiota composition. Microbiota transplantation showed that helminth-induced, Th2-dependent alterations of the gut microbiota are sufficient to confer protection against obesity. Collectively, these results indicate that helminth infection protects against HFD-induced obesity by Th2-dependent, M2 macrophage-mediated alterations of the intestinal microbiota. CONCLUSION: Our findings provide new mechanistic insights into the complex interplay between helminth infection, the immune system and the gut microbiota in a HFD-induced obesity model and holds promise for gut microbiome-targeted immunotherapy in obesity prevention.

Breast Milk and Microbiota in the Premature Gut: A Method of Preventing Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating inflammatory condition of the intestine, which affects premature infants and causes untold damage. Its pathogenesis has to do with how colonizing bacteria interact with the immature newborn intestine. An immature innate immune response with increased TLR-4 on the cell surface and increased signaling molecules, such as NF-κB, can cause excessive inflammation. This is in conjunction with a decrease in the appearance of regulatory molecules which effect the control of innate responses. This condition is so devastating that it must be prevented and not treated. Fortunately, breast milk and probiotics can affect the condition leading to reduced inflammation. How does this effect work? We have shown that breast milk tryptophan and Bifidobacterium infantis result in a metabolite (indole-3-lactic acid) response, which is anti-inflammatory via inhibition of the aryl hydrocarbon receptor transcription factor which stimulates an IL-8 response. We have also shown that breast milk complex carbohydrates interacting with Bacteroides fragilis can cause short-chain fatty acids which exert anti-inflammatory effects on the newborn intestine. These breast milk metabolites could help prevent NEC if shown to be effective clinically.

Short chain fatty acids produced by colonizing intestinal commensal bacterial interaction with expressed breast milk are anti-inflammatory in human immature enterocytes.

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother's expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.

Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine.

BACKGROUND: Necrotizing enterocolitis (NEC), a necrotic inflammation of the intestine, represents a major health problem in the very premature infant. Although prevention is difficult, the combination of ingestion of maternal-expressed breastmilk in conjunction with a probiotic provides the best protection. In this study, we establish a mechanism for breastmilk/probiotic protection. METHODS: Ultra-high-performance liquid chromatography-tandem mass spectrometry of Bifidobacterium longum subsp. infantis (B. infantis) secretions was used to identify an anti-inflammatory molecule. Indole-3-lactic acid (ILA) was then tested in an established human immature small intestinal cell line, necrotizing colitis enterocytes, and other immature human enteroids for anti-inflammatory effects and to establish developmental function. ILA was also examined in immature and mature enterocytes. RESULTS: We have identified ILA, a metabolite of breastmilk tryptophan, as the anti-inflammatory molecule. This molecule is developmentally functional in immature but not mature intestinal enterocytes; ILA reduces the interleukin-8 (IL-8) response after IL-1ß stimulus. It interacts with the transcription factor aryl hydrocarbon receptor (AHR) and prevents transcription of the inflammatory cytokine IL-8. CONCLUSIONS: This molecule produced by B. infantis (ATCC No. 15697) interaction with ingested breastmilk functions in a complementary manner and could become useful in the treatment of all at-risk premature infants for NEC if safety and clinical studies are performed.

Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace.

Intestinal helminth infections elicit Th2-type immunity, which influences host immune responses to additional threats, such as allergens, metabolic disease, and other pathogens. Th2 immunity involves a shift of the CD4+ T-cell population from type-0 to type-2 (Th2) with increased abundance of interleukin (IL)-4 and IL-13. This study sought to investigate if existing gut-restricted intestinal helminth infections impact bacterial-induced acute airway neutrophil recruitment. C57BL/6 mice were divided into four groups: uninfected; helminth-Heligmosomoides polygyrus infected; Pseudomonas aeruginosa infected; and coinfected. Mice infected with H. polygyrus were incubated for 2 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage, blood, and lung samples were analyzed. Interestingly, infection with gut-restricted helminths resulted in immunological and structural changes in the lung. These changes include increased lung CD4+ T cells, increased Th2 cytokine expression, and airway goblet cell hyperplasia. Furthermore, coinfected mice exhibited significantly more airspace neutrophil infiltration at 6 hours following P. aeruginosa infection and exhibited an improved rate of survival compared with bacterial infected alone. These results suggest that chronic helminth infection of the intestines can influence and enhance acute airway neutrophil responses to P. aeruginosa infection.

The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

Helminth infection protects against high fat diet-induced obesity via induction of alternatively activated macrophages.

Epidemiological studies indicate an inverse correlation between the prevalence of the so-called western diseases, such as obesity and metabolic syndrome, and the exposure to helminths. Obesity, a key risk factor for many chronic health problems, is rising globally and is accompanied by low-grade inflammation in adipose tissues. The precise mechanism by which helminths modulate metabolic syndrome and obesity is not fully understood. We infected high fat diet (HFD)-induced obese mice with the intestinal nematode parasite Heligmosomoides polygyrus and observed that helminth infection resulted in significantly attenuated obesity. Attenuated obesity corresponded with marked upregulation of uncoupling protein 1 (UCP1), a key protein involved in energy expenditure, in adipose tissue, suppression of glucose and triglyceride levels, and alteration in the expression of key genes involved in lipid metabolism. Moreover, the attenuated obesity in infected mice was associated with enhanced helminth-induced Th2/Treg responses and M2 macrophage polarization. Adoptive transfer of helminth-stimulated M2 cells to mice that were not infected with H. polygyrus resulted in a significant amelioration of HFD-induced obesity and increased adipose tissue browning. Thus, our results provide evidence that the helminth-dependent protection against obesity involves the induction of M2 macrophages.

The importance of appropriate initial bacterial colonization of the intestine in newborn, child, and adult health.

The fetus does not reside in a sterile intrauterine environment and is exposed to commensal bacteria from the maternal gut/blood stream that cross the placenta and enter the amniotic fluid. This intestinal exposure to colonizing bacteria continues at birth and during the first year of life and has a profound influence on lifelong health. Why is this important? Intestinal crosstalk with colonizing bacteria in the developing intestine affects the infant's adaptation to extrauterine life (immune homeostasis) and provides protection against disease expression (allergy, autoimmune disease, obesity, etc.) later in life. Colonizing intestinal bacteria are critical to the normal development of host defense. Disrupted colonization (dysbiosis) due to maternal dysbiosis, cesarean section delivery, use of perinatal antibiotics, or premature delivery may adversely affect the gut development of host defense and predispose to inflammation rather than to homeostasis, leading to increased susceptibility to disease later in life. Babies born by cesarean section have a higher incidence of allergy, type 1 diabetes, and obesity. Infants given repeated antibiotic regimens during the first year of life are more likely to have asthma as adolescents. This research breakthrough helps to explain the shift in disease paradigms from infections to immune-mediated in children from developed countries. This review will develop this research breakthrough.

The symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis inhibits IL-1ß-induced inflammation in human fetal enterocytes via toll receptors 2 and 4.

Colonizing bacteria interacting with the immature, unlike the mature, human intestine favors inflammation over immune homeostasis. As a result, ten percent of premature infants under 1500 grams weight develop an inflammatory necrosis of the intestine after birth, e.g., necrotizing enterocolitis (NEC). NEC is a major health problem in this population causing extensive morbidity and mortality and an enormous expenditure of health care dollars. NEC can be prevented by giving preterm infants their mother's expressed breast milk or ingesting selective probiotic organisms. Vaginally delivered, breast fed newborns develop health promoting bacteria ("pioneer" bacteria) which preferentially stimulate intestinal host defense and anti-inflammation. One such "pioneer" organism is Bacteroides fragilis with a polysaccharide (PSA) on its capsule. B. fragilis has been shown developmentally in intestinal lymphocytes and dendritic cells to produce a balanced T-helper cell (TH1/TH2) response and to reduce intestinal inflammation by activity through the TLR2 receptor stimulating IL-10 which inhibits IL-17 causing inflammation. No studies have been done on the role of B. fragilis PSA on fetal enterocytes and its increased inflammation. Accordingly, using human and mouse fetal intestinal models, we have shown that B. fragilis with PSA and PSA alone inhibits IL-1ß-induced IL-8 inflammation in fetal and NEC intestine. We have also begun to define the mechanism for this unique inflammation noted in fetal intestine. We have shown that B. fragilis PSA anti-inflammation requires both the TLR2 and TLR4 receptor and is in part mediated by the AP1 transcription factor (TLR2) which is developmentally regulated. These observations may help to devise future preventative treatments of premature infants against NEC.

Bacterial Colonization of the Newborn Gut, Immune Development, and Prevention of Disease.

We now know that the fetus does not reside in a sterile intrauterine environment but is exposed to commensal bacteria from the maternal gut which cross the placenta and infiltrate the amniotic fluid. This exposure to colonizing bacteria continues at birth and during the first year of life, and it has a profound influence on lifelong health. Why is this important? Cross talk with colonizing bacteria in the developing neonatal intestine helps in the initial adaptation of the infant to extrauterine life, particularly in acquiring immune homeostasis, and provides protection against disease expression (e.g., allergy, autoimmune disease, and obesity) later in life. Colonizing intestinal bacteria are critical to the development of host defense during the neonatal period. Disrupted colonization (dysbiosis) due to cesarean section delivery, perinatal antibiotics, or premature delivery may adversely affect the development of host defense mechanisms in the gut and predispose to inflammation leading to increased susceptibility to disease later in life. Clinical evidence suggests that babies born by cesarean section have higher incidence rates of allergy, type 1 diabetes, and obesity. Infants given repeated antibiotic regimens are more likely to have asthma as adolescents. This observation helps to explain the disease paradigm shift in children from developed countries.

Human fetal intestinal epithelial cells metabolize and incorporate branched chain fatty acids in a structure specific manner.

BACKGROUND: Branched chain fatty acids (BCFA) are constituents of gastrointestinal (GI) tract in healthy newborn human infants, reduce the incidence of necrotizing enterocolitis (NEC) in a neonatal rat model, and are incorporated into small intestine cellular lipids in vivo. We hypothesize that BCFA are taken up, metabolized and incorporated into human fetal cells in vitro. METHODS: Human H4 cells, a fetal non-transformed primary small intestine cell line, were incubated with albumin-bound non-esterified anteiso-17:0, iso-16:0, iso-18:0 and/or iso-20:0, and FA profiles in lipid fractions were analyzed. RESULTS: All BCFA were readily incorporated as major constituents of cellular lipids. Anteiso-17:0 was preferentially taken up, and was most effective among BCFA tested in displacing normal (n-) FA. The iso BCFA were preferred in reverse order of chain length, with iso-20:0 appearing at lowest level. BCFA incorporation in phospholipids (PL) followed the same order of preference, accumulating 42% of FA as BCFA with no overt morphological signs of cell death. Though cholesterol esters (CE) are at low cellular concentration among lipid classes examined, CE had the greatest affinity for BCFA, accumulating 65% of FA as BCFA. BCFA most effectively displaced lower saturated FA. Iso-16:0, iso-18:0 and anteiso-17:0 were both elongated and chain shortened by ±C2. Iso-20:0 was chain shortened to iso-18:0 and iso-16:0 but not elongated. CONCLUSIONS: Nontransformed human fetal intestinal epithelial cells incorporate high levels of BCFA when they are available and metabolize them in a structure specific manner. These findings imply that specific pathways for handling BCFA are present in the lumen-facing cells of the human fetal GI tract that is exposed to vernix-derived BCFA in late gestation.