Microbial-induced Redox Imbalance in the Neonatal Lung Is Ameliorated by Live Biotherapeutics.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants. Hyperoxia exposure and microbial dysbiosis are contributors to BPD development. However, the mechanisms linking pulmonary microbial dysbiosis to worsening lung injury are unknown. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates oxidative stress responses and modulates hyperoxia-induced lung injury. We hypothesized that airway dysbiosis would attenuate Nrf2-dependent antioxidant function, resulting in a more severe phenotype of BPD. Here, we show that preterm infants with a Gammaproteobacteria-predominant dysbiosis have increased endotoxin in tracheal aspirates, and mice monocolonized with the representative Gammaproteobacteria Escherichia coli show increased tissue damage compared with germ-free (GF) control mice. Furthermore, we show Nrf2-deficient mice have worse lung structure and function after exposure to hyperoxia when the airway microbiome is augmented with E. coli. To confirm the disease-initiating potential of airway dysbiosis, we developed a novel humanized mouse model by colonizing GF mice with tracheal aspirates from human infants with or without severe BPD, producing gnotobiotic mice with BPD-associated and non-BPD-associated lung microbiomes. After hyperoxia exposure, BPD-associated mice demonstrated a more severe BPD phenotype and increased expression of Nrf2-regulated genes, compared with GF and non-BPD-associated mice. Furthermore, augmenting Nrf2-mediated antioxidant activity by supporting colonization with Lactobacillus species improved dysbiotic-augmented lung injury. Our results demonstrate that a lack of protective pulmonary microbiome signature attenuates an Nrf2-mediated antioxidant response, which is augmented by a respiratory probiotic blend. We anticipate antioxidant pathways will be major targets of future microbiome-based therapeutics for respiratory disease.

Altered hepatic and intestinal homeostasis in a neonatal murine model of short-term total parenteral nutrition and antibiotics.

Parenteral nutrition (PN) prevents starvation and supports metabolic requirements intravenously when patients are unable to be fed enterally. Clinically, infants are frequently provided PN in intensive care settings along with exposure to antibiotics (ABX) to minimize infection during care. Unfortunately, neonates experience extremely high rates of hepatic complications. Adult rodent and piglet models of PN are well-established but neonatal models capable of leveraging the considerable transgenic potential of the mouse remain underdeveloped. Utilizing our newly established neonatal murine PN mouse model, we administered ABX or controlled drinking water to timed pregnant dams to disrupt the maternal microbiome. We randomized mouse pups to PN or sham surgery controls +/- ABX exposure. ABX or short-term PN decreased liver and brain organ weights, intestinal length, and mucosal architecture (vs. controls). PN significantly elevated evidence of hepatic proinflammatory markers, neutrophils and macrophage counts, bacterial colony-forming units, and evidence of cholestasis risk, which was blocked by ABX. However, ABX uniquely elevated metabolic regulatory genes resulting in accumulation of hepatocyte lipids, triglycerides, and elevated tauro-chenoxycholic acid (TCDCA) in serum. Within the gut, PN elevated the relative abundance of Akkermansia, Enterococcus, and Suterella with decreased Anaerostipes and Lactobacillus compared with controls, whereas ABX enriched Proteobacteria. We conclude that short-term PN elevates hepatic inflammatory stress and risk of cholestasis in early life. Although concurrent ABX exposure protects against hepatic immune activation during PN, the dual exposure modulates metabolism and may contribute toward early steatosis phenotype, sometimes observed in infants unable to wean from PN.NEW & NOTEWORTHY This study successfully established a translationally relevant, murine neonatal parenteral nutrition (PN) model. Short-term PN is sufficient to induce hepatitis-associated cholestasis in a neonatal murine model that can be used to understand disease in early life. The administration of antibiotics during PN protects animals from bacterial translocation and proinflammatory responses but induces unique metabolic shifts that may predispose the liver toward early steatosis.

Chorioamnionitis and neonatal outcomes.

Chorioamnionitis or intrauterine inflammation is a frequent cause of preterm birth. Chorioamnionitis can affect almost every organ of the developing fetus. Multiple microbes have been implicated to cause chorioamnionitis, but "sterile" inflammation appears to be more common. Eradication of microorganisms has not been shown to prevent the morbidity and mortality associated with chorioamnionitis as inflammatory mediators account for continued fetal and maternal injury. Mounting evidence now supports the concept that the ensuing neonatal immune dysfunction reflects the effects of inflammation on immune programming during critical developmental windows, leading to chronic inflammatory disorders as well as vulnerability to infection after birth. A better understanding of microbiome alterations and inflammatory dysregulation may help develop better treatment strategies for infants born to mothers with chorioamnionitis.

The gut microbiome of extremely preterm infants randomized to the early progression of enteral feeding.

BACKGROUND: Early progression of feeding could influence the development of the gut microbiome. METHODS: We collected fecal samples from extremely preterm infants randomized to receive either early (feeding day 2) or delayed (feeding day 5) feeding progression. After study completion, we compared samples obtained at three different time points (week 1, week 2, and week 3) to determine longitudinal differences in specific taxa between the study groups using unadjusted and adjusted negative binomial and zero-inflated mixed models. Analyses were adjusted for a mode of delivery, breastmilk intake, and exposure to antibiotics. RESULTS: We analyzed 137 fecal samples from 51 infants. In unadjusted and adjusted analyses, we did not observe an early transition to higher microbial diversity within samples (i.e., alpha diversity) or significant differences in microbial diversity between samples (i.e., beta diversity) in the early feeding group. Our longitudinal, single-taxon analysis found consistent differences in the genera Lactococcus, Veillonella, and Bilophila between groups. CONCLUSIONS: Differences in single-taxon analyses independent of the mode of delivery, exposure to antibiotics, and breastmilk feeding suggest potential benefits of early progression of enteral feeding volumes. However, this dietary intervention does not appear to increase the diversity of the gut microbiome in the first 28 days after birth. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02915549. IMPACT: Early progression of enteral feeding volumes with human milk reduces the duration of parenteral nutrition and the need for central venous access among extremely preterm infants. Early progression of enteral feeding leads to single-taxon differences in longitudinal analyses of the gut microbiome, but it does not appear to increase the diversity of the gut microbiome in the first 28 days after birth. Randomization in enteral feeding trials creates appealing opportunities to evaluate the effects of human milk diets on the gut microbiome.

Recent advances in understanding the ecology of the lung microbiota and deciphering the gut-lung axis.

A rapidly expanding new field of lung research has been produced by the emergence of culture-independent next-generation sequencing technologies. While pulmonary microbiome research lags behind the exploration of the microbiome in other organ systems, the field is maturing and has recently produced multiple exciting discoveries. In this mini-review, we will explore recent advances in our understanding of the lung microbiome and the gut-lung axis from an ecological perspective.

Perinatal maternal antibiotic exposure augments lung injury in offspring in experimental bronchopulmonary dysplasia.

During the newborn period, intestinal commensal bacteria influence pulmonary mucosal immunology via the gut-lung axis. Epidemiological studies have linked perinatal antibiotic exposure in human newborns to an increased risk for bronchopulmonary dysplasia, but whether this effect is mediated by the gut-lung axis is unknown. To explore antibiotic disruption of the newborn gut-lung axis, we studied how perinatal maternal antibiotic exposure influenced lung injury in a hyperoxia-based mouse model of bronchopulmonary dysplasia. We report that disruption of intestinal commensal colonization during the perinatal period promotes a more severe bronchopulmonary dysplasia phenotype characterized by increased mortality and pulmonary fibrosis. Mechanistically, metagenomic shifts were associated with decreased IL-22 expression in bronchoalveolar lavage and were independent of hyperoxia-induced inflammasome activation. Collectively, these results demonstrate a previously unrecognized influence of the gut-lung axis during the development of neonatal lung injury, which could be leveraged to ameliorate the most severe and persistent pulmonary complication of preterm birth.

TGR5 signaling mitigates parenteral nutrition-associated liver disease.

Bile acid receptors regulate the metabolic and immune functions of circulating enterohepatic bile acids. This process is disrupted by administration of parenteral nutrition (PN), which may induce progressive hepatic injury for unclear reasons, especially in the newborn, leading to PN-associated liver disease. To explore the role of bile acid signaling on neonatal hepatic function, we initially observed that Takeda G protein receptor 5 (TGR5)-specific bile acids were negatively correlated with worsening clinical disease markers in the plasma of human newborns with prolonged PN exposure. To test our resulting hypothesis that TGR5 regulates critical liver functions to PN exposure, we used TGR5 receptor deficient mice (TGR5-/-). We observed PN significantly increased liver weight, cholestasis, and serum hepatic stress enzymes in TGR5-/- mice compared with controls. Mechanistically, PN reduced bile acid synthesis genes in TGR5-/-. Serum bile acid composition revealed that PN increased unconjugated primary bile acids and secondary bile acids in TGR5-/- mice, while increasing conjugated primary bile acid levels in TGR5-competent mice. Simultaneously, PN elevated hepatic IL-6 expression and infiltrating macrophages in TGR5-/- mice. However, the gut microbiota of TGR5-/- mice compared with WT mice following PN administration displayed highly elevated levels of Bacteroides and Parabacteroides, and possibly responsible for the elevated levels of secondary bile acids in TGR5-/- animals. Intestinal bile acid transporters expression was unchanged. Collectively, this suggests TGR5 signaling specifically regulates fundamental aspects of liver bile acid homeostasis during exposure to PN. Loss of TGR5 is associated with biochemical evidence of cholestasis in both humans and mice on PN.NEW & NOTEWORTHY Parenteral nutrition is associated with deleterious metabolic outcomes in patients with prolonged exposure. Here, we demonstrate that accelerated cholestasis and parental nutrition-associated liver disease (PNALD) may be associated with deficiency of Takeda G protein receptor 5 (TGR5) signaling. The microbiome is responsible for production of secondary bile acids that signal through TGR5. Therefore, collectively, these data support the hypothesis that a lack of established microbiome in early life or under prolonged parenteral nutrition may underpin disease development and PNALD.

Fungi form interkingdom microbial communities in the primordial human gut that develop with gestational age.

Fungal and bacterial commensal organisms play a complex role in the health of the human host. Expansion of commensal ecology after birth is a critical period in human immune development. However, the initial fungal colonization of the primordial gut remains undescribed. To investigate primordial fungal ecology, we performed amplicon sequencing and culture-based techniques of first-pass meconium, which forms in the intestine prior to birth, from a prospective observational cohort of term and preterm newborns. Here, we describe fungal ecologies in the primordial gut that develop complexity with advancing gestational age at birth. Our findings suggest homeostasis of fungal commensals may represent an important aspect of human biology present even before birth. Unlike bacterial communities that gradually develop complexity, the domination of the fungal communities of some preterm infants by Saccromycetes, specifically Candida, may suggest a pathologic association with preterm birth.-Willis, K. A., Purvis, J. H., Myers, E. D., Aziz, M. M., Karabayir, I., Gomes, C. K., Peters, B. M., Akbilgic, O., Talati, A. J., Pierre, J. F. Fungi form interkingdom microbial communities in the primordial human gut that develop with gestational age.

Clinical trial of a probiotic and herbal supplement for lung health.

Introduction: Dysbiosis of the gut microbiome may augment lung disease via the gut-lung axis. Proteobacteria may contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic inflammation. To study the effects of probiotics across the gut-lung axis, we sought to determine if a Lactobacillus probiotic and herbal blend was safe and well-tolerated in healthy volunteers and asthmatic patients. Methods: We conducted a 1-month randomized, open-label clinical trial in Cork, Ireland with healthy and asthmatic patients who took the blend twice a day. The primary endpoint was safety with exploratory endpoints including quality of life, lung function, gut microbiome ecology, and inflammatory biomarkers. Results: All subjects tolerated the blend without adverse events. Asthmatic subjects who took the blend showed significant improvements in lung function as measured by forced expiratory volume and serum short chain fatty acid levels from baseline to Week 4. The gut microbiome of asthmatic subjects differed significantly from controls, with the most prominent difference in the relative abundance of the proteobacteria Escherichia coli. Administration of the probiotic maintained overall microbial community architecture with the only significant difference being an increase in absolute abundance of the probiotic strains measured by strain-specific PCR. Conclusion: This study supports the safety and efficacy potential of a Lactobacillus probiotic plus herbal blend to act on the gut-lung axis. However, due to the lack of a control group, a longer blinded, placebo-controlled study will be warranted to confirm the efficacy improvements observed in this trial. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT05173168.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair. In Brief: Using a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota. Highlights: Supraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs).Intestinal AMP expression has an inverse relationship with the severity of lung injury.AMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury.AMPs may mediate a gut-lung axis that modulates lung injury.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

BACKGROUND: Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical, host-derived regulators of the microbiota. However, mechanisms that support microbiota homeostasis in response to inflammatory stimuli, such as supraphysiologic oxygen, remain unclear. RESULTS: We show that supraphysiologic oxygen exposure to neonatal mice, or direct exposure of intestinal organoids to supraphysiologic oxygen, suppresses the intestinal expression of AMPs and alters intestinal microbiota composition. Oral supplementation of the prototypical AMP lysozyme to hyperoxia-exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. CONCLUSIONS: Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury in newborns. Together, these data support that intestinal AMPs modulate lung injury and repair. Video Abstract.

The fungal intestinal microbiota predict the development of bronchopulmonary dysplasia in very low birthweight newborns.

RATIONALE: Bronchopulmonary dysplasia (BPD) is the most common morbidity affecting very preterm infants. Gut fungal and bacterial microbial communities contribute to multiple lung diseases and may influence BPD pathogenesis. METHODS: We performed a prospective, observational cohort study comparing the multikingdom fecal microbiota of 144 preterm infants with or without moderate to severe BPD by sequencing the bacterial 16S and fungal ITS2 ribosomal RNA gene. To address the potential causative relationship between gut dysbiosis and BPD, we used fecal microbiota transplant in an antibiotic-pseudohumanized mouse model. Comparisons were made using RNA sequencing, confocal microscopy, lung morphometry, and oscillometry. RESULTS: We analyzed 102 fecal microbiome samples collected during the second week of life. Infants who later developed BPD showed an obvious fungal dysbiosis as compared to infants without BPD (NoBPD, p = 0.0398, permutational multivariate ANOVA). Instead of fungal communities dominated by Candida and Saccharomyces, the microbiota of infants who developed BPD were characterized by a greater diversity of rarer fungi in less interconnected community architectures. On successful colonization, the gut microbiota from infants with BPD augmented lung injury in the offspring of recipient animals. We identified alterations in the murine intestinal microbiome and transcriptome associated with augmented lung injury. CONCLUSIONS: The gut fungal microbiome of infants who will develop BPD is dysbiotic and may contribute to disease pathogenesis.

Efficacy of a Probiotic and Herbal Supplement in Models of Lung Inflammation.

BACKGROUND: Gut microbiome dysbiosis is associated with lung disease through the gut-lung axis. Abundant proteobacteria increase MMP-9 and contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic lung disease. We sought to determine if a scientifically formulated probiotic and herbal supplement could attenuate neutrophilic inflammation and improve lung structure and function in models of lung inflammation. METHODS: For in vitro experiments, epithelial cells exposed to proteobacteria were treated with resB-a blend of three probiotic Lactobacillus strains and turmeric, holy basil, and vasaka herbal extracts. For in vivo experimentation, mice exposed to pulmonary proteobacteria-derived lipopolysaccharide were treated by gavage with resB. RESULTS: In vitro, the bacterial and herbal components of resB decreased activity of the MMP-9 pathway. Mice exposed to LPS and pre- and post-treated with resB had decreased neutrophil recruitment and inflammatory biomarkers in bronchoalveolar lavage fluid, serum, and lung tissue compared to untreated mice. CONCLUSIONS: This study describes the mechanisms and efficacy of probiotic and herbal blend in pre-clinical models of lung injury and inflammation.

Necrotizing enterocolitis and the gut-lung axis.

The recently recognized connection between the gut microbiota and pulmonary disease has been termed the gut-lung axis. However, broader connections link the gut and the lungs and these organ systems are tightly interrelated in both homeostasis and disease. This concept is often ignored in the compartmentalized treatment of pulmonary or gastrointestinal disease. In newborns, the most severe gastrointestinal complication of prematurity, necrotizing enterocolitis, and the most severe pulmonary complication, bronchopulmonary dysplasia, both produce significant systemic morbidity. In this review, we highlight the often neglected pathophysiology of the gut-lung axis contributes to increased risk of bronchopulmonary dysplasia in premature infants with necrotizing enterocolitis.

The gut mycobiome of healthy mice is shaped by the environment and correlates with metabolic outcomes in response to diet.

As an active interface between the host and their diet, the gut microbiota influences host metabolic adaptation; however, the contributions of fungi have been overlooked. Here, we investigate whether variations in gut mycobiome abundance and composition correlate with key features of host metabolism. We obtained animals from four commercial sources in parallel to test if differing starting mycobiomes can shape host adaptation in response to processed diets. We show that the gut mycobiome of healthy mice is shaped by the environment, including diet, and significantly correlates with metabolic outcomes. We demonstrate that exposure to processed diet leads to persistent differences in fungal communities that significantly associate with differential deposition of body mass in male mice compared to mice fed standardized diet. Fat deposition in the liver, transcriptional adaptation of metabolically active tissues and serum metabolic biomarker levels are linked with alterations in fungal community diversity and composition. Specifically, variation in fungi from the genera Thermomyces and Saccharomyces most strongly associate with metabolic disturbance and weight gain. These data suggest that host-microbe metabolic interactions may be influenced by variability in the mycobiome. This work highlights the potential significance of the gut mycobiome in health and has implications for human and experimental metabolic studies.

The closed eye harbours a unique microbiome in dry eye disease.

Dry eye affects millions of individuals. In experimental models, dry eye disease is associated with T helper cell 17-mediated inflammation of the ocular surface that may cause persistent damage to the corneal epithelium. However, the initiating and perpetuating factors associated with chronic inflammation of the ocular surface remain unclear. The ocular microbiota alters ocular surface inflammation and may influence dry eye disease development and progression. Here, we collected serial samples of tears on awakening from sleep, closed eye tears, during a randomized clinical trial of a non-pharmaceutical dry eye therapy and used 16S rRNA metabarcoding to characterize the microbiome. We show the closed dry eye microbiome is distinct from the healthy closed eye microbiome, and that the microbiome remains distinct despite daily saline eye wash upon awakening. The ocular microbiome was described only recently, and this report implicates a distinct microbiome in ocular disease development. Our findings suggest an interplay between microbial commensals and inflammation on the ocular surface. This information may inform future studies of the pathophysiological mechanisms of dry eye disease.