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.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Circulating Microbiota in Cardiometabolic Disease.

The rapid expansion of microbiota research has significantly advanced our understanding of the complex interactions between gut microbiota and cardiovascular, metabolic, and renal system regulation. Low-grade chronic inflammation has long been implicated as one of the key mechanisms underlying cardiometabolic disease risk and progression, even before the insights provided by gut microbiota research in the past decade. Microbial translocation into the bloodstream can occur via different routes, including through the oral and/or intestinal mucosa, and may contribute to chronic inflammation in cardiometabolic disease. Among several gut-derived products identifiable in the systemic circulation, bacterial endotoxins and metabolites have been extensively studied, however recent advances in microbial DNA sequencing have further allowed us to identify highly diverse communities of microorganisms in the bloodstream from an -omics standpoint, which is termed "circulating microbiota." While detecting microorganisms in the bloodstream was historically considered as an indication of infection, evidence on the circulating microbiota is continually accumulating in various patient populations without clinical signs of infection and even in otherwise healthy individuals. Moreover, both quantitative and compositional alterations of the circulating microbiota have recently been implicated in the pathogenesis of chronic inflammatory conditions, potentially through their immunostimulatory, atherogenic, and cardiotoxic properties. In this mini review, we aim to provide recent evidence on the characteristics and roles of circulating microbiota in several cardiometabolic diseases, such as type 2 diabetes, cardiovascular disease, and chronic kidney disease, with highlights of our emerging findings on circulating microbiota in patients with end-stage kidney disease undergoing hemodialysis.

SMAD2 and SMAD3 differentially regulate adiposity and the growth of subcutaneous white adipose tissue.

Adipose tissue is the primary site of energy storage, playing important roles in health. While adipose research largely focuses on obesity, fat also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety and total energy homeostasis. SMAD2/3 proteins are downstream mediators of activin signaling, which regulate critical preadipocyte and mature adipocyte functions. Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity. The direct contributions of Smad2 and Smad3 in adipose tissues, however, are unknown. Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 on diet-induced adiposity using Smad2 or Smad3 "floxed" mice intercrossed with Adiponectin-Cre mice. Additionally, we examined visceral and subcutaneous preadipocyte differentiation efficiency in vitro. Almost all wild type subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A suppressed differentiation of preadipocytes from both depots. Smad2 conditional knockout (Smad2cKO) mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3 conditional knockout (Smad3cKO) male mice displayed a trend of reduced body weight on high-fat diet. On both diets, Smad3cKO mice displayed an adipose depot-selective phenotype, with a significant reduction in subcutaneous fat mass but not visceral fat mass. Our data suggest that Smad3 is an important contributor to the maintenance of subcutaneous white adipose tissue in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation.

Circulating Microbial Signatures and Cardiovascular Death in Patients With ESRD.

INTRODUCTION: Patients with end-stage renal disease (ESRD) experience disproportionately high cardiovascular morbidity and mortality. Accumulating evidence suggests a role for the circulating microbiome in the pathogenesis of cardiovascular disease; however, little is known about its association with premature cardiovascular mortality in ESRD. METHODS: In a pilot case-control study of 17 hemodialysis patients who died of a cardiovascular event and 17 matched hemodialysis controls who remained alive during a median follow-up of 2.0 years, we compared the levels and composition of circulating microbiome, including Bacteria, Archaea, and Fungi, in serum samples by quantitative polymerase chain reaction and 16S or Internal Transcribed Spacer (ITS) ribosomal RNA (rRNA) sequencing, respectively. Associations of the circulating cell-free microbial signatures with clinical parameters and cardiovascular death were examined using the Spearman rank correlation and multivariable conditional logistic regression, respectively. RESULTS: Both 16S and ITS rRNA were detectable in all (except 3 for ITS) examined patients' serum samples. Despite no significant difference in 16S rRNA levels and α diversity between cases and controls, taxonomic analysis demonstrated differential community membership between groups, with significantly greater Actinobacteria and less Proteobacteria observed in cases than in controls at the phylum level. Proportions of Actinobacteria and Proteobacteria phyla were significantly correlated with plasma nuclear factor erythroid 2-related factor 2 (Nrf2) levels (rho = -0.41 and 0.42, P = 0.015 and 0.013, respectively) and marginally associated with risk of cardiovascular death (adjusted odds ratios [95% confidence intervals] = 1.12 [0.98-1.29] and 0.88 [0.76-1.02] for 1% increase, respectively). CONCLUSION: Alterations of the circulating cell-free microbial signatures may be associated with higher premature cardiovascular mortality in ESRD.

Immune checkpoint blockade reprograms systemic immune landscape and tumor microenvironment in obesity-associated breast cancer.

Immune checkpoint blockade (ICB) has improved outcomes in some cancers. A major limitation of ICB is that most patients fail to respond, which is partly attributable to immunosuppression. Obesity appears to improve immune checkpoint therapies in some cancers, but impacts on breast cancer (BC) remain unknown. In lean and obese mice, tumor progression and immune reprogramming were quantified in BC tumors treated with anti-programmed death-1 (PD-1) or control. Obesity augments tumor incidence and progression. Anti-PD-1 induces regression in lean mice and potently abrogates progression in obese mice. BC primes systemic immunity to be highly responsive to obesity, leading to greater immunosuppression, which may explain greater anti-PD-1 efficacy. Anti-PD-1 significantly reinvigorates antitumor immunity despite persistent obesity. Laminin subunit beta-2 (Lamb2), downregulated by anti-PD-1, significantly predicts patient survival. Lastly, a microbial signature associated with anti-PD-1 efficacy is identified. Thus, anti-PD-1 is highly efficacious in obese mice by reinvigorating durable antitumor immunity. VIDEO ABSTRACT.

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.

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.