Enterohepatic Shunt-Driven Cholemia Predisposes to Liver Cancer.

BACKGROUND & AIMS: Pathogenesis of hepatocellular carcinoma (HCC), which kills millions annually, is poorly understood. Identification of risk factors and modifiable determinants and mechanistic understanding of how they impact HCC are urgently needed. METHODS: We sought early prognostic indicators of HCC in C57BL/6 mice, which we found were prone to developing this disease when fed a fermentable fiber-enriched diet. Such markers were used to phenotype and interrogate stages of HCC development. Their human relevance was tested using serum collected prospectively from an HCC/case-control cohort. RESULTS: HCC proneness in mice was dictated by the presence of congenitally present portosystemic shunt (PSS), which resulted in markedly elevated serum bile acids (BAs). Approximately 10% of mice from various sources exhibited PSS/cholemia, but lacked an overt phenotype when fed standard chow. However, PSS/cholemic mice fed compositionally defined diets, developed BA- and cyclooxygenase-dependent liver injury, which was exacerbated and uniformly progressed to HCC when diets were enriched with the fermentable fiber inulin. Such progression to cholestatic HCC associated with exacerbated cholemia and an immunosuppressive milieu, both of which were required in that HCC was prevented by impeding BA biosynthesis or neutralizing interleukin-10 or programmed death protein 1. Analysis of human sera revealed that elevated BA was associated with future development of HCC. CONCLUSIONS: PSS is relatively common in C57BL/6 mice and causes silent cholemia, which predisposes to liver injury and HCC, particularly when fed a fermentable fiber-enriched diet. Incidence of silent PSS/cholemia in humans awaits investigation. Regardless, measuring serum BA may aid HCC risk assessment, potentially alerting select individuals to consider dietary or BA interventions.

Selective IgA Deficiency in Spontaneously Hypertensive Rats With Gut Dysbiosis.

BACKGROUND: The spontaneously hypertensive rat (SHR) is extensively used to study hypertension. Gut microbiota dysbiosis is a notable feature in SHR for reasons unknown. Immunoglobulin A (IgA) is a major host factor required for gut microbiota homeostasis. We hypothesized that inadequate IgA contributes to gut microbiota dysbiosis in SHR. METHODS: IgA was measured in feces, cecum, serum, liver, gut-associated lymphoid tissue, and milk from SHR and Wistar Kyoto rats. IgA regulatory factors like IgM, IgG, and pIgR (polymeric immunoglobulin receptor) were analyzed. IgA and IgG antibodies and blood pressure (BP) were measured before and after administrating a bacterial antigen (ie, flagellin). RESULTS: Compared with Wistar Kyoto rats, SHR displayed remarkably near-deficient IgA levels accompanied by compensatory increases in serum IgM and IgG and gut-liver pIgR expression. Inadequate milk IgA in SHR emphasized this immune defect stemmed from the neonatal stage. Reduced IgA+ B cells in circulation and Peyer patches indicated a possible reason for the lower IgA in SHR. Noteworthy, a genetic insufficiency was unlikely because administering flagellin to SHR induced anti-flagellin IgA antibodies. This immune response surprisingly accelerated hypertension development in SHR, suggesting IgA quiescence may help maintain lower BP. CONCLUSIONS: This study is the first to reveal IgA deficiency in SHR as one host factor associated with gut microbiota dysbiosis and invigorates future research to determine the pathophysiological role of IgA in hypertension.

Immunoglobulin A, an Active Liaison for Host-Microbiota Homeostasis.

Mucosal surfaces in the gastrointestinal tract are continually exposed to native, commensal antigens and susceptible to foreign, infectious antigens. Immunoglobulin A (IgA) provides dual humoral responses that create a symbiotic environment for the resident gut microbiota and prevent the invasion of enteric pathogens. This review features recent immunological and microbial studies that elucidate the underlying IgA and microbiota-dependent mechanisms for mutualism at physiological conditions. IgA derailment and concurrent microbiota instability in pathological diseases are also discussed in detail. Highlights of this review underscore that the source of IgA and its structural form can dictate microbiota reactivity to sustain a diverse niche where both host and bacteria benefit. Other important studies emphasize IgA insufficiency can result in the bloom of opportunistic pathogens that encroach the intestinal epithelia and disseminate into circulation. The continual growth of knowledge in these subjects can lead to the development of therapeutics targeting IgA and/or the microbiota to treat life threatening diseases.

Reconstitution of the host holobiont in germ-free born male rats acutely increases bone growth and affects marrow cellular content.

Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiological interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-wk-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, whereas the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short-chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyrate. Furthermore, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders.

IL-10 Receptor Neutralization-Induced Colitis in Mice: A Comprehensive Guide.

Interleukin-10 (IL-10) and its receptor (IL-10R) have been foremost targets to understand inflammatory bowel disease (IBD) pathogenesis. For the past several decades, IL-10-deficient (Il10-/- ) mice were considered one of the best models to study immune-mediated colitis. Several physiologic limitations with this model, e.g., delayed and varied disease onset, have hindered investigators in testing new clinical therapies for IBD. In this article, we provide comprehensive guidance for using anti-IL-10R monoclonal antibody (αIL-10R mAb) neutralization as a superior alternative model to study IBD. This article describes the feasibility of using αIL-10R mAb to induce chronic colitis (within 4 weeks), perform time-dependent mechanistic studies, and assess the efficacy of IBD therapeutics. This article also delineates protocols for in-house assays to critically assess colitis and associated inflammatory parameters. Overall, we underscore αIL-10R mAb neutralization as a relevant immune-mediated murine colitis model to study human Crohn's disease. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Induction of chronic colitis in mice via αIL-10R mAb neutralization Basic Protocol 2: Biochemical evaluation of αIL-10R mAb neutralization-induced chronic colitis Support Protocol 1: Stool analysis and scoring Support Protocol 2: Swiss roll method.

SARS-CoV-2, ACE2 expression, and systemic organ invasion.

A novel coronavirus disease, COVID-19, has created a global pandemic in 2020, posing an enormous challenge to healthcare systems and affected communities. COVID-19 is caused by severe acute respiratory syndrome (SARS)-coronavirus-2 (CoV-2) that manifests as bronchitis, pneumonia, or a severe respiratory illness. SARS-CoV-2 infects human cells via binding a "spike" protein on its surface to angiotensin-converting enzyme 2 (ACE2) within the host. ACE2 is crucial for maintaining tissue homeostasis and negatively regulates the renin-angiotensin-aldosterone system (RAAS) in humans. The RAAS is paramount for normal function in multiple organ systems including the lungs, heart, kidney, and vasculature. Given that SARS-CoV-2 internalizes via ACE2, the resultant disruption in ACE2 expression can lead to altered tissue function and exacerbate chronic diseases. The widespread distribution and expression of ACE2 across multiple organs is critical to our understanding of the varied clinical outcomes of COVID-19. This perspective review based on the current literature was prompted to show how disruption of ACE2 by SARS-CoV-2 can affect different organ systems.

Enterobactin induces the chemokine, interleukin-8, from intestinal epithelia by chelating intracellular iron.

Iron is an indispensable nutrient for both mammals and microbes. Bacteria synthesize siderophores to sequester host iron, whereas lipocalin 2 (Lcn2) is the host defense protein that prevent this iron thievery. Enterobactin (Ent) is a catecholate-type siderophore that has one of the strongest known affinities for iron. Intestinal epithelial cells (IECs) are adjacent to large microbial population and are in contact with microbial products, including Ent. We undertook this study to investigate whether a single stimulus of Ent could affect IEC functions. Using three human IEC cell-lines with differential basal levels of Lcn2 (i.e. HT29 < DLD-1 < Caco-2/BBe), we demonstrated that iron-free Ent could induce a dose-dependent secretion of the pro-inflammatory chemokine, interleukin 8 (IL-8), in HT29 and DLD-1 IECs, but not in Caco-2/BBe. Ent-induced IL-8 secretion was dependent on chelation of the labile iron pool and on the levels of intracellular Lcn2. Accordingly, IL-8 secretion by Ent-treated HT29 cells could be substantially inhibited by either saturating Ent with iron or by adding exogenous Lcn2 to the cells. IL-8 production by Ent could be further potentiated when co-stimulated with other microbial products (i.e. flagellin, lipopolysaccharide). Water-soluble microbial siderophores did not induce IL-8 production, which signifies that IECs are specifically responding to the lipid-soluble Ent. Intriguingly, formyl peptide receptor (FPR) antagonists (i.e. Boc2, cyclosporine H) abrogated Ent-induced IL-8, implicating that such IEC response could be, in part, dependent on FPR. Taken together, these results demonstrate that IECs sense Ent as a danger signal, where its recognition results in IL-8 secretion.

Distinct iron homeostasis in C57BL/6 and Balb/c mouse strains.

C57BL/6 (BL6) and Balb/c mice exhibit prototypical Th1- and Th2-dominant immune predispositions, respectively. Iron is a proinflammatory metal ion; however, limited information is documented on the differences in iron homeostasis between BL6 and Balb/c strains. The objective of this study was to investigate the extent to which strain-level differences in these mice dictates the regulation of iron homeostasis during physiologic and inflammatory conditions. At basal levels, Balb/c mice displayed significantly higher levels of iron in systemic circulation and tissue compared to BL6 mice. Moreover, Balb/c mice had greater iron absorption as indicated by higher gene expressions of duodenal DcytB, DMT1, Fpn, SFT, and Heph. Similarly, hepatic Tf, TfR1, TfR2, and DMT1 expressions were augmented in Balb/c mice. Interestingly, there was no change in hepatic Hamp expression between the two strains, suggesting that the disparity in their maintenance of iron is independent of hepcidin. Additionally, the basal levels of intracellular labile iron pool in Balb/c intestinal epithelial cells, and bone marrow-derived macrophages and neutrophils, were higher compared to BL6 mice. When mice were challenged with lipopolysaccharide, the acute inflammatory response in BL6 mice was more pronounced than in Balb/c mice, as indicated by the more rapid development of hypoferremia and upregulation of serum IL-6 and TNF-α levels in BL6 mice. In conclusion, this study underscores that iron homeostasis is distinct between BL6 and Balb/c strains under both physiologic and inflammatory conditions.

Vancomycin prevents fermentable fiber-induced liver cancer in mice with dysbiotic gut microbiota.

Owing to their health benefits, dietary fermentable fibers, such as refined inulin, are increasingly fortified in processed foods to enhance their nutritional value. However, we previously demonstrated that when inulin was fed to Toll-like receptor 5 deficient (T5KO) mice susceptible to dysbiosis, a subset of them developed cholestasis and subsequently liver cancer in a gut microbiota-dependent manner. Therefore, we hypothesized that clearance of bacterial taxa, and thereby gut metabolites, involved in the onset and progression to liver cancer could abate the disease in these mice. Such a reshaping of microbiota by vancomycin treatment was sufficient to halt the development of liver cancer in inulin-fed T5KO mice; however, this intervention did not remedy disease penetrance for cholestatic liver injury and its sequelae, including hyperbilirubinemia, hypolipidemia, cholemia and liver fibrosis. Selective depletion of gut bacterial communities was observed in vancomycin-treated mice, including Gram-positive Lachnospiraceae and Ruminococcaceae belonging to the phylum Firmicutes, Bifidobacteria of the phylum Actinobacteria, which ferment fibers, and Clostridium cluster XIVa, which produce secondary bile acids. Lack of liver cancer in vancomycin-treated mice strongly correlated with the substantial loss of secondary bile acids in circulation. Although cholemia was unabated by vancomycin, the composition of serum bile acids shifted toward an abundance of hydrophilic primary bile acids, denoted by the increase in conjugated-to-unconjugated bile acid ratio. Taken together, the present study suggests that microbiotal regulation of bile acid metabolism is one of the critical mediators of fermentable fiber-induced liver cancer in dysbiotic mice.

Fermentable fibers induce rapid macro- and micronutrient depletion in Toll-like receptor 5-deficient mice.

Functional fermentable fibers are considered essential for a healthy diet. Recently, we demonstrated that gut microbiota dysbiotic mice fed an inulin-containing diet (ICD) developed hepatocellular carcinoma (HCC) within 6 mo. In particular, a subset of Toll-like receptor 5-deficient (T5KO) mice prone to HCC exhibited rapid onset of hyperbilirubinemia (HB) and cholemia; these symptoms provide rationale that ICD induces cholestasis. Our objective in the present study was to determine whether inulin-fed T5KO-HB mice exhibit other known consequences of cholestasis, including essential fatty acid and fat-soluble vitamin deficiencies. Here, we measured hepatic fatty acids and serum vitamin A and D levels from wild-type (WT), T5KO low bilirubin (LB) and T5KO-HB mice fed ICD for 4 wk. Additionally, hepatic RNAseq and proteomics were performed to ascertain other metabolic alterations. Compared with WT and T5KO-LB, T5KO-HB mice exhibited steatorrhea, i.e., ~50% increase in fecal lipids. This could contribute to the significant reduction of linoleate in hepatic neutral lipids in T5KO-HB mice. Additionally, serum vitamins A and D were ~50% reduced in T5KO-HB mice, which was associated with metabolic compromises. Overall, our study highlights that fermentable fiber-induced cholestasis is further characterized by depletion of macro-and micronutrients.NEW & NOTEWORTHY Feeding a dietary, fermentable fiber diet to a subset of Toll-like receptor 5 deficient (T5KO) mice induces early onset hyperbilirubinemia and cholemia that later manifests to hepatocellular carcinoma (HCC). Our study highlights that fermentable fiber-induced cholestasis is characterized with modest macro- and micronutrient deficiencies that may further contribute to hepatic biliary disease. Compared with chemical induction, immunization, surgery, or genetic manipulation, these findings provide a novel approach to study the cholestatic subtype of HCC.

Altered nutrient status reprograms host inflammation and metabolic health via gut microbiota.

The metabolism of macro- and micronutrients is a complex and highly regulated biological process. An imbalance in the metabolites and their signaling networks can lead to nonresolving inflammation and consequently to the development of chronic inflammatory-associated diseases. Therefore, identifying the accumulated metabolites and altered pathways during inflammatory disorders would not only serve as "real-time" markers but also help in the development of nutritional therapeutics. In this review, we explore recent research that has delved into elucidating the effects of carbohydrate/calorie restriction, protein malnutrition, lipid emulsions and micronutrient deficiencies on metabolic health and inflammation. Moreover, we describe the integrated stress response in terms of amino acid starvation and lipemia and how this modulates new age diseases such as inflammatory bowel disease and atherosclerosis. Lastly, we explain the latest research on metaflammation and inflammaging. This review focuses on multiple signaling pathways, including, but not limited to, the FGF21-ß-hydroxybutryate-NLRP3 axis, the GCN2-eIF2α-ATF4 pathway, the von Hippel-Lindau/hypoxia-inducible transcription factor pathway and the TMAO-PERK-FoxO1 axis. Additionally, throughout the review, we explain how the gut microbiota responds to altered nutrient status and also how antimicrobial peptides generated from nutrient-based signaling pathways can modulate the gut microbiota. Collectively, it must be emphasized that metabolic starvation and inflammation are strongly regulated by both environmental (i.e., nutrition, gut microbiome) and nonenvironmental (i.e., genetics) factors, which can influence the susceptibility to inflammatory disorders.