Germ-free status but not subacute polychlorinated biphenyl (PCB) exposure altered hepatic phosphatidylcholine and ether-phosphatidylcholine levels in mice.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that pose a current ecosystem and human health concern. PCB exposure impacts the gut microbiome in animal models, suggesting a mechanistic link between PCB exposure and adverse health outcomes. The presence and absence of the microbiome and exposure to PCBs independently affect the lipid composition in the liver, which in turn affects the PCB disposition in target tissues, such as the liver. Here, we investigated microbiome × subacute PCB effects on the hepatic lipid composition of conventional and germ-free female mice exposed to 0, 6, or 30 mg/kg body weight of an environmental PCB mixture in sterile corn oil once daily for 3 consecutive days. Hepatic triacylglyceride and polar lipid levels were quantified using mass spectrometric methods following the subacute PCB exposure. The lipidomic analysis revealed no PCB effect on the hepatic levels. No microbiome effect was observed on levels of triacylglyceride and most polar lipid classes. The total hepatic levels of phosphatidylcholine (PC) and ether-phosphatidylcholine (ePC) lipids were lower in germ-free mice than the conventional mice from the same exposure group. Moreover, levels of several unsaturated PCs, such as PC(36:5) and PC(42:10), and ePCs, such as ePC(36:2) and ePC(4:2), were lower in germ-free than conventional female mice. Based on a KEGG pathway meta-analysis of RNA sequencing data, the ether lipid metabolism pathway is altered in the germ-free mouse liver. In contrast to the liver, extractable lipid levels, determined gravimetrically, differed in several tissues from naïve conventional vs. germ-free mice. Overall, microbiome × subacute PCB exposure effects on hepatic lipid composition are unlikely to affect PCB distribution into the mouse liver. Further studies are needed to assess how the different extractable lipid levels in other tissues alter PCB distribution in conventional vs. germ-free mice.

Targeting PD-L2-RGMb overcomes microbiome-related immunotherapy resistance.

The gut microbiota is a crucial regulator of anti-tumour immunity during immune checkpoint inhibitor therapy. Several bacteria that promote an anti-tumour response to immune checkpoint inhibitors have been identified in mice1-6. Moreover, transplantation of faecal specimens from responders can improve the efficacy of anti-PD-1 therapy in patients with melanoma7,8. However, the increased efficacy from faecal transplants is variable and how gut bacteria promote anti-tumour immunity remains unclear. Here we show that the gut microbiome downregulates PD-L2 expression and its binding partner repulsive guidance molecule b (RGMb) to promote anti-tumour immunity and identify bacterial species that mediate this effect. PD-L1 and PD-L2 share PD-1 as a binding partner, but PD-L2 can also bind RGMb. We demonstrate that blockade of PD-L2-RGMb interactions can overcome microbiome-dependent resistance to PD-1 pathway inhibitors. Antibody-mediated blockade of the PD-L2-RGMb pathway or conditional deletion of RGMb in T cells combined with an anti-PD-1 or anti-PD-L1 antibody promotes anti-tumour responses in multiple mouse tumour models that do not respond to anti-PD-1 or anti-PD-L1 alone (germ-free mice, antibiotic-treated mice and even mice colonized with stool samples from a patient who did not respond to treatment). These studies identify downregulation of the PD-L2-RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy.

Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice.

The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.

Microbiota-derived 3-IAA influences chemotherapy efficacy in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second most deadly cancer by 2040, owing to the high incidence of metastatic disease and limited responses to treatment1,2. Less than half of all patients respond to the primary treatment for PDAC, chemotherapy3,4, and genetic alterations alone cannot explain this5. Diet is an environmental factor that can influence the response to therapies, but its role in PDAC is unclear. Here, using shotgun metagenomic sequencing and metabolomic screening, we show that the microbiota-derived tryptophan metabolite indole-3-acetic acid (3-IAA) is enriched in patients who respond to treatment. Faecal microbiota transplantation, short-term dietary manipulation of tryptophan and oral 3-IAA administration increase the efficacy of chemotherapy in humanized gnotobiotic mouse models of PDAC. Using a combination of loss- and gain-of-function experiments, we show that the efficacy of 3-IAA and chemotherapy is licensed by neutrophil-derived myeloperoxidase. Myeloperoxidase oxidizes 3-IAA, which in combination with chemotherapy induces a downregulation of the reactive oxygen species (ROS)-degrading enzymes glutathione peroxidase 3 and glutathione peroxidase 7. All of this results in the accumulation of ROS and the downregulation of autophagy in cancer cells, which compromises their metabolic fitness and, ultimately, their proliferation. In humans, we observed a significant correlation between the levels of 3-IAA and the efficacy of therapy in two independent PDAC cohorts. In summary, we identify a microbiota-derived metabolite that has clinical implications in the treatment of PDAC, and provide a motivation for considering nutritional interventions during the treatment of patients with cancer.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Products of gut microbial Toll/interleukin-1 receptor domain NADase activities in gnotobiotic mice and Bangladeshi children with malnutrition.

Perturbed gut microbiome development has been linked to childhood malnutrition. Here, we characterize bacterial Toll/interleukin-1 receptor (TIR) protein domains that metabolize nicotinamide adenine dinucleotide (NAD), a co-enzyme with far-reaching effects on human physiology. A consortium of 26 human gut bacterial strains, representing the diversity of TIRs observed in the microbiome and the NAD hydrolase (NADase) activities of a subset of 152 bacterial TIRs assayed in vitro, was introduced into germ-free mice. Integrating mass spectrometry and microbial RNA sequencing (RNA-seq) with consortium membership manipulation disclosed that a variant of cyclic-ADPR (v-cADPR-x) is a specific product of TIR NADase activity and a prominent, colonization-discriminatory, taxon-specific metabolite. Guided by bioinformatic analyses of biochemically validated TIRs, we find that acute malnutrition is associated with decreased fecal levels of genes encoding TIRs known or predicted to generate v-cADPR-x, as well as decreased levels of the metabolite itself. These results underscore the need to consider microbiome TIR NADases when evaluating NAD metabolism in the human holobiont.

Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells.

Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.

High-Fat Diet Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites.

BACKGROUND AND AIMS: Dietary fat intake is associated with increased risk of colorectal cancer (CRC). We examined the role of high-fat diet (HFD) in driving CRC through modulating gut microbiota and metabolites. METHODS: HFD or control diet was fed to mice littermates in CRC mouse models of an azoxymethane (AOM) model and Apcmin/+ model, with or without antibiotics cocktail treatment. Germ-free mice for fecal microbiota transplantation were used for validation. Gut microbiota and metabolites were detected using metagenomic sequencing and high-performance liquid chromatography-mass spectrometry, respectively. Gut barrier function was determined using lipopolysaccharides level and transmission electron microscopy. RESULTS: HFD promoted colorectal tumorigenesis in both AOM-treated mice and Apcmin/+ mice compared with control diet-fed mice. Gut microbiota depletion using antibiotics attenuated colon tumor formation in HFD-fed mice. A significant shift of gut microbiota composition with increased pathogenic bacteria Alistipessp.Marseille-P5997 and Alistipessp.5CPEGH6, and depleted probiotic Parabacteroides distasonis, along with impaired gut barrier function was exhibited in HFD-fed mice. Moreover, HFD-modulated gut microbiota promotes colorectal tumorigenesis in AOM-treated germ-free mice, indicating gut microbiota was essential in HFD-associated colorectal tumorigenesis. Gut metabolites alteration, including elevated lysophosphatidic acid, which was confirmed to promote CRC cell proliferation and impair cell junction, was also observed in HFD-fed mice. Moreover, transfer of stools from HFD-fed mice to germ-free mice without interference increased colonic cell proliferation, impaired gut barrier function, and induced oncogenic genes expression. CONCLUSIONS: HFD drives colorectal tumorigenesis through inducing gut microbial dysbiosis, metabolomic dysregulation with elevated lysophosphatidic acid, and gut barrier dysfunction in mice.

Synthetic Microbiomes on the Rise-Application in Deciphering the Role of Microbes in Host Health and Disease.

The intestinal microbiota conveys significant benefits to host physiology. Although multiple chronic disorders have been associated with alterations in the intestinal microbiota composition and function, it is still unclear whether these changes are a cause or a consequence. Hence, to translate microbiome research into clinical application, it is necessary to provide a proof of causality of host-microbiota interactions. This is hampered by the complexity of the gut microbiome and many confounding factors. The application of gnotobiotic animal models associated with synthetic communities allows us to address the cause-effect relationship between the host and intestinal microbiota by reducing the microbiome complexity on a manageable level. In recent years, diverse bacterial communities were assembled to analyze the role of microorganisms in infectious, inflammatory, and metabolic diseases. In this review, we outline their application and features. Furthermore, we discuss the differences between human-derived and model-specific communities. Lastly, we highlight the necessity of generating novel synthetic communities to unravel the microbial role associated with specific health outcomes and disease phenotypes. This understanding is essential for the development of novel non-invasive targeted therapeutic strategies to control and modulate intestinal microbiota in health and disease.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

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.

Microbes exploit death-induced nutrient release by gut epithelial cells.

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.

Gnotobiotic Operations and Assembly for Development of Germ-Free Animal Model of Laser-Induced Choroidal Neovascularization.

Purpose: Compelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD. Methods: We developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase-polymerase chain reaction. Results: The experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment. Conclusions: We established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure. Translational Relevance: To our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host-microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.

Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors.

Bacterial ADP-ribosyltransferases (ADPRTs) have been described as toxins involved in pathogenesis through the modification of host proteins. Here, we report that ADPRTs are not pathogen restricted but widely prevalent in the human gut microbiome and often associated with phage elements. We validated their biochemical activity in a large clinical isolate collection and further examined Bxa, a highly abundant ADPRT in Bacteroides. Bxa is expressed, secreted, and enzymatically active in Bacteroides and can ADP-ribosylate non-muscle myosin II proteins. Addition of Bxa to epithelial cells remodeled the actin cytoskeleton and induced secretion of inosine. Bxa-encoding B. stercoris can use inosine as a carbon source and colonizes the gut to significantly greater numbers than a bxa-deleted strain in germ-free and altered Schaedler flora (ASF) mice. Colonization correlated with increased inosine concentrations in the feces and tissues. Altogether, our results show that ADPRTs are abundant in the microbiome and act as bacterial fitness factors.

Colesevelam Reduces Ethanol-Induced Liver Steatosis in Humanized Gnotobiotic Mice.

Alcohol-related liver disease is associated with intestinal dysbiosis. Functional changes in the microbiota affect bile acid metabolism and result in elevated serum bile acids in patients with alcohol-related liver disease. The aim of this study was to identify the potential role of the bile acid sequestrant colesevelam in a humanized mouse model of ethanol-induced liver disease. We colonized germ-free (GF) C57BL/6 mice with feces from patients with alcoholic hepatitis and subjected humanized mice to the chronic-binge ethanol feeding model. Ethanol-fed gnotobiotic mice treated with colesevelam showed reduced hepatic levels of triglycerides and cholesterol, but liver injury and inflammation were not decreased as compared with non-treated mice. Colesevelam reduced hepatic cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1) protein expression, although serum bile acids were not lowered. In conclusion, our findings indicate that colesevelam treatment mitigates ethanol-induced liver steatosis in mice.

The microbiome affects liver sphingolipids and plasma fatty acids in a murine model of the Western diet based on soybean oil.

Studies in mice using germfree animals as controls for microbial colonization have shown that the gut microbiome mediates diet-induced obesity. Such studies use diets rich in saturated fat, however, Western diets in the United States America are enriched in soybean oil, composed of unsaturated fatty acids, either linoleic or oleic acid. Here, we addressed whether the microbiome is a variable in fat metabolism in mice on a soybean oil diet. We used conventionally-raised, low-germ, and germfree mice fed for 10 weeks diets either high or low in high-linoleic-acid soybean oil as the sole source of fat. Conventional and germfree mice gained relative fat weight and all mice consumed more calories on the high fat vs. low fat soybean oil diet. Plasma fatty acid levels were generally dependent on diet, with microbial colonization status affecting iso-C18:0, C20:3n-6, C14:0, and C15:0 levels. Colonization status, but not diet, impacted levels of liver sphingolipids including ceramides, sphingomyelins, and sphinganine. Our results confirm that absorbed fatty acids are mainly a reflection of the diet and that microbial colonization influences liver sphingolipid pools regardless of diet.

Feeding diversified protein sources exacerbates hepatic insulin resistance via increased gut microbial branched-chain fatty acids and mTORC1 signaling in obese mice.

Animal models of human diseases are classically fed purified diets that contain casein as the unique protein source. We show that provision of a mixed protein source mirroring that found in the western diet exacerbates diet-induced obesity and insulin resistance by potentiating hepatic mTORC1/S6K1 signaling as compared to casein alone. These effects involve alterations in gut microbiota as shown by fecal microbiota transplantation studies. The detrimental impact of the mixed protein source is also linked with early changes in microbial production of branched-chain fatty acids (BCFA) and elevated plasma and hepatic acylcarnitines, indicative of aberrant mitochondrial fatty acid oxidation. We further show that the BCFA, isobutyric and isovaleric acid, increase glucose production and activate mTORC1/S6K1 in hepatocytes. Our findings demonstrate that alteration of dietary protein source exerts a rapid and robust impact on gut microbiota and BCFA with significant consequences for the development of obesity and insulin resistance.

Embryonic macrophages function during early life to determine invariant natural killer T cell levels at barrier surfaces.

It is increasingly recognized that immune development within mucosal tissues is under the control of environmental factors during early life. However, the cellular mechanisms that underlie such temporally and regionally restrictive governance of these processes are unclear. Here, we uncover an extrathymic pathway of immune development within the colon that is controlled by embryonic but not bone marrow-derived macrophages, which determines the ability of these organs to receive invariant natural killer T (iNKT) cells and allow them to establish local residency. Consequently, early-life perturbations of fetal-derived macrophages result in persistent decreases of mucosal iNKT cells and is associated with later-life susceptibility or resistance to iNKT cell-associated mucosal disorders. These studies uncover a host developmental program orchestrated by ontogenically distinct macrophages that is regulated by microbiota, and they reveal an important postnatal function of macrophages that emerge in fetal life.

Spontaneous and Induced Tumors in Germ-Free Animals: A General Review.

Cancer, bacteria, and immunity relationships are much-debated topics in the last decade. Microbiome's importance for metabolic and immunologic modulation of the organism adaptation and responses has become progressively evident, and models to study these relationships, especially about carcinogenesis, have acquired primary importance. The availability of germ-free (GF) animals, i.e., animals born and maintained under completely sterile conditions avoiding the microbiome development offers a unique tool to investigate the role that bacteria can have in carcinogenesis and tumor development. The comparison between GF animals with the conventional (CV) counterpart with microbiome can help to evidence conditions and mechanisms directly involving bacterial activities in the modulation of carcinogenesis processes. Here, we review the literature about spontaneous cancer and cancer modeling in GF animals since the early studies, trying to offer a practical overview on the argument.

Modeling microbe-host interaction in the pathogenesis of Crohn's disease.

Alterations in the gut microbiota structure and function are thought to play an important role in the pathogenesis of Crohn's disease (CD). The rapid advancement of high-throughput sequencing technologies led to the identification of microbiome risk signatures associated with distinct disease phenotypes and progressing disease entities. Functional validation of the identified microbiome signatures is essential to understand the underlying mechanisms of microbe-host interactions. Germfree mouse models are available to study the functional role of disease-conditioning complex gut microbial ecosystems (dysbiosis) or pathobionts (single bacteria) in the pathogenesis of CD-like inflammation. Here, we discuss the clinical and mechanistic relevance and limitations of gnotobiotic mouse models in the context of CD. In addition, we will address the role of diet as an essential external factor modulating microbiome changes, potentially underlying disease initiation and development.