Germ-free Mice Research Articles

Intestinal dysbiosis exacerbates susceptibility to the anti-NMDA receptor encephalitis-like phenotype by changing blood brain barrier permeability and immune homeostasis

Changes in the intestinal microbiota have been observed in patients with anti-N-methyl-D-aspartate receptor encephalitis (NMDARE). However, whether and how the intestinal microbiota is involved in the pathogenesis of NMDARE susceptibility needs to be demonstrated. Here, we first showed that germ-free (GF) mice that underwent fecal microbiota transplantation (FMT) from NMDARE patients, whose fecal microbiota exhibited low short-chain fatty acid content, decreased abundance of Lachnospiraceae, and increased abundance of Verrucomicrobiota, Akkermansia, Parabacteroides, Oscillospirales, showed significant behavioral deficits. Then, these FMT mice were actively immunized with an amino terminal domain peptide from the GluN1 subunit (GluN1356–385) to mimic the pathogenic process of NMDARE. We found that FMT mice showed an increased susceptibility to an encephalitis-like phenotype characterized by more clinical symptoms, greater pentazole (PTZ)-induced susceptibility to seizures, and higher levels of T2 weighted image (T2WI) hyperintensities following immunization. Furthermore, mice with dysbiotic microbiota had impaired blood–brain barrier integrity and a proinflammatory condition. In NMDARE-microbiota recipient mice, the levels of Evan’s blue (EB) dye extravasation increased, ZO-1 and claudin-5 expression decreased, and the levels of proinflammatory cytokines (IL-1, IL-6, IL-17, TNF-α and LPS) increased. Finally, significant brain inflammation, mainly in hippocampal and cortical regions, with modest neuroinflammation, immune cell infiltration, and reduced expression of NMDA receptors were observed in NMDARE microbiota recipient mice following immunization. Overall, our findings demonstrated that intestinal dysbiosis increased NMDARE susceptibility, suggesting a new target for limiting the occurrence of the severe phenotype of NMDARE.

Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life

BackgroundPerinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel diseases (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2′-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis.ResultsIn pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring.ConclusionsOur findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

Microbial exposure during early life regulates development of bile duct inflammation

Objectives The early life microbiome has been linked to inflammatory diseases in adulthood and a role for the microbiome in bile duct inflammation is supported by both human and murine studies. We utilized the NOD.c3c4 mouse model that develops a spontaneous immune-driven biliary disease with a known contribution of the microbiome to evaluate the temporal effects of the early life microbiome. Materials and methods Germ-free (GF) NOD.c3c4 mice were conventionalized into a specific pathogen free environment at birth (conventionally raised, CONV-R) or at weaning (germ-free raised, GF-R) and compared with age and gender-matched GF and conventional (CONV) NOD.c3c4 mice. At 9 weeks of age, liver pathology was assessed by conventional histology and flow cytometry immunophenotyping. Results Neonatal exposure to microbes (CONV-R) increased biliary inflammation to similar levels as regular conventional NOD.c3c4 mice, while delayed exposure to microbes (GF-R) restrained the biliary inflammation. Neutrophil infiltration was increased in all conventionalized mice compared to GF. An immunophenotype in the liver similar to CONV was restored in both CONV-R and GF-R compared to GF mice displaying a proportional increase of B cells and reduction of T cells in the liver. Conclusions Microbial exposure during early life has a temporal impact on biliary tract inflammation in the NOD.c3c4 mouse model suggesting that age-sensitive interaction with commensal microbes have long-lasting effects on biliary immunity that can be of importance for human cholangiopathies.

A key genetic factor governing arabinan utilization in the gut microbiome alleviates constipation

Impaired gastrointestinal motility is associated with gut dysbiosis. Probiotics, such as Bifidobacteria, can improve this bowel disorder; however, efficacy is strain-dependent. We determine that a genetic factor, the abfA cluster governing arabinan utilization, in Bifidobacterium longum impacts treatment efficacy against functional constipation (FC). In mice with FC, B.longum, but not an abfA mutant, improved gastrointestinal transit time, an affect that was dependent upon dietary arabinan. abfA genes were identified in other commensal bacteria, whose effects in ameliorating murine FC were similarly abfA-dependent. In a double-blind, randomized, placebo-controlled clinical trial, supplementation with abfA-cluster-carrying B.longum, but not an abfA-deficient strain, enriched arabinan-utilization residents, increased beneficial metabolites, and improved FC symptoms. Across human cohorts, abfA-cluster abundance can predict FC, and transplantation of abfA cluster-enriched human microbiota to FC-induced germ-free mice improved gut motility. Collectively, these findings demonstrate a role for microbial abfA cluster in ameliorating FC, establishing principles for genomics-directed probiotic therapies.

OA susceptibility in mice is partially mediated by the gut microbiome, is transferrable via microbiome transplantation and is associated with immunophenotype changes

ObjectivesThe Murphy Roths Large (MRL)/MpJ ‘superhealer’ mouse strain is protected from post-traumatic osteoarthritis (OA), although no studies have evaluated the microbiome in the context of this protection. This study characterised...

Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in mice

BackgroundIntestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn’s disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1i∆IEC) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1∆PC) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs.ResultsDepletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1∆PC or Phb1i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate.ConclusionsThese results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn’s disease patients harboring epithelial cell mitochondrial dysfunction.BykqUrdWkjvtZT9W9KFZmHVideo

Protective effect of microbiota-derived short chain fatty acids on vascular dysfunction in mice with systemic lupus erythematosus induced by toll like receptor 7 activation

Our objective was to investigate whether short-chain fatty acids (SCFAs), specifically acetate and butyrate, could prevent vascular dysfunction and elevated blood pressure (BP) in mice with systemic lupus erythematosus (SLE) induced by TLR7 activation using imiquimod (IMQ). Treatment with both SCFAs and dietary fibers rich in resistant starch (RS) or inulin-type fructans (ITF) effectively prevented the development of hypertension and cardiac hypertrophy. Additionally, these treatments improved aortic relaxation induced by acetylcholine and mitigated vascular oxidative stress. Acetate and butyrate treatments also contributed to the maintenance of colonic integrity, reduced endotoxemia, and decreased the proportion of helper T (Th)17 cells in mesenteric lymph nodes (MLNs), blood, and aorta in TLR7-induced SLE mice. The observed changes in MLNs were correlated with increased levels of GPR43 mRNA in mice treated with acetate and increased GPR41 levels along with decreased histone deacetylase (HDAC)− 3 levels in mice treated with butyrate. Notably, the effects attributed to acetate, but not butyrate, were nullified when co-administered with the GPR43 antagonist GLPG-0974. T cell priming and differentiation into Th17 cells in MLNs, as well as increased Th17 cell infiltration, were linked to aortic endothelial dysfunction and hypertension subsequent to the transfer of faecal microbiota from IMQ-treated mice to germ-free (GF) mice. These effects were counteracted in GF mice through treatment with either acetate or butyrate. To conclude, these findings underscore the potential of SCFA consumption in averting hypertension by restoring balance to the interplay between the gut, immune system, and vascular wall in SLE induced by TLR7 activation.

Mechanism of Qizhen decoction-mediated maturation of DC cells to activate the IL-12/JAK2/STAT4 pathway to sensitise PD-1 inhibitors in the treatment of colorectal cancer

Ethnopharmacological relevanceTraditional Chinese medicine has been utilized to treat colorectal cancer (CRC). Qizhen decoction (QZD), a potential compound prescription of traditional Chinese medicine, possesses multiple biological activities. It has been used to treat CRC in clinical practice and has been proven to be effective. Aim of the studyTo investigate the impact of QZD supported by intestinal flora in combination with PD-1 inhibitor on colorectal cancer, and to elucidate the mechanism by which QZD enhances the sensitivity of PD-1 inhibitor against colorectal cancer. Materials and methodsObservation of Intestinal Flora Mediating the Effect of QZD Combined with PD-1 Inhibitor in the Treatment of Colorectal Cancer. We used Flow cytometry and qPCR to detect the effect of QZD combined with PD-1 inhibitor on the activation of effector T cells in a wild mouse model of colorectal cancer. In wild and germ-free mouse models, the differences in inflammatory factors, pathological change, body mass, colorectal length, and tumour load were observed. In the study of the mechanism of QZD combined with PD-1 inhibitor in the treatment of colorectal cancer, the study evaluated the abundance of Akkermansia, the phenotypes of effector T cells and DC cells, as well as inflammatory factors in each group of mice to determine whether Akkermansia played a role in activating DC cells. Based on the JAK2/TYK2/STAT4 pathway, the mechanism of PD-1 inhibitor sensitisation by QZD in colorectal cancer was further investigated. ResultsWe found that QZD combined with PD-1 inhibitor could improve the therapeutic effect on colorectal cancer by inducing more critical immune functions. QZD promotes increased Akkermansia abundance in the gut. Akkermansia promotes maturation of DC cells, and mature DC cells activate the IL-12/JAK2/STAT4 pathway, which significantly activates effector T cells. Akkermansia is key to QZD combined with PD-1 inhibitor-mediated immunity exerting a therapeutic effect on colorectal cancer. ConclusionThe mechanism of action of the QZD sensitizing PD-1 inhibitor is to promote the maturation of DC cells to release IL-12 and activate the JAK2/STAT4 pathway to induce effector T cell activation by increasing the abundance of Akkermansia.

Microbe-derived uremic solutes enhance thrombosis potential in the host.

Alterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

Gut microbiota regulates postprandial GLP-1 response via ileal bile acid-TGR5 signaling

ABSTRACT The gut microbiota interacts with intestinal epithelial cells through microbial metabolites to regulate the release of gut hormones. We investigated whether the gut microbiota affects the postprandial glucagon-like peptide-1 (GLP-1) response using antibiotic-treated mice and germ-free mice. Gut microbiome depletion completely abolished postprandial GLP-1 response in the circulation and ileum in a lipid tolerance test. Microbiome depletion did not influence the GLP-1 secretory function of primary ileal cells in response to stimulators in vitro, but dramatically changed the postprandial dynamics of endogenous bile acids, particularly ω-muricholic acid (ωMCA) and hyocholic acid (HCA). The bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) but not farnesoid X receptor (FXR), participated in the regulation of postprandial GLP-1 response in the circulation and ileum, and ωMCA or HCA stimulated GLP-1 secretion via TGR5. Finally, fecal microbiota transplantation or ωMCA and HCA supplementation restored postprandial GLP-1 response. In conclusion, gut microbiota is indispensable for maintaining the postprandial GLP-1 response specifically in the ileum, and bile acid (ωMCA and HCA)-TGR5 signaling is involved in this process. This study helps to understand the essential interplay between the gut microbiota and host in regulating postprandial GLP-1 response and opens the foundation for new therapeutic targets.

Dysbiotic microbiome induce cardiac impairment by attenuating brain-derived neurotrophic factor in inflammatory bowel diseases

Abstract Background & Aims Mounting evidence suggests that inflammatory bowel disease (IBD) is associated with increased incidences of cardiovascular diseases (CVD), including myocardial infarction and heart failure. However, the molecular mechanisms by which IBD predisposes patients to CVD are poorly understood. This study aimed to identify the role of dysbiosis in gut-heart crosstalk. Methods Chronic colitis-induced cardiac impairment was assessed in mice subjected to dextran sodium sulfate (DSS), rats treated with trinitrobenzenesulfonic acid, and IL10-/-mice which develop spontaneous colitis after weaning. Fecal microbiota of wild type or IL10-/- mice were transplanted to C57BL/6J mice treated with microbiome-depleting antibiotic cocktail and germ-free mice to determine if dysbiosis afflicts the heart. Heart function was evaluated by echocardiography. Heart transcriptomes were profiled by RNA-sequencing. Gut microbiota profiling was performed by 16S rRNA sequencing. Adverse effect of brain-derived neutrophic factor (BDNF) deficiency on the heart was evaluated in cardiac-specific BDNF knockout mice (BdnfΔheart). Results Impairment of heart function, evidenced by decreased left ventricle ejection fraction (LVEF), increased LV mass, and elevated serum B-type natriuretic peptide, was observed in all 3 models of colitis, providing direct evidence that chronic colitis deteriorates the heart (Figure 1). RNA-sequencing revealed loss of Bdnf and alterations of multiple genes critical to heart homeostasis in the myocardium. Many of the changes, including Bdnf reduction, were found in microbiota-depleted C57BL/6J mice and germ-free mice received dysbiotic fecal microbiome, compared to controls, suggesting that dysbiosis impaired the heart by suppressing Bdnf. BdnfΔheart mice had impaired heart function, including decreased contractility and EF, increased fibrosis, and impaired coronary microcirculation (lower CD31), compared to Bdnf[2lox] mice. Mechanistically, miR-155 in the heart and exosomal miR-29b in the plasma were significantly elevated by chronic colitis. miR-155 and miR-29b mimics suppressed BDNF in cardiomyocytes both in vitro and in vivo. Administration of miR-155 inhibitors or exosomes packaged with miR-29b antagonists ameliorated BDNF reduction in the heart of IL10-/- mice. BDNF supplementation markedly improved heart function in DSS mice. Conclusions Chronic colitis impairs the heart through dysbiosis/miR-29b/miR-155/BDNF signaling axis. Targeting this pathway is a promising strategy to overcome gut dysbiosis harmful for the heart in IBD.

Heart failure stimulates colorectal tumour growth through gut microbial alterations of specific populations

Abstract Background/purpose The field of reverse cardio-oncology has received growing attention in the last years, since epidemiological studies showed that patients with HF are at increased risk of developing cancer. In addition, HF can stimulate tumour growth directly in distant organs, including colon cancer. The exact mechanisms underlying this phenomenon, however, remain largely unknown. The connection between the microbiome and colorectal cancer (CRC) has been studied extensively and microbial dysbiosis in CRC is linked to cancer progression. Recent studies have also shown that HF patients display microbial dysbiosis. We hypothesized that microbial dysbiosis might contribute to HF-induced CRC tumour growth. Methods C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16s rRNA sequencing, and pooled for faecal microbial transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane (AOM)/Dextran sodium sulphate (DSS). The CRC mice were transplanted with faeces from MI or sham mice. Disease progression was monitored using the disease severity score and endoscopy. After ten weeks mice were sacrificed, and tumour number and proliferation were quantified. All experimental procedures were performed in accordance with the European Union guidelines (CCD_8141, the Netherlands, LA_1400637, Belgium). Results MI-induced HF resulted in a decrease in microbial diversity (Shannon diversity p=0.018), characterized by a decrease in short chain fatty acid-producing bacteria, including the genera Blautia (p<0.01) and Roseburia (p<0.01). In addition, caecal butyrate levels were decreased in MI mice (p=0.029). These changes were largely comparable to microbial changes observed in HF patients and CRC patients. Interestingly, faecal transplantation with faeces from MI mice resulted in faster growth of CRC tumours in mice treated with AOM/DSS. This was demonstrated by a higher endoscopy score (7.9 ±2.5 vs. 10 ±2.4, p=0.038), indicative of worse disease progression, increased tumour counts (22 ±5.3 vs. 29 ±6, p<0.01), and a higher percentage of Ki67 positive cells in tumour tissue (p=0.012), relative to AOM/DSS treated mice transplanted with faeces from sham mice. Sequencing data revealed several differences in the microbial composition of mice transplanted with faeces from MI mice compared to mice transplanted with faeces from sham mice. Conclusion Our results demonstrate that HF induces microbial dysbiosis in mice. In addition, we show, for the first time, using a faecal transplantation model, that HF-induced microbial dysbiosis is causally involved in CRC tumour growth in a mouse model of CRC. Targeting the microbiome may present a tool to mitigate HF associated co-morbidities, especially cancer.Graphical abstract

Murine cartilage microbial DNA deposition occurs rapidly following the introduction of a gut microbiome and changes with obesity, aging, and knee osteoarthritis

Cartilage microbial DNA patterns have been recently characterized in osteoarthritis (OA). The objectives of this study were to evaluate the gut origins of cartilage microbial DNA, to characterize cartilage microbial changes with age, obesity, and OA in mice, and correlate these to gut microbiome changes. We used 16S rRNA sequencing performed longitudinally on articular knee cartilage from germ-free (GF) mice following oral microbiome inoculation and cartilage and cecal samples from young and old wild-type mice with/without high-fat diet-induced obesity (HFD) and with/without OA induced by destabilization of the medial meniscus (DMM) to evaluate gut and cartilage microbiota. Microbial diversity was assessed, groups compared, and functional metagenomic profiles reconstructed. Findings were confirmed in an independent cohort by clade-specific qPCR. We found that cartilage microbial patterns developed at 48 h and later timepoints following oral microbiome inoculation of GF mice. Alpha diversity was increased in SPF mouse cartilage samples with age (P = 0.013), HFD (P = 5.6E-4), and OA (P = 0.029) but decreased in cecal samples with age (P = 0.014) and HFD (P = 1.5E-9). Numerous clades were altered with aging, HFD, and OA, including increases in Verrucomicrobia in both cartilage and cecal samples. Functional analysis suggested changes in dihydroorotase, glutamate-5-semialdehyde dehydrogenase, glutamate-5-kinase, and phosphoribosylamine-glycine ligase, in both cecum and cartilage, with aging, HFD, and OA. In conclusion, cartilage microbial DNA patterns develop rapidly after the introduction of a gut microbiome and change in concert with the gut microbiome during aging, HFD, and OA in mice. DMM-induced OA causes shifts in both cartilage and cecal microbiome patterns independent of other factors.

Sterility testing of germ-free mouse colonies.

In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided.

Gut microbiota regulate wheat starch‐induced metabolic disorders via control of colonic SCD1

AbstractUnbalanced carbohydrate diets are linked to metabolic disorders; however, there is limited information on the role of gut microbiota. We explored differences in mice gut microbiota and metabolic phenotypes resulting from diets with different carbohydrate sources. To investigate the metabolic dysfunction and mechanism of the wheat flour and starch on metabolic disorders, we analyzed the changes of phenotype and biomarkers in rice and wheat flour diet in C57BL/6 mice (specific pathogen‐free) and fecal transplantation from antibiotic‐treated or germ‐free (GF) mice model. Rice‐fed mice presented normal metabolic reactions, whereas wheat‐fed mice exhibited increased body weight, insulin resistance, serum triglycerides, and fasting glucose levels. Wheat starch‐fed mice displayed lower fecal microbiota alpha diversity, with a reduced abundance of Bacteroidetes, than high‐fat diet (HFD)‐fed and chow‐fed mice. Fecal transplantation from antibiotic‐treated or GF mice revealed that metabolic disruption induced by wheat diet feeding was mediated through gut microbiota and stearoyl‐CoA desaturase 1 (SCD1) signaling in the colon. Taken together, wheat‐based diets induce gut microbiota alterations, causing obesity and metabolic diseases through molecular activity linked to colonic SCD1. Therefore, colonic SCD1 could be a therapeutic target for patients with metabolic diseases who consume high‐carbohydrate diets.

Tissue-resident B cells orchestrate macrophage polarisation and function

B cells play a central role in humoral immunity but also have antibody-independent functions. Studies to date have focused on B cells in blood and secondary lymphoid organs but whether B cells reside in non-lymphoid organs (NLO) in homeostasis is unknown. Here we identify, using intravenous labeling and parabiosis, a bona-fide tissue-resident B cell population in lung, liver, kidney and urinary bladder, a substantial proportion of which are B-1a cells. Tissue-resident B cells are present in neonatal tissues and also in germ-free mice NLOs, albeit in lower numbers than in specific pathogen-free mice and following co-housing with ‘pet-store’ mice. They spatially co-localise with macrophages and regulate their polarization and function, promoting an anti-inflammatory phenotype, in-part via interleukin-10 production, with effects on bacterial clearance during urinary tract infection. Thus, our data reveal a critical role for tissue-resident B cells in determining the homeostatic ‘inflammatory set-point’ of myeloid cells, with important consequences for tissue immunity.

Gut-Brain-axis: effect of basil oil on the gut microbiota and its contribution to the anticonvulsant properties

BackgroundEpilepsy is a chronic neurological condition that disrupts the normal functioning of the brain and it is characterized by seizures. Research suggests the involvement of the Gut-Brain axis in epilepsy. This study seeks to determine the role of the gut microbiota in the anticonvulsant effect of basil oil (BO) using antibiotic-depleted and altered germ-free mice against naïve mice in Pentylenetetrazole (PTZ) induced seizure model. There is an ever growing interest in improvement of treatment outcomes in epilepsy and also in the development of newer therapeutic options, especially in the population of patients that do not attain seizure relief from available antiseizure medications (ASMs). According to research, gut microbiota can alter brain function and development. Increasing evidence suggests disrupting the delicate symbiotic balance existing between the gut and brain results in disease conditions. Also, the oil from Ocimum basilicum L., (BO) has been proven scientifically to significantly block clonic seizures induced by PTZ and picrotoxin in seizure models.MethodsThe microbiota of mice were depleted or altered by administering cocktail antibiotics and individual antibiotics respectively. DNA was isolated from mouse stool, and then the 16S ribosomal ribonucleic acid (16S rRNA) gene was quantitatively amplified using reverse transcription-polymerase chain reaction (RT-PCR). Amplicons were sequenced to determine the phylogenetic make-up of the bacteria involved. Metabolic profiles of the serum and stool of mice were determined using Proton (1H) Nuclear Magnetic Resonance (NMR) spectroscopy.ResultsCocktail antibiotic pre-treatment significantly reversed the anticonvulsant effect of BO by increasing frequency and duration of seizures but did not affect latency to seizure. In mice pre-treated with single antibiotics, the anticonvulsant effect of BO was lost as latency to seizures, frequency and duration of seizures increased compared to mice that received only BO. Assessment of the phylogenetic make-up of the microbiota in antibiotic pre-treated mice showed a distorted composition of the microbiota compared to the control group.ConclusionDepletion of the microbiota significantly reversed the anticonvulsant actions of BO. The concentrations of short chain fatty acids (SCFAs) was higher in stool than in the serum of the mice. Administration of BO probably does not influence the microbial composition within the mouse microbiota. The elevated ratio of Firmicutes to Bacteroidetes in microbiota-depleted groups might have contributed to the reversal of anticonvulsant actions of BO.

275 Awardee Talk: Gnotobiotic Porcine Models to Better Understand Gut Microbiome-Host Interactions in Human and Porcine Hosts

Abstract Better understanding the interactions between the gut microbiome and its host is at the forefront of research conducted in areas related to human health as well as livestock production. The complexity of the gut microbiome as well as the numerous environmental and host factors that influence the structure and function of the microbial community complicates the study of host-gut microbiome interactions under natural conditions. To this end, germ-free animal models which have been transplanted with target microbiomes and raised under controlled experimental conditions provide a unique opportunity to mitigate some of the inherent difficulties related to studying host-microbiome interactions in conventional animals as well as in humans. Due to its high degree of similarity to humans at the anatomical, biochemical, and immunological levels and its importance in the livestock industry, the domestic pig (Sus scrofa domesticus) is an excellent animal model which has a unique ‘dual-purpose’ potential for use in both human health and livestock production research. Using high-throughput DNA sequencing technologies, we have demonstrated some of the advantages of using human microbiota-associated (HMA) porcine models compared with commonly used HMA mouse models to study the human gut microbiome. Specifically, we identified that a significantly greater percentage of taxa from child, adult, and senior donors were able to colonize (69% to 85% in pigs versus 41% to 49% in mice) and persist (28% to 57% in piglets versus 21% to 28% in mice) in the HMA piglets compared with the HMA mice, and that this was primarily driven by a greater colonization success of Firmicutes members in the pigs compared with the mice. In addition, we have characterized human infant microbiota-associated porcine models at both the taxonomic and functional levels to better understand the HMA piglet as a model for infant microbiome investigations. We have also utilized the porcine microbiota-associated (PMA) pig model to understand the establishment and development of the porcine gut microbiome. This model has great potential to dissect the mechanistic relationship between the host and its microbiome in pigs. Additionally, this model can be used to understand how prebiotics and direct-fed microbials might interact with the gut microbiome and influence porcine health and well-being. Despite some of the challenges to the widespread use of gnotobiotic porcine models, such as high overhead costs and labor involved in animal care, the porcine model holds a lot of promise to help better understand host-gut microbiome interactions.

High Soluble Fiber Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites in Mice

Dietary fibers are mainly fermented by the gut microbiota, but their roles in colorectal cancer (CRC) are largely unclear. Here, we investigated the associations of different fibers with colorectal tumorigenesis in mice. Apcmin/+ mice and C57BL/6 mice with azoxymethane (AOM) injection were used as CRC mouse models. Mice were fed with mixed high-fiber diet (20% soluble fiber and 20% insoluble fiber), high-inulin diet, high-guar gum diet, high-cellulose diet, or diets with different inulin dose. Germ-free mice were used for validation. Fecal microbiota and metabolites were profiled by shotgun metagenomic sequencing and liquid chromatography-mass spectrometry, respectively. Mixed high-fiber diet promoted colorectal tumorigenesis with increased tumor number and tumor load in AOM-treated and Apcmin/+ mice. Antibiotics use abolished the pro-tumorigenic effect of mixed high-fiber diet, while transplanting stools from mice fed with mixed high-fiber diet accelerated tumor growth in AOM-treated germ-free mice. We therefore characterized the contribution of soluble and insoluble fiber in CRC separately. Our results revealed that soluble fiber inulin or guar gum, but not insoluble fiber cellulose, promoted colorectal tumorigenesis in AOM-treated and Apcmin/+ mice. Soluble fiber induced gut dysbiosis with Bacteroides uniformis enrichment and Bifidobacterium pseudolongum depletion, accompanied by increased fecal butyrate and serum bile acids and decreased inosine. We also identified a positive correlation between inulin dosage and colorectal tumorigenesis. Moreover, transplanting stools from mice fed with high-inulin diet increased colonic cell proliferation and oncogene expressions in germ-free mice. High-dose soluble but not insoluble fiber potentiates colorectal tumorigenesis in a dose-dependent manner by dysregulating gut microbiota and metabolites in mice.

The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. Although the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle immunization, the microbiome suppresses Ig and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA lipid nanoparticle vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for continued therapeutic development and deployment of these vaccines.