Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice.

Streptococcus anginosus (S. anginosus) was enriched in the gastric mucosa of patients with gastric cancer (GC). Here, we show that S. anginosus colonized the mouse stomach and induced acute gastritis. S. anginosus infection spontaneously induced progressive chronic gastritis, parietal cell atrophy, mucinous metaplasia, and dysplasia in conventional mice, and the findings were confirmed in germ-free mice. In addition, S. anginosus accelerated GC progression in carcinogen-induced gastric tumorigenesis and YTN16 GC cell allografts. Consistently, S. anginosus disrupted gastric barrier function, promoted cell proliferation, and inhibited apoptosis. Mechanistically, we identified an S. anginosus surface protein, TMPC, that interacts with Annexin A2 (ANXA2) receptor on gastric epithelial cells. Interaction of TMPC with ANXA2 mediated attachment and colonization of S. anginosus and induced mitogen-activated protein kinase (MAPK) activation. ANXA2 knockout abrogated the induction of MAPK by S. anginosus. Thus, this study reveals S. anginosus as a pathogen that promotes gastric tumorigenesis via direct interactions with gastric epithelial cells in the TMPC-ANXA2-MAPK axis.

Establishing or Exaggerating Causality for the Gut Microbiome: Lessons from Human Microbiota-Associated Rodents.

Human diseases are increasingly linked with an altered or "dysbiotic" gut microbiota, but whether such changes are causal, consequential, or bystanders to disease is, for the most part, unresolved. Human microbiota-associated (HMA) rodents have become a cornerstone of microbiome science for addressing causal relationships between altered microbiomes and host pathology. In a systematic review, we found that 95% of published studies (36/38) on HMA rodents reported a transfer of pathological phenotypes to recipient animals, and many extrapolated the findings to make causal inferences to human diseases. We posit that this exceedingly high rate of inter-species transferable pathologies is implausible and overstates the role of the gut microbiome in human disease. We advocate for a more rigorous and critical approach for inferring causality to avoid false concepts and prevent unrealistic expectations that may undermine the credibility of microbiome science and delay its translation.

Segmented Filamentous Bacteria Prevent and Cure Rotavirus Infection.

Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.

Microbiome Influences Prenatal and Adult Microglia in a Sex-Specific Manner.

Microglia are embryonically seeded macrophages that contribute to brain development, homeostasis, and pathologies. It is thus essential to decipher how microglial properties are temporally regulated by intrinsic and extrinsic factors, such as sexual identity and the microbiome. Here, we found that microglia undergo differentiation phases, discernable by transcriptomic signatures and chromatin accessibility landscapes, which can diverge in adult males and females. Remarkably, the absence of microbiome in germ-free mice had a time and sexually dimorphic impact both prenatally and postnatally: microglia were more profoundly perturbed in male embryos and female adults. Antibiotic treatment of adult mice triggered sexually biased microglial responses revealing both acute and long-term effects of microbiota depletion. Finally, human fetal microglia exhibited significant overlap with the murine transcriptomic signature. Our study shows that microglia respond to environmental challenges in a sex- and time-dependent manner from prenatal stages, with major implications for our understanding of microglial contributions to health and disease.

Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation.

The severity of T cell-mediated gastrointestinal (GI) diseases such as graft-versus-host disease (GVHD) and inflammatory bowel diseases correlates with a decrease in the diversity of the host gut microbiome composition characterized by loss of obligate anaerobic commensals. The mechanisms underpinning these changes in the microbial structure remain unknown. Here, we show in multiple specific pathogen-free (SPF), gnotobiotic, and germ-free murine models of GI GVHD that the initiation of the intestinal damage by the pathogenic T cells altered ambient oxygen levels in the GI tract and caused dysbiosis. The change in oxygen levels contributed to the severity of intestinal pathology in a host intestinal HIF-1α- and a microbiome-dependent manner. Regulation of intestinal ambient oxygen levels with oral iron chelation mitigated dysbiosis and reduced the severity of the GI GVHD. Thus, targeting ambient intestinal oxygen levels may represent a novel, non-immunosuppressive strategy to mitigate T cell-driven intestinal diseases.

A Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis.

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Defining Genetic Variation in Widely Used Congenic and Backcrossed Mouse Models Reveals Varied Regulation of Genes Important for Immune Responses.

Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.

Microbial influences on severity and sex bias of systemic autoimmunity.

Commensal microbes have the capacity to affect development and severity of autoimmune diseases. Germ-free (GF) animals have proven to be a fine tool to obtain definitive answers to the queries about the microbial role in these diseases. Moreover, GF and gnotobiotic animals can be used to dissect the complex symptoms and determine which are regulated (enhanced or attenuated) by microbes. These include disease manifestations that are sex biased. Here, we review comparative analyses conducted between GF and Specific-Pathogen Free (SPF) mouse models of autoimmunity. We present data from the B6;NZM-Sle1NZM2410/AegSle2NZM2410/AegSle3NZM2410/Aeg-/LmoJ (B6.NZM) mouse model of systemic lupus erythematosus (SLE) characterized by multiple measurable features. We compared the severity and sex bias of SPF, GF, and ex-GF mice and found variability in the severity and sex bias of some manifestations. Colonization of GF mice with the microbiotas taken from B6.NZM mice housed in two independent institutions variably affected severity and sexual dimorphism of different parameters. Thus, microbes regulate both the severity and sexual dimorphism of select SLE traits. The sensitivity of particular trait to microbial influence can be used to further dissect the mechanisms driving the disease. Our results demonstrate the complexity of the problem and open avenues for further investigations.

Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response.

Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.

Mucosal vaccination in a murine gnotobiotic model of Giardia lamblia infection.

Giardia lamblia is an important protozoan cause of diarrheal disease worldwide, delayed development and cognitive impairment in children in low- and middle-income countries, and protracted post-infectious syndromes in developed regions. G. lamblia resides in the lumen and at the epithelial surface of the proximal small intestine but is not mucosa invasive. The protozoan parasite is genetically diverse with significant genome differences across strains and assemblages. Animal models, particularly murine models, have been instrumental in defining mechanisms of host defense against G. lamblia, but mice cannot be readily infected with most human pathogenic strains. Antibiotic pretreatment can increase susceptibility, suggesting that the normal microbiota plays a role in controlling G. lamblia infection in mice, but the broader implications on susceptibility to diverse strains are not known. Here, we have used gnotobiotic mice to demonstrate that robust intestinal infection can be achieved for a broad set of human-pathogenic strains of the genetic assemblages A and B. Furthermore, gnotobiotic mice were able to eradicate infection with a similar kinetics to conventional mice after trophozoite challenge. Germ-free mice could also be effectively immunized by the mucosal route with a protective antigen, α1-giardin, in a manner dependent on CD4 T cells. These results indicate that the gnotobiotic mouse model is powerful for investigating acquired host defenses in giardiasis, as the mice are broadly susceptible to diverse G. lamblia strains yet display no apparent defects in mucosal immunity needed for controlling and eradicating this lumen-dwelling pathogen.

Crohn's disease-associated Escherichia coli LF82 in the gut damage of germ-free honeybees: A laboratory study.

Escherichia coli LF82 (LF82) is associated with Crohn's disease. The simplicity and genetic maneuverability of honeybees' gut microbiota make them suitable for studying host-microbe interactions. To understand the interaction between LF82 and host gut, LF82 was used to infect germ-free honeybees (Apis mellifera) orally. We found that LF82 successfully colonized the gut and shortened the lifespan of germ-free bees. LF82 altered the gut structure and significantly increased gut permeability. RT-qPCR showed that LF82 infection activated anti-infective immune pathways and upregulated the mRNAs levels of antimicrobial peptides in the gut of germ-free bees. The gut transcriptome showed that LF82 significantly upregulated genes involved in Notch signaling, adhesion junctions, and Toll and Imd signaling pathways and downregulated genes involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, protein digestion and absorption, and tyrosine metabolism. In conclusion, the human-derived enteropathogenic bacterium LF82 can successfully colonize the gut of germ-free honeybees and cause enteritis-like changes, which provides an ideal model organism for revealing the pathogenesis of bacterial-associated diseases.

Role of bacteria and microbial metabolites in immune modulation during early life.

Host-microbiome interplay from birth is essential for immune imprinting and tuning. Live gut microbes and microbial-derived metabolites regulate the development and modulation of the immune system, but whether microbial metabolites solely are sufficient to induce immune maturation remains unclear. Sterile faecal filtrates (FFT) were generated from murine gut contents. Newborn germ-free (GF) mice were treated twice daily with FFT (GF-FFT) or saline (GF-NaCl) from post-natal day 5 until 4 weeks of age. A third group of GF neonates were conventionalized by the transfer of caecal microbiota with live gut microbes. Host immune compartments were comprehensively immunophenotyped and systemically analysed in all available immune-related organs using flow cytometry. Oral FFT was associated with reduced survival among neonates (n = 7/19; 36.8% mortality), while saline treatment was well tolerated (n = 1/17, 5.9% mortality). Four-week-old FFT-treated pups were comparable in body weight to GF-NaCl, and the major B-cell, conventional T-cell and unconventional T-cell subsets were unchanged from saline-treated mice. Live bacteria administered during early life induced clear changes in proportions of B cells, T cells and T-cell subsets in all mucosal tissues and secondary lymphoid organs compared to GF-FFT, including restoration of intestinal natural killer T (NKT) cells with characteristics similar to conventional pups. Our findings show that oral administration of a FFT made of microbial metabolites, antigens and bacteriophages alone is insufficient to induce normal immune development elicited by the presence of live bacteria. Reduced survival during neonatal FFT treatment suggests a potential bioactive attribute of sterile faecal filtrates.

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.

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.

Investigating the impact of chlorine dioxide in shrimp-rearing water on stomach microbiome, gill transcriptome, and infection-related mortality in shrimp.

AIMS: This study aimed to assess the effects of chlorine dioxide (ClO2) in water on whiteleg shrimp Penaeus vannamei, evaluating its impact on stomach microbiota, gill transcriptome, and pathogens. METHODS AND RESULTS: ClO2 was added to the aquarium tanks containing the shrimp. The application of ClO2 to rearing water was lethal to shrimp at concentrations above 1.2 ppm. On the other hand, most of the shrimp survived at 1.0 ppm of ClO2. Microbiome analysis showed that ClO2 administration at 1.0 ppm significantly reduced the α-diversity of bacterial community composition in the shrimp stomach, and this condition persisted for at least 7 days. Transcriptome analysis of shrimp gill revealed that ClO2 treatment caused massive change of the gene expression profile including stress response genes. However, after 7 days of the treatment, the gene expression profile was similar to that of shrimp in the untreated control group, suggesting a recovery to the normal state. This 1.0-ppm ClO2 significantly reduced shrimp mortality in artificial challenges with an AHPND-causing Vibrio parahaemolyticus and white spot syndrome virus, which were added to rearing water. CONCLUSIONS: The use of ClO2 at appropriate concentrations effectively eliminates a significant portion of the bacteria in the shrimp stomach and pathogens in the water. The results of this study provide fundamental knowledge on the disinfection of pathogens in water using ClO2 and the creation of semi germ-free shrimp, which has significantly decreased microbiome in the stomach.

Impacts of maternal microbiota and microbial metabolites on fetal intestine, brain, and placenta.

BACKGROUND: The maternal microbiota modulates fetal development, but the mechanisms of these earliest host-microbe interactions are unclear. To investigate the developmental impacts of maternal microbial metabolites, we compared full-term fetuses from germ-free and specific pathogen-free mouse dams by gene expression profiling and non-targeted metabolomics. RESULTS: In the fetal intestine, critical genes mediating host-microbe interactions, innate immunity, and epithelial barrier were differentially expressed. Interferon and inflammatory signaling genes were downregulated in the intestines and brains of the fetuses from germ-free dams. The expression of genes related to neural system development and function, translation and RNA metabolism, and regulation of energy metabolism were significantly affected. The gene coding for the insulin-degrading enzyme (Ide) was most significantly downregulated in all tissues. In the placenta, genes coding for prolactin and other essential regulators of pregnancy were downregulated in germ-free dams. These impacts on gene expression were strongly associated with microbially modulated metabolite concentrations in the fetal tissues. Aryl sulfates and other aryl hydrocarbon receptor ligands, the trimethylated compounds TMAO and 5-AVAB, Glu-Trp and other dipeptides, fatty acid derivatives, and the tRNA nucleobase queuine were among the compounds strongly associated with gene expression differences. A sex difference was observed in the fetal responses to maternal microbial status: more genes were differentially regulated in male fetuses than in females. CONCLUSIONS: The maternal microbiota has a major impact on the developing fetus, with male fetuses potentially more susceptible to microbial modulation. The expression of genes important for the immune system, neurophysiology, translation, and energy metabolism are strongly affected by the maternal microbial status already before birth. These impacts are associated with microbially modulated metabolites. We identified several microbial metabolites which have not been previously observed in this context. Many of the potentially important metabolites remain to be identified.

Intestinal Microbiota Increases Cell Proliferation of Colonic Mucosa in Human-Flora-Associated (HFA) Mice.

Intestinal epithelium renewal strictly depends on fine regulation between cell proliferation, differentiation, and apoptosis. While murine intestinal microbiota has been shown to modify some epithelial cell kinetics parameters, less is known about the role of the human intestinal microbiota. Here, we investigated the rate of intestinal cell proliferation in C3H/HeN germ-free mice associated with human flora (HFA, n = 8), and in germ-free (n = 15) and holoxenic mice (n = 16). One hour before sacrifice, all mice were intraperitoneally inoculated with 5-bromodeoxyuridine (BrdU), and the number of BrdU-positive cells/total cells (labelling index, LI), both in the jejunum and the colon, was evaluated by immunohistochemistry. Samples were also observed by scanning electron microscopy (SEM). Moreover, the microbiota composition in the large bowel of the HFA mice was compared to that of of human donor's fecal sample. No differences in LI were found in the small bowels of the HFA, holoxenic, and germ-free mice. Conversely, the LI in the large bowel of the HFA mice was significantly higher than that in the germ-free and holoxenic counterparts (p = 0.017 and p = 0.048, respectively). In the holoxenic and HFA mice, the SEM analysis disclosed different types of bacteria in close contact with the intestinal epithelium. Finally, the colonic microbiota composition of the HFA mice widely overlapped with that of the human donor in terms of dominant populations, although Bifidobacteria and Lactobacilli disappeared. Despite the small sample size analyzed in this study, these preliminary findings suggest that human intestinal microbiota may promote a high proliferation rate of colonic mucosa. In light of the well-known role of uncontrolled proliferation in colorectal carcinogenesis, these results may deserve further investigation in a larger population study.

Guidelines for microbiome studies in renal physiology.

Gut microbiome research has increased dramatically in the last decade, including in renal health and disease. The field is moving from experiments showing mere association to causation using both forward and reverse microbiome approaches, leveraging tools such as germ-free animals, treatment with antibiotics, and fecal microbiota transplantations. However, we are still seeing a gap between discovery and translation that needs to be addressed, so that patients can benefit from microbiome-based therapies. In this guideline paper, we discuss the key considerations that affect the gut microbiome of animals and clinical studies assessing renal function, many of which are often overlooked, resulting in false-positive results. For animal studies, these include suppliers, acclimatization, baseline microbiota and its normalization, littermates and cohort/cage effects, diet, sex differences, age, circadian differences, antibiotics and sweeteners, and models used. Clinical studies have some unique considerations, which include sampling, gut transit time, dietary records, medication, and renal phenotypes. We provide best-practice guidance on sampling, storage, DNA extraction, and methods for microbial DNA sequencing (both 16S rRNA and shotgun metagenome). Finally, we discuss follow-up analyses, including tools available, metrics, and their interpretation, and the key challenges ahead in the microbiome field. By standardizing study designs, methods, and reporting, we will accelerate the findings from discovery to translation and result in new microbiome-based therapies that may improve renal health.

The gut microbiota is important for the maintenance of blood-cerebrospinal fluid barrier integrity.

The gut microbiota communicates with the brain through several pathways including the vagus nerve, immune system, microbial metabolites and through the endocrine system. Pathways along the humoral/immune gut microbiota-brain axis are composed of a series of vascular and epithelial barriers including the intestinal epithelial barrier, gut-vascular barrier, blood-brain barrier and blood-cerebrospinal fluid barrier. Of these barriers, the relationship between the gut microbiota and blood-cerebrospinal fluid barrier is yet to be fully defined. Here, using a germ-free mouse model, we aimed to assess the relationship between the gut microbiota and the integrity of the blood-cerebrospinal fluid barrier, which is localized to the choroid plexus epithelium. Using confocal microscopy, we visualized the tight junction protein zonula occludens-1, an integral aspect of choroid plexus integrity, as well as the choroid plexus fenestrated capillaries. Quantification of tight junction proteins via network analysis led to the observation that there was a decrease in the zonula occludens-1 network organization in germ-free mice; however, we did not observe any differences in capillary structure. Taken together, these data indicate that the blood-cerebrospinal fluid barrier is another barrier along the gut microbiota-brain axis. Future studies are required to elucidate its relative contribution in signalling from microbiota to the brain.