Evaluating a potential model to analyze the function of the gut microbiota of the giant panda.

To contribute to the conservation of endangered animals, the utilization of model systems is critical to analyze the function of their gut microbiota. In this study, the results of a fecal microbial transplantation (FMT) experiment with germ-free (GF) mice receiving giant panda or horse fecal microbiota showed a clear clustering by donor microbial communities in GF mice, which was consistent with the results of blood metabolites from these mice. At the genus level, FMT re-established approximately 9% of the giant panda donor microbiota in GF mice compared to about 32% for the horse donor microbiota. In line with this, the difference between the panda donor microbiota and panda-mice microbiota on whole-community level was significantly larger than that between the horse donor microbiota and the horse-mice microbiota. These results were consistent with source tracking analysis that found a significantly higher retention rate of the horse donor microbiota (30.9%) than the giant panda donor microbiota (4.0%) in GF mice where the microbiota remained stable after FMT. Further analyzes indicated that the possible reason for the low retention rate of the panda donor microbiota in GF mice was a low relative abundance of Clostridiaceae in the panda donor microbiota. Our results indicate that the donor microbiota has a large effect on GF mice microbiota after FMT.

Colonization of fecal microbiota from patients with neonatal necrotizing enterocolitis exacerbates intestinal injury in germfree mice subjected to necrotizing enterocolitis-induction protocol via alterations in butyrate and regulatory T cells.

BACKGROUND: Necrotizing enterocolitis (NEC) remains a life-threatening disease in neonates. Numerous studies have shown a correlation between the intestinal microbiota and NEC, but the causal link remains unclear. This study aimed to demonstrate the causal role of gut microbiota in NEC and explore potential mechanisms involved. METHODS: Eighty-one fecal samples from patients with NEC and eighty-one matched controls (matched to the NEC infants by gestational age, birth weight, date of birth, mode of delivery and feeding patterns) were collected. To explore if altered gut microbiota contributes to the pathogenesis of NEC, fecal microbiota transplantation (FMT) was carried out in germ-free (GF) mice prior to a NEC-induction protocol that included exposure to hypoxia and cold stress. Butyric acid was also administered to demonstrate its role in NEC. The fecal microbiota from patients and mice were analyzed by 16S rRNA gene sequencing analysis. Short chain fatty acid (SCFA) levels were measured by gas chromatography-mass spectrometry (GC-MS). The ontogeny of T cells and regulatory T cells (Tregs) in lamina propria mononuclear cells (LPMC) from the ileum of patients and mice were isolated and analyzed by flow cytometry.The transcription of inflammatory cytokines was quantified by qRT-PCR. RESULTS: NEC patients had increased Proteobacteria and decreased Firmicutes and Bacteroidetes compared to fecal control samples, and the level of butyric acid in the NEC group was lower than the control group. FMT in GF mice with samples from NEC patients achieved a higher histological injury scores when compared to mice that received FMT with control samples. Alterations in microbiota and butyrate levels were maintained in mice following FMT. The ratio of Treg/CD4+T (Thelper) cells was reduced in both NEC patients and mice modeling NEC following FMT. CONCLUSIONS: The microbiota was found to have NEC and the microbial butyrate-Treg axis was identified as a potential mechanism for the observed effects.

Proteomics analysis of the gut-brain axis in a gut microbiota-dysbiosis model of depression.

Major depressive disorder (MDD) is a serious mental illness. Increasing evidence from both animal and human studies suggested that the gut microbiota might be involved in the onset of depression via the gut-brain axis. However, the mechanism in depression remains unclear. To explore the protein changes of the gut-brain axis modulated by gut microbiota, germ-free mice were transplanted with gut microbiota from MDD patients to induce depression-like behaviors. Behavioral tests were performed following fecal microbiota transplantation. A quantitative proteomics approach was used to examine changes in protein expression in the prefrontal cortex (PFC), liver, cecum, and serum. Then differential protein analysis and weighted gene coexpression network analysis were used to identify microbiota-related protein modules. Our results suggested that gut microbiota induced the alteration of protein expression levels in multiple tissues of the gut-brain axis in mice with depression-like phenotype, and these changes of the PFC and liver were model specific compared to chronic stress models. Gene ontology enrichment analysis revealed that the protein changes of the gut-brain axis were involved in a variety of biological functions, including metabolic process and inflammatory response, in which energy metabolism is the core change of the protein network. Our data provide clues for future studies in the gut-brain axis on protein level and deepen the understanding of how gut microbiota cause depression-like behaviors.

Carrageenan oligosaccharides and associated carrageenan-degrading bacteria induce intestinal inflammation in germ-free mice.

Carrageenans (CGNs) are widely used in foods and pharmaceuticals although their safety remains controversial. To investigate the effects of CGNs and CGN-degrading bacteria in the human colon, we screened for CGN degradation by human fecal microbiota, and for inflammatory response to CGNs and/or CGN-degrading bacteria in germ free mice. Thin-layer chromatography indicated that high molecular weight (MW) CGNs (≥100 kDa) remained undegraded in the presence of human fecal microbiota, whereas low MW CGNs, i.e., κ-carrageenan oligosaccharides (KCO, ~4.5 kDa) were degraded when exposed to seven of eight human fecal samples, although sulfate groups were not removed during degradation. Bacteroides xylanisolvens and Escherichia coli isolates from fecal samples apparently degraded KCO synergistically, with B. xylanisolvens serving as the primary degrader. Combined treatment of KCO with KCO-degrading bacteria led to greater pro-inflammatory effects in the colon and rectum of germ-free mice than either KCO or bacteria alone. Similarly, p-p38-, CD3-, and CD79a-positive immune cells were more abundant in combined treatment group mice than in either single treatment group. Our study shows that KCO-degrading bacteria and the low MW products of KCO can promote proinflammatory effects in mice, and represent two key markers for evaluating CGN safety in foods or medicines.

Corrigendum: Effect of a Humanized Diet Profile on Colonization Efficiency and Gut Microbial Diversity in Human Flora-Associated Mice.

[This corrects the article DOI: 10.3389/fnut.2021.633738.].

Fecal microbiota from children with vitamin A deficiency impair colonic barrier function in germ-free mice: The possible role of alterative bile acid metabolites.

OBJECTIVE: This study explores the effects of fecal microbiota from children with vitamin A (VA) deficiency on colonic mucosal barrier function. METHODS: The composition of gut microbes was identified in children with different VA levels, then feces from children with normal VA or VA deficiency was collected separately and transplanted into germ-free (GF) mice, respectively. Three weeks after transplantation, the colon morphology, colonic tight junction proteins, gut microbes, and metabolites were evaluated. RESULTS: In children, Bifidobacterium and Bacteroides were positively correlated with VA levels. Colonization of VA deficiency fecal microbiota markedly impaired colonic development in GF mice, down-regulated colonic tight junction-related proteins occludin and claudin-1, and reduced immunoglobulin A secretion. Furthermore, fecal microbiota transplantation with different VA levels altered composition of gut microbes and bile acid metabolism pathways in GF mice. CONCLUSION: These data suggest that fecal microbiota from children with VA deficiency attenuates colonic barrier function in GF mice, which may be achieved by changing the bile acid metabolic pathways.

Effect of a Humanized Diet Profile on Colonization Efficiency and Gut Microbial Diversity in Human Flora-Associated Mice.

Human flora-associated (HFA) mouse models allow us to design interventions for human disease research to test specific hypotheses and explore the complex commensal microbiome while avoiding the ethical limitations of using humans as models to directly study intestinal flora diseases. However, few studies have investigated the effect of a humanized diet profile (coarse-feed diet; CFD) on colonization efficiency and gut microbial diversity in HFA mice. We tested the colonization efficiency and gut microbial diversity in germ-free Kunming (KM) mice fed a CFD or a purified feed diet (PFD) at 1, 2, and 4 weeks. Although the colonization efficiencies differed significantly (67.50-70.00% vs. 72.69-85.96%) in the HFA mice, the colonization efficiency of the PFD-fed HFA mice (85.96%) was significantly higher than that of the CFD-fed mice (69.61%) at 2 weeks. At 4 weeks, the colonization efficiency of the PFD-fed mice (72.69%) was comparable to that of the CFD-fed mice (70.00%). Additionally, the gut microbial diversity of the CFD-fed HFA mice was similar to that of a human fecal donor. Regarding the Kyoto Encyclopedia of Genes and Genomes colonic microbiota metabolic pathways, the CFD-fed HFA mice showed more similarities to the human donor than to the PFD-fed mice in amino sugar and nucleotide sugar metabolism, biosynthesis of amino acids, carbon metabolism, purine metabolism, and phosphotransferase systems. In conclusion, the humanized diet profiles of the CFD and PFD could help establish human microbiotas in mice. Constructing HFA mouse models fed a CFD for 4 weeks may be useful in researching human-derived intestinal diseases.

Spatial heterogeneity of bacterial colonization across different gut segments following inter-species microbiota transplantation.

BACKGROUND: The microbiota presents a compartmentalized distribution across different gut segments. Hence, the exogenous microbiota from a particular gut segment might only invade its homologous gut location during microbiota transplantation. Feces as the excreted residue contain most of the large-intestinal microbes but lack small-intestinal microbes. We speculated that whole-intestinal microbiota transplantation (WIMT), comprising jejunal, ileal, cecal, and colonic microbiota, would be more effective for reshaping the entire intestinal microbiota than conventional fecal microbiota transplantation fecal microbiota transplantation (FMT). RESULTS: We modeled the compartmentalized colonization of the gut microbiota via transplanting the microbiota from jejunum, ileum, cecum, and colon, respectively, into the germ-free mice. Transplanting jejunal or ileal microbiota induced more exogenous microbes' colonization in the small intestine (SI) of germ-free mice rather than the large intestine (LI), primarily containing Proteobacteria, Lactobacillaceae, and Cyanobacteria. Conversely, more saccharolytic anaerobes from exogenous cecal or colonic microbiota, such as Bacteroidetes, Prevotellaceae, Lachnospiraceae, and Ruminococcaceae, established in the LI of germ-free mice that received corresponding intestinal segmented microbiota transplantation. Consistent compartmentalized colonization patterns of microbial functions in the intestine of germ-free mice were also observed. Genes related to nucleotide metabolism, genetic information processing, and replication and repair were primarily enriched in small-intestinal communities, whereas genes associated with the metabolism of essential nutrients such as carbohydrates, amino acids, cofactors, and vitamins were mainly enriched in large-intestinal communities of germ-free mice. Subsequently, we compared the difference in reshaping the community structure of germ-free mice between FMT and WIMT. FMT mainly transferred LI-derived microorganisms and gene functions into the recipient intestine with sparse SI-derived microbes successfully transplanted. However, WIMT introduced more SI-derived microbes and associated microbial functions to the recipient intestine than FMT. Besides, WIMT also improved intestinal morphological development as well as reduced systematic inflammation responses of recipients compared with FMT. CONCLUSIONS: Segmented exogenous microbiota transplantation proved the spatial heterogeneity of bacterial colonization along the gastrointestinal tract, i.e., the microbiota from one specific location selectively colonizes its homologous gut region. Given the lack of exogenous small-intestinal microbes during FMT, WIMT may be a promising alternative for conventional FMT to reconstitute the microbiota across the entire intestinal tract. Video Abstract.

Integrated phosphoproteomic and metabolomic profiling reveals perturbed pathways in the hippocampus of gut microbiota dysbiosis mice.

The dysbiosis of gut microbiota is an important environmental factor that can induce mental disorders, such as depression, through the microbiota-gut-brain axis. However, the underlying pathogenic mechanisms are complex and not completely understood. Here we utilized mass spectrometry to identify the global phosphorylation dynamics in hippocampus tissue in germ-free mice and specific pathogen-free mice (GF vs SPF), fecal microbiota transplantation (FMT) model ("depression microbiota" and the "healthy microbiota" recipient mice). As a result, 327 phosphosites of 237 proteins in GF vs SPF, and 478 phosphosites of 334 proteins in "depression microbiota" vs "healthy microbiota" recipient mice were identified as significant. These phosphorylation dysregulations were consistently associated with glutamatergic neurotransmitter system disturbances. The FMT mice exhibited disturbances in lipid metabolism and amino acid metabolism in both the periphery and brain through integrating phosphoproteomic and metabolomic analysis. Moreover, CAMKII-CREB signaling pathway, in response to these disturbances, was the primary common perturbed cellular process. In addition, we demonstrated that the spliceosome, never directly implicated in mental disorders previously, was a substantially neuronal function disrupted by gut microbiota dysbiosis, and the NCBP1 phosphorylation was identified as a novel pathogenic target. These results present a new perspective to study the pathologic mechanisms of gut microbiota dysbiosis related depression and highlight potential gut-mediated therapies for depression.

Microbial regulation of a lincRNA-miRNA-mRNA network in the mouse hippocampus.

Aim: To comprehensively understand microbiota-regulated lincRNA-miRNA-mRNA networks in psychiatric disorders. Materials & methods: Integrated analyses of lincRNAs, mRNAs and miRNAs, obtained by microarray analysis of hippocampus from specific pathogen-free, germ-free and colonized germ-free mice, were performed. Results: Expression of 139 mRNAs, seven miRNAs and one lincRNA was restored following colonization. The restored transcripts were mainly involved in CREB and Ras/MAPK signaling pathways. RNA transcription and post-transcriptional regulation were the primary perturbed functions. Finally, 12 lincRNAs, six miRNAs and 47 mRNAs were included in a lincRNA-miRNA-mRNA network, and lincRNA0926-miR-190a-5p-Celf4 interactions may play a pivotal role in this regulatory network. Conclusion: This study provides clues for understanding the molecular basis of gut microbiota-brain interactions in depressive- and anxiety-like behaviors.

Extracellular Matrix and Oxidative Phosphorylation: Important Role in the Regulation of Hypothalamic Function by Gut Microbiota.

BACKGROUND: In previous studies, our team examined the gut microbiota of healthy individuals and depressed patients using fecal microbiota transplantation of germ-free (GF) mice. Our results showed that depression-like and anxiety-like behavioral phenotypes of host mice were increased, but the molecular mechanism by which gut microbiota regulate host behavioral phenotypes is still unclear. METHODS: To investigate the molecular mechanism by which gut microbiota regulate host brain function, adult GF mice were colonized with fecal samples derived from healthy control (HC) individuals or patients with major depressive disorder (MDD). Transcriptomic profiling of hypothalamus samples was performed to detect differentially expressed genes (DEGs). qRT-PCR was used for validation experiments. RESULTS: Colonization germ-free (CGF) mice had 243 DEGs compared with GF mice. The most enriched KEGG pathways associated with upregulated genes were "protein digestion and absorption," "extracellular matrix (ECM)-receptor interaction," and "focal adhesion." MDD mice had 642 DEGs compared with HC mice. The most enriched KEGG pathways associated with upregulated genes in MDD mice were also "protein digestion and absorption," "ECM-receptor interaction," and "focal adhesion." Meanwhile, the most enriched KEGG pathway associated with downregulated genes in these mice was "oxidative phosphorylation," and genes related to this pathway were found to be highly correlated in PPI network analysis. CONCLUSION: In summary, our findings suggested that regulation of ECM is a key mechanism shared by different gut microbiota and that inhibition of energy metabolism in the hypothalamus by gut microbiota derived from MDD patients is a potential mechanism of behavioral regulation and depression.

Commensal Microbiota Regulation of Metabolic Networks During Olfactory Dysfunction in Mice.

INTRODUCTION: Recently, an increasing number of studies have focused on commensal microbiota. These microorganisms have been suggested to impact human health and disease. However, only a small amount of data exists to support the assessment of the influences that commensal microbiota exert on olfactory function. METHODS: We used a buried food pellet test (BFPT) to investigate and compare olfactory functions in adult, male, germ-free (GF) and specific-pathogen-free (SPF) mice, then examined and compared the metabolomic profiles for olfactory bulbs (OBs) isolated from GF and SPF mice to uncover the mechanisms associated with olfactory dysfunction. RESULTS: We found that the absence of commensal microbiota was able to influence olfactory function and the metabolic signatures of OBs, with 38 metabolites presenting significant differences between the two groups. These metabolites were primarily associated with disturbances in glycolysis, the tricarboxylic acid (TCA) cycle, amino acid metabolism, and purine catabolism. Finally, the commensal microbiota regulation of metabolic networks during olfactory dysfunction was identified, based on an integrated analysis of metabolite, protein, and mRNA levels. CONCLUSION: This study demonstrated that the absence of commensal microbiota may impair olfactory function and disrupt metabolic networks. These findings provide a new entry-point for understanding olfactory-associated disorders and their potential underlying mechanisms.

Faecal microbiota transplantation from metabolically compromised human donors accelerates osteoarthritis in mice.

OBJECTIVES: Emerging evidence suggests that the microbiome plays an important role in the pathogenesis of osteoarthritis (OA). We aimed to test the two-hit model of OA pathogenesis and potentiation in which one 'hit' is provided by an adverse gut microbiome that activates innate immunity; the other 'hit' is underlying joint damage. METHODS: Medical history, faecal and blood samples were collected from human healthy controls (OA-METS-, n=4), knee OA without metabolic syndrome (OA+METS-, n=7) and knee OA with metabolic syndrome (OA+METS+, n=9). Each group of human faecal samples, whose microbial composition was identified by 16S rRNA sequencing, was pooled and transplanted into germ-free mice 2 weeks prior to meniscal/ligamentous injury (MLI) (n≥6 per group). Eight weeks after MLI, mice were evaluated for histological OA severity and synovitis, systemic inflammation and gut permeability. RESULTS: Histological OA severity following MLI was minimal in germ-free mice. Compared with the other groups, transplantation with the OA+METS+ microbiome was associated with higher mean systemic concentrations of inflammatory biomarkers (interleukin-1ß, interleukin-6 and macrophage inflammatory protein-1α), higher gut permeability and worse OA severity. A greater abundance of Fusobacterium and Faecalibaterium and lesser abundance of Ruminococcaceae in transplanted mice were consistently correlated with OA severity and systemic biomarkers concentrations. CONCLUSION: The study clearly establishes a direct gut microbiome-OA connection that sets the stage for a new means of exploring OA pathogenesis and potentially new OA therapeutics. Alterations of Fusobacterium, Faecalibaterium and Ruminococcaceae suggest a role of these particular microbes in exacerbating OA.

Publisher Correction: Research on oral microbiota of monozygotic twins with discordant caries experience - in vitro and in vivo study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hippocampus-specific regulation of long non-coding RNA and mRNA expression in germ-free mice.

Gut microbiota can affect multiple brain functions and cause behavioral alterations through the microbiota-gut-brain axis. In our previous study, we found that the absence of gut microbiota can influence the expression of microRNAs and mRNAs in the hippocampal region of the germ-free (GF) mice. Long non-coding RNAs (lncRNAs) are increasingly being recognized as an important functional transcriptional regulator in the brain. In the present study, we aim to identify possible biological pathways and functional networks for lncRNA-associated transcript of the gut microbiota in relation to the brain function. The profiles of lncRNA and mRNA from specific pathogen-free (SPF), colonized GF (CGF), and GF mice were generated using the Agilent Mouse LncRNA Array v2.0. Differentially expressed (DE) lncRNAs and mRNAs were identified, and lncRNA target genes were also predicted. Ingenuity pathway analysis (IPA) was performed to analyze related signaling pathways and biological functions associated with these dysregulated mRNAs and target genes. Validation with quantitative real-time PCR was performed on several key genes. Compared with SPF mice a total of 2230 DE lncRNAs were found in GF mice. Among these, 1355 were upregulated and 875 were downregulated. After comparing the target genes of DE lncRNAs with mRNA datasets, 669 overlapping genes were identified. IPA core analyses revealed that most of these genes were highly associated with cardiac hypertrophy, nuclear factors of activated T cells (NFAT) gonadotropin-releasing hormone (GnRH), calcium, and cAMP-response element-binding protein (CREB) signaling pathways. Additionally, mRNA expression levels of APP, CASP9, IGFBP2, PTGDS, and TGFBR2 genes that are involved in central nervous system functions were significantly changed in the GF mouse hippocampus. Through this study, for the first time, we describe the effect of gut microbiota on the hippocampal lncRNA regulation. This will help in enhancing the overall knowledge about microbiota-gut-brain axis.

Absence of gut microbiota affects lipid metabolism in the prefrontal cortex of mice.

Objectives: Lipid metabolism is closely associated with many important biological functions. Here, we conducted this study to explore the effects of gut microbiota on the lipid metabolism in the prefrontal cortex of mice. Methods: Germ-free (GF) mice, specific pathogen-free (SPF) and colonized GF (CGF) mice were used in this study. The open field test (OFT), forced swimming test (FST) and novelty suppressed feeding test (NSFT) were conducted to assess the changes in general behavioral activity. The liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was used to obtain the lipid metabolites. Both one-way analysis of variance (one-way ANOVA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to obtain the key differential lipid metabolites. Results: The behavioral tests showed that compared to SPF mice, GF mice had more center distance, more center time, less immobility time and less latency to familiar food. Meanwhile, 142 key differential lipid metabolites between SPF mice and GF mice were identified. These lipid metabolites mainly belonged to glycerophospholipids, glycerolipids, sphingolipids, and saccharolipids. The gut microbiota colonization did not reverse these changed behavioral phenotypes, but could restore 25 key differential lipid metabolites. Discussion: These results showed that the absence of gut microbiota could influence host behaviors and lipid metabolism. Our findings could provide original and valuable data for future studies to further investigate the microbiota-gut-brain axis.

Intestinal lysozyme liberates Nod1 ligands from microbes to direct insulin trafficking in pancreatic beta cells.

Long-range communication between intestinal symbiotic bacteria and extra-intestinal organs can occur through circulating bacterial signal molecules, through neural circuits, or through cytokines or hormones from host cells. Here we report that Nod1 ligands derived from intestinal bacteria act as signal molecules and directly modulate insulin trafficking in pancreatic beta cells. The cytosolic peptidoglycan receptor Nod1 and its downstream adapter Rip2 are required for insulin trafficking in beta cells in a cell-autonomous manner. Mechanistically, upon recognizing cognate ligands, Nod1 and Rip2 localize to insulin vesicles, recruiting Rab1a to direct insulin trafficking through the cytoplasm. Importantly, intestinal lysozyme liberates Nod1 ligands into the circulation, thus enabling long-range communication between intestinal microbes and islets. The intestine-islet crosstalk bridged by Nod1 ligands modulates host glucose tolerance. Our study defines a new type of inter-organ communication based on circulating bacterial signal molecules, which has broad implications for understanding the mutualistic relationship between microbes and host.

Absence of gut microbiota during early life affects anxiolytic Behaviors and monoamine neurotransmitters system in the hippocampal of mice.

The gut microbiome is composed of an enormous number of microorganisms, generally regarded as commensal bacteria. Resident gut bacteria are an important contributor to health and significant evidence suggests that the presence of healthy and diverse gut microbiota is important for normal cognitive and emotional processing. Here we measured the expression of monoamine neurotransmitter-related genes in the hippocampus of germ-free (GF) mice and specific-pathogen-free (SPF) mice to explore the effect of gut microbiota on hippocampal monoamine functioning. In total, 19 differential expressed genes (Htr7, Htr1f, Htr3b, Drd3, Ddc, Maob, Tdo2, Fos, Creb1, Akt1, Gsk3a, Pik3ca, Pla2g5, Cyp2d22, Grk6, Ephb1, Slc18a1, Nr4a1, Gdnf) that could discriminate between the two groups were identified. Interestingly, GF mice displayed anxiolytic-like behavior compared to SPF mice, which were not reversed by colonization with gut microbiota from SPF mice. Besides, colonization of adolescent GF mice by gut microbiota was not sufficient to reverse the altered gene expression associated with their GF status. Taking these findings together, the absence of commensal microbiota during early life markedly affects hippocampal monoamine gene-regulation, which was associated with anxiolytic behaviors and monoamine neurological signs.

Sema3A - mediated modulation of NR1D1 expression may be involved in the regulation of axonal guidance signaling by the microbiota.

AIMS: The microbiota has a profound impact on host development and function. Axon guidance is essential for the formation of neural circuits and plays an important role in neurological diseases and behavioral disorders. However, the impact of the microbiota on axon guidance signaling is unclear. MAIN METHODS: Gnotobiotic models-germ free mice were applied to explore behavioral phenotypes and possible molecular mechanisms that were evaluated by Realtime-PCR and western blot analysis. Primary cultures of mouse cortical neurons were performed to demonstrate the role of Sema3A on NR1D1 expression. KEY FINDINGS: The results showed that the microbiota modulates host behavior, and that colonization is not sufficient to normalize behavioral alterations in germ-free (GF) mice. Five genes, Sema3A, Sema3E, EphB2, Slit3 and Robo1, were differentially expressed in GF and specific pathogen-free (SPF) mice. Furthermore, colonization did not completely reverse the differential expression, which was consistent with the behavioral phenotypes in colonization germ-free (CGF) mice. The transcript and protein levels of Sema3A, and of its membrane-bound co-receptor NRP1, were increased in GF mice. Interestingly, Sema3A inhibited the expression of NR1D1, which was blocked by a RhoA/ROCK pathway agonist in primary cortical neurons. The NR1D1 and ROCK2 expression levels were reduced in GF and CGF mice compared with SPF mice, consistent with the increased expression of Sema3A. SIGNIFICANCE: Our findings suggest that the microbiota regulates axon guidance signaling in the prefrontal cortex. Furthermore, this effect appears to involve the inhibition of NR1D1 expression by Sema3A through the RhoA/ROCK pathway.

Oral bacteria colonize and compete with gut microbiota in gnotobiotic mice.

The oral microbiota is associated with oral diseases and digestive systemic diseases. Nevertheless, the causal relationship between them has not been completely elucidated, and colonisation of the gut by oral bacteria is not clear due to the limitations of existing research models. The aim of this study was to develop a human oral microbiota-associated (HOMA) mouse model and to investigate the ecological invasion into the gut. By transplanting human saliva into germ-free (GF) mice, a HOMA mouse model was first constructed. 16S rRNA gene sequencing was used to reveal the biogeography of oral bacteria along the cephalocaudal axis of the digestive tract. In the HOMA mice, 84.78% of the detected genus-level taxa were specific to the donor. Principal component analysis (PCA) revealed that the donor oral microbiota clustered with those of the HOMA mice and were distinct from those of specific pathogen-free (SPF) mice. In HOMA mice, OTU counts decreased from the stomach and small intestine to the distal gut. The distal gut was dominated by Streptococcus, Veillonella, Haemophilus, Fusobacterium, Trichococcus and Actinomyces. HOMA mice and human microbiota-associated (HMA) mice along with the GF mice were then cohoused. Microbial communities of cohoused mice clustered together and were significantly separated from those of HOMA mice and HMA mice. The Source Tracker analysis and network analysis revealed more significant ecological invasion from oral bacteria in the small intestines, compared to the distal gut, of cohoused mice. In conclusion, a HOMA mouse model was successfully established. By overcoming the physical and microbial barrier, oral bacteria colonised the gut and profiled the gut microbiota, especially in the small intestine.