SPF Mice Research Articles

Odd chain fatty acid metabolism in mice after a high fat diet.

Epidemiological studies show that higher circulating levels of odd chain saturated fatty acids (FA: C15:0 and C17:0) are associated with lower risk of metabolic disease. These odd chain saturated fatty acids (OCSFA) are produced by α-oxidation in peroxisomes, de novo lipogenesis, from the diet and by gut microbiota. Although present at low concentrations, they are of interest as potential targets to reduce metabolic disease risk. To determine whether OCSFA are affected by obesogenic diets, we have investigated whether high dietary fat intake affects the frequency of OCSFA-producing gut microbiota, liver lipid metabolism genes and circulating OCSFA. FA concentrations were determined in liver and serum from pathogen-free SPF C57BL/6 J mice fed either standard chow or a high fat diet (HFD; 60% calories as fat) for four and twelve weeks. Post-mortem mouse livers were analysed histologically for fat deposition by gas chromatography–mass spectrometry for FA composition and by qPCR for the lipid metabolic genes fatty acid desaturase 2 (FADS2), stearoyl CoA desaturase 1 (SCD1), elongation of long-chain fatty acids family member 6 (ELOVL6) and 2-hydroxyacyl-CoA lyase 1 (HACL). Gut microbiota in faecal pellets from the ileum were analysed by 16S RNA sequencing. A significant depletion of serum and liver C15:0 (>50%; P < 0.05) and liver C17:0 (>35%; P < 0.05) was observed in HFD-fed SPF mice in parallel with hepatic fat accumulation after four weeks. In addition, liver gene expression (HACL1, ELOVL6, SCD1 and FADS2) was lower (>50%; P < 0.05) and the relative abundance of beneficial C3:0-producing gut bacteria such as Akkermansia, Lactobacillus, Bifidobacterium was lower after HFD in SPF mice. In summary, high dietary fat intake reduces serum and liver OCSFA, OCSFA-producing gut microbiota and is associated with impaired liver lipid metabolism. Further studies are required to identify whether there is any beneficial effect of OCSFA and C3:0-producing gut bacteria to counter metabolic disease.

Characteristics of an In Vitro Mesenteric Lymph Node Cell Suspension Model and Its Possible Association with In Vivo Functional Evaluation.

In a previous study, we uncovered three immune-responsive patterns of gut microbes using an in vitro mesenteric lymph node cell suspension model, abbreviated as the MLN model hereafter. We used Akkermansia muciniphila and Clostridium butyricum as the first group directly inducing an immune response, Bifidobacterium sp. and Bacteroides sp. as the second group evoking an immune response with the help of stimuli (anti-CD3 and anti-CD28 antibodies), and Lactobacillus sp. as the third group blunting the immune response with or without stimuli. Our group previously clarified the immune-activation characteristics of A. muciniphila and linked its in vivo immune induction effect in GF and SPF mice under homeostasis. In the present study, we supplemented the characteristics of C. butyricum and B. bifidum in the in vitro MLN model and addressed the specific elements of the model. Finally, we used an in vivo TNBS-challenge model to show the functional differences between these species with different response patterns in vitro. The results showed that C. butyricum and B. bifidum evoked an immune response in vitro in a dose-dependent and strain-unique manner. Although TLR2, rather than TLR4, is indispensable for immune activation in the present in vitro model, it may not involve interaction between TLR2 and bacterial ligands. Like the PBMC model, the present in vitro MLN model is highly dependent on cell resources and should be given more attention when used to conduct a quantitative comparison. Finally, a mixture of two strong immunogenic strains, A. muciniphila and C. butyricum, significantly increased the mortality of TNBS-challenged (2,4,6-trinitrobenzene sulfonic acid, TNBS) mice, indicating a possible link between the in vitro MLN model and in vivo functional evaluation. However, more evidence is needed to clarify the associations and underlying mechanisms.

Analysis of the Therapeutic Effect of Changyanning on Intestinal Flora in Inflammatory Bowel Disease.

Research Purposes. Inflammatory bowel disease (IBD) is an autoimmune disease coinduced by genes, environment, and immune response, mainly including ulcerative colitis (UC) and Crohn's disease (CD). There are a large number and variety of intestinal bacteria in the human intestinal tract. These bacteria maintain a balance with the human environment and participate in the normal physiological processes of the human body. They play a unique role in defending against pathogen invasion, maintaining the homeostasis of the human immune system and metabolizing substances. Intestinal flora imbalance may be one of the pathogenic factors of IBD, and restoring the disturbed intestinal flora has become a research hotspot in the prevention and treatment of IBD. Changyanning is mainly composed of Dijincao grass, yellow hairy ear grass, camphor tree roots, maple leaves, and so on. Clinical studies have shown that Changyanning alone or in combination has a significant effect on ulcerative colitis, but the treatment mechanism is not yet clear. In this study, we established an IBD animal model to explore the therapeutic mechanism of Changyanning on inflammatory bowel disease and its effect on intestinal flora. Research Methods. Male C57BL/6 SPF mice were given free access to 4% dextran sulfate sodium (DSS) solution for 7 days to establish an ulcerative colitis model. After the model was established, different doses of Changyanning tablets, Changyanning granules, and sulfasalazine were given by gavage for 7 days. The relieving effects of the above drugs on the symptoms of inflammatory bowel disease were evaluated by evaluating the mouse/rat body weight, survival rate, disease activity index, colon length, pathological tissue score, and other indicators. Results. The DSS-induced IBD mouse model showed significant increases in weight loss, DAI score, and pathological score. Both Changyanning tablets and granules can relieve the weight loss of mice, restore the colon length, and protect the colon tissue structure of mice. In reducing DAI and pathological scores in mice, Changyanning granules had a better effect. In conclusion, Changyanning can significantly improve the quality of life of IBD model animals, relieve intestinal inflammatory response, and relieve colonic edema, ulceration, and necrosis. The results show that Changyanning has a certain therapeutic effect on IBD. This study also provides experimental evidence for the application of Changyanning in the treatment of IBD, which is of great significance to its clinical application. The trail is registered in ChiCTR with number (TRN) ChiCTR2000028830.

Correlation between the regulation of intestinal bacteriophages by green tea polyphenols and the flora diversity in SPF mice.

Green tea polyphenols (GTP) play an important role in shaping the gut microbiome, comprising a range of densely colonizing microorganisms, including bacteriophages. Previous studies focused on the effect of GTP on the bacteria in the gut microbiota. However, little is known about the role of GTP in the bacteriophage composition of healthy intestines. In this study, SPF male C57BL/6J mice were divided into a polyphenol-free diet group and a tea polyphenol diet group where drinking water was supplemented with 0.1% GTP for 28 days. The ultra-deep metagenomic sequencing of virus-like particle preparations and bacterial 16S rRNA sequencing were performed on mouse stool samples. Changes in the gut bacteriome, bacteriophages, and bacterial-bacteriophage correlations were then compared between the groups. The results revealed an abundance of Firmicutes, a significant decrease in Bacteroidetes, and a significant increase in the ratio of F/B after GTP exposure. The GTP altered the abundance (relative abundance > 1.00%) of Bifidobacterium (regulation rate of 89.78% and the abundance up-regulated by 0.89%) and Akkermansia (regulation rate of 99.70% and the abundance down-regulated by 1.77%). The abundance of Faecalibaculum (regulation rate of 60.17%) increased by 24.38% following GTP treatment. The GTP also altered the abundance of Salmonella phage (regulation rate of 98.64% and the abundance up-regulated by 3.16%) and that of Gordonia_phage_Yakult (regulation rate of 99.99% and the abundance down-regulated by 5.44%). It significantly increased the intestine's lytic phages and reduced the temperate phages by 29.22%. The dominant microorganisms (relative abundance >1.00%) of Bifidobacterium and Dubosiella had a significantly negative relationship with the Faecalibacterium phage and a significantly positive relationship with the Lactobacillus prophage. Exposure to GTP positively promoted changes in the gut bacteriophage community and interaction network in the microbial community of the SPF mice. These findings highlight the importance of "profitable" bacteriophage-bacteria relationships and reveal a potential mechanism of GTP towards the regulation of intestinal flora via intestinal phage communities.

Effects of thymulin 5cH in granuloma evolution and B1 cell differentiation: an experimental model to understand its biological mechanisms

In previous studies, we found that thymulin (a thymic hormone), when prepared in homeopathic 5cH potency, had the property to improve the productive performance of broiler chickens infected with reovirus, as well as modulate the development of Ehrlich tumor and granuloma inflammatory lesions in mice by immune-mediated mechanisms. The aim of the present work was to study the immunomodulatory mechanisms of thymulin 5cH in a granuloma experimental model, by subcutaneous inoculation of BCG in mice, focusing the B-1 cells and zinc involvement in this process. Three groups of male Balb/c SPF mice (group A treated with thymulin 5cH, group B treated with thymulin 5cH incubated in Chelex ® - a zinc chelant - and group C, control, treated with vehicle) were inoculated with BCG in the left footpad and subcutaneous granuloma and spleen were harvested for histomorphometry analysis, after 7, 14 and 21 days. Ziehl-Neelsen, HE and Prussia Blue staining methods were used. Flow cytometry was also used in the same times to characterize and quantify peritoneal cells. Positive cells for CD11b (activated phagocytes, B-1 cells), CD19 (B-1 and B-2 cells), CD23 (negative B-1 cells, positive B2 cells) and CD5 (B-1a cells) were analyzed in a FACS Calibur (BD) device. Statistical analysis was performed using Kruskal - Wallis / Dunn for nonparametric evaluations and ANOVA / Tuckey-Krammer for the parametric ones. The X² method was used to evaluate the cell count in flow cytometry. P values ≤ 0.05 were considered statistically significant. Mice treated with thymulin 5cH presented higher macrophage activity and increase in the follicular area were seen in spleen after 7 days. Increase in gross lesion diameter and decrease in local BCG infection were seen after 21 days. At this time, the flow cytometry demonstrated the increase in peritoneal phagocytes derived from B-1 cells in thymulin 5cH treated mice, independently of Chelex ® incubation. The incubation of thymulin 5cH with Chelex ® blocked its effects only upon the number of B2 cells in the peritoneum and reduces Mn levels in the medicine solution. We conclude that thymulin 5CH modulates the BCG-induced granuloma through more than one mechanism, especially by peritoneal B1 cell differentiation into phagocytes.

Potential targets of Euodiae Fructus in treatment of insomnia based on network pharmacology

The acupoint application of Euodiae Fructus at Yongquan(KI1) can significantly improve the sleep quality of patients with insomnia with berberine as the main effective component for the efficacy. Nineteen active compounds and 203 drug targets were screened out from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). After comparison with GeneCards and Online Mendelian Inheritance in Man(OMIM), 24 common genes of diseases and drugs were obtained. STRING 11.0 was used to construct a protein-protein interaction(PPI) network of the overlapping genes, and Matthews correlation coefficient(MCC) was employed to screen the core genes, which were then subjected to enrichment analysis with gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG). The results revealed that the main compounds of Euodiae Fructus, such as berberine and rutaecarpine, participated in the biological processes(such as neurotransmitter receptor activity) by regulating C-reactive protein(CRP), estrogen receptor 1(ESR1), 5-hydroxytryptamine(5-HT) receptor, and interleukin-6(IL-6) to exert sedative, anxiolytic, and antidepressant effects. Sixty 4-week-old SPF mice were randomly divided into a control group, a model group, a positive drug(diazepam tablets) group, and low-, medium-, and high-dose berberine groups. Medication with corresponding drugs was performed for one week. The results demonstrated that berberine was potent in reducing the activities and standing times of mice, down-regulating the levels of CRP and IL-6 mRNA in the hypothalamus, and up-regulating the expression of 5-HT(P&lt;0.01); however, no significant effect on ESR1 was observed. The network of Euodiae Fructus in treating insomnia was constructed by network pharmacology and verified by tests. The findings indicated that the therapeutic efficacy of Euodiae Fructus in treating insomnia was achieved by participating in multiple biological processes, such as neurotransmitter receptor activity, which provided a scientific basis for its clinical application.

The development of peripheral intraepithelial lymphocytes in vivo is influenced by microbial metabolites

Abstract Peripheral CD4+CD8αα+TCRαβ+ intraepithelial lymphocytes (pIELs) are a specialised T cell population that require the presence of intestinal microbes for their development, however the microbes and factors involved are not well characterised. pIELs are closely associated with intestinal epithelial cells (IECs) that line the gastrointestinal tract, but the contribution of IECs in shaping pIEL development is also not well defined. We investigated a number of microbial metabolites for their ability to influence pIEL development, and whether this is mediated by microbe-IEC-pIEL interactions. In this study we utilised flow cytometry to characterise pIEL frequencies in GF mice administered with different microbial metabolites, in SPF mice from different colonies and animal facilities, and in GF mice monocolonised with potential pIEL-inducing commensal species. qPCR of IEC RNA isolated from these mice was performed to identify candidate genes expressed by IECs that may influence pIEL development. We identified a common bacterial metabolite that partially restores pIEL development in GF mice, and a novel pIEL-inducing bacterial species. pIEL frequency of SPF mice varied between different sources, and qPCR identified IECs expressed several T-cell-influencing cytokines that correlated with pIEL frequency in vivo. Our results identify a common microbial metabolite that contributes to pIEL development that may mediate IEC expression of T-cell-influencing cytokines. Our work further elucidates the mechanisms of host tolerance triggered by commensal microbes, which will facilitate the development of better strategies to prevent and treat intestinal infections and inflammatory diseases.

High-dimensional single cell mass cytometry analysis of the murine hematopoietic system reveals signatures induced by ageing and physiological pathogen challenges

BackgroundImmune ageing is a result of repetitive microbial challenges along with cell intrinsic or systemic changes occurring during ageing. Mice under ‘specific-pathogen-free’ (SPF) conditions are frequently used to assess immune ageing in long-term experiments. However, physiological pathogenic challenges are reduced in SPF mice. The question arises to what extent murine experiments performed under SPF conditions are suited to analyze immune ageing in mice and serve as models for human immune ageing. Our previous comparisons of same aged mice with different microbial exposures, unambiguously identified distinct clusters of immune cells characteristic for numerous previous pathogen encounters in particular in pet shop mice.ResultsWe here performed single cell mass cytometry assessing splenic as secondary and bone marrow as primary lymphoid organ-derived leukocytes isolated from young versus aged SPF mice in order to delineate alterations of the murine hematopoietic system induced during ageing. We then compared immune clusters from young and aged SPF mice to pet shop mice in order to delineate alterations of the murine hematopoietic system induced by physiological pathogenic challenges and those caused by cell intrinsic or systemic changes during ageing. Notably, distinct immune signatures were similarly altered in both pet shop and aged SPF mice in comparison to young SPF mice, including increased frequencies of memory T lymphocytes, effector-cytokine producing T cells, plasma cells and mature NK cells. However, elevated frequencies of CD4+ T cells, total NK cells, granulocytes, pDCs, cDCs and decreased frequencies of naïve B cells were specifically identified only in pet shop mice. In aged SPF mice specifically the frequencies of splenic IgM+ plasma cells, CD8+ T cells and CD4+ CD25+ Treg were increased as compared to pet shop mice and young mice.ConclusionsOur study dissects firstly how ageing impacts both innate and adaptive immune cells in primary and secondary lymphoid organs. Secondly, it partly distinguishes murine intrinsic immune ageing alterations from those induced by physiological pathogen challenges highlighting the importance of designing mouse models for their use in preclinical research including vaccines and immunotherapies.

Microbiota-derived short chain fatty acids modulate microglia and promote Aβ plaque deposition.

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aβ deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aβ plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aβ plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aβ plaques upon SCFA supplementation, microglia contained less intracellular Aβ. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aβ deposition likely via modulation of the microglial phenotype.

The microbiome does not influence cartilage degradation following joint destabilisation in mice when performed under stringently controlled conditions

Purpose: The influence of gut bacteria in the modulation of disease severity of OA is poorly understood. Reports to date on the role of the microbiome in germ free (GF) models of OA are conflicting and haven’t always been stringently controlled. In the present study, surgical induction of OA by destabilisation of the medial meniscus (DMM) was used to examine the potential influence of the microbiome on cartilage damage severity. Methods: Male C57BL/6J mice used in the present study were bred and housed in the gnotobiotic facility at the Kennedy Institute. Microbiome analysis by 16S rRNA amplicon sequencing was performed on stool samples from mice prior to DMM surgery. At 10 weeks of age, mice were randomly selected for sham (n=6-8) or DMM (n=17-20) surgery, which was performed in sterile Class II cabinets. Knees from operated mice were harvested at 8 or 12wks post DMM. Faecal samples were collected post surgery and at the end of the experiment to test GF status. Joints were sectioned and severity of cartilage degradation was assessed in a blinded method according to OARSI guidelines. No mice were excluded from data analysis. Differences were analysed by ANOVA with Tukey post test correction and P values less than 0.05 were considered statistically significant. Control (specific pathogen free, SPF) male C57BL/6J mice (originally derived from the GF founders) were bred under SPF conditions but housed in an environment to mimic the GF conditions in terms of bedding, food (irradiated) and water, and underwent sham (n=4-6) or DMM (n=11-13) surgery at 10 weeks for analysis after 8 and 12wks. Results: All mice were confirmed GF at the time of surgery but 50% tested positive for microbes at the end of the surgical procedure suggesting contamination at the time of surgery (indicated in red dots). Proteoglycan loss and cartilage erosion were evident in SPF and GF mice 12 wks after surgery. Cartilage damage following DMM was significantly higher than sham in both groups, but was not different between GF and SPF mice. Cartilage damage seen at 8wks was modest and only reached significance over sham-operated in GF mice. No significant differences in scores between the GF and SPF DMM groups were demonstrated. No apparent relationship between those animals that became contaminated at the time of surgery and disease scores was seen. Conclusions: The present study shows that significant cartilage degradation occurs in GF mice undergoing DMM at both timepoints. The disease severity was not altered by the absence of a microbiome, nor apparently when mice lost their GF status during the experimental course. When SPF mice were housed under tightly matched cage conditions to the GF mice, disease appeared indistinguishable. The absence of a gut microbiome does not appear to protect the cartilage from erosion after joint destabilisation.

A9 DORSAL ROOT GANGLIA NEURONAL RESPONSES AND SUBSTANCE P PRODUCTION ARE HIGHER IN MALE MICE

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to display visceral hypersensitivity, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as women report more abdominal pain than men. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventionally raised (SPF) or germ-free (GF) mice (7–18 weeks old). To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered i) the percentage of responding neurons ii) the intensity of the neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by a mixture of G-protein coupled receptors agonist (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next measured the neuronal production of substance P and calcitonin gene-related peptide (CGRP), two neuropeptides associated with nociception, in response to capsaicin (1.25µM) or GPCR agonists (100µM) by ELISA and EIA, respectively. Results The percentage of neurons responding to capsaicin and GPCR agonists was similar in male and female SPF and GF mice. However, the intensity of the neuronal response was higher in SPF male compared to SPF female in response to capsaicin (125nM: p=0.0336; 1.25µM: p=0.033) but not to GPCR agonists. Neuronal activation was similar in GF and SPF mice of both sexes after administration of capsaicin or GPCR agonists. Furthermore, substance P and CGRP production by sensory neurons induced by capsaicin or GPCR agonists was similar in SPF and GF mice, regardless of sex. However, while the response to capsaicin was similar, the GPCR agonists-induced production of substance P was higher in SPF male mice compared to SPF females (p=0.003). The GPCR agonists-induced production of CGRP was similar in SPF male and female mice. Conclusions Our data suggest that at the level of DRG neurons, the absence of gut microbiota does not predispose to visceral hypersensitivity. The intensity of DRG neuronal responses to capsaicin and the GPCR agonists-induced production of substance P are higher in male compared to female mice, in contrast to previously published studies in various models of acute and chronic pain. Further studies are thus needed to investigate the role of sex in visceral sensitivity. Funding Agencies CIHR

A238 ABSENCE OF AGE-RELATED MEMORY DECLINE IN GERM-FREE MICE

Abstract Background Age-associated deterioration of cognitive function and memory capacity occur in a variety of mammals, from humans to rodents. For example, significant memory deficits have been reported in conventionally raised (SPF) old mice compared to conventionally raised young mice submitted to a spatial memory task (Prevot et al., Mol Neuropsychiatry 2019). Microbiota to brain signaling is now well established in mice, but the extent to which this influences age-related memory decline is unknown. Aims Our project aims to determine whether the intestinal microbiota contributes to age-related changes in brain function. We address the hypothesis that age-related cognitive decline is attenuated in the absence of the intestinal microbiota. Methods We studied locomotor behavior and spatial memory performance in young germ-free (GF) mice (2–3 months of age, n=24) and senescent GF mice (13–27 months old, n=22) maintained in axenic conditions, and compared them to conventionally raised (SPF) mice. We used the Y-maze test based on a spontaneous alternations task to assess cognition, with alternation rate as a proxy of spatial working memory performance. The locomotor activity was measured using the open-field test. Results GF old mice traveled less distance (458.9 cm) than GF young mice (875.7 cm, p &amp;lt; 0.001) but these differences in locomotor activity did not influence spatial memory performance. Indeed, both GF old and GF young mice had an identical alternation rate of 73.3% (p &amp;gt; 0.05). This contrasted with the memory impairment found in old SPF mice that displayed lower alternation rate of 58.3%, compared to that found in young SPF mice (76.2%, p = 0.13). Conclusions We conclude that the absence of age-related memory decline in germ-free mice is consistent with a role for the microbiota in the cognitive decline associated with aging, likely through action on the immune system, well documented in SPF mice (Thevaranjan et al., Cell Host &amp; Microbe 2017). We propose that novel microbiota-targeted therapeutic strategies may delay or prevent the cognitive decline of aging. Funding Agencies CIHRBalsam Family Foundation

A231 THE MICROBIOTA-NOCICEPTORS-MICROGLIA AXIS CONTROLS THE DEVELOPMENT AND MAINTENANCE OF CHRONIC VISCERAL HYPERSENSITIVITY

Abstract Background Pain is the most common cause of disability in inflammatory bowel disease (IBD). Current medical interventions control the debilitating clinical symptoms by reducing gastrointestinal (GI) inflammation. Despite successful treatment of active disease, abdominal pain persists during remission, suggesting a high level of plasticity in pain-sensing circuits (hyperalgesic priming) caused by inflammation. What drives this remodelling has remained elusive. We have identified microglia as active players of hyperalgesic priming in IBD. Furthermore, it was recently shown that commensal bacteria control the maturation of microglia in the CNS, suggesting that dysbiosis could influence visceral sensitivity through regulating colonic nociceptors-microglia interaction. Here we test the hypothesis that microbiome-nociceptors-microglia interactions control visceral sensitivity and pain in IBD. Aims We investigated the role of the microbiota in the developmental regulation of colonic nociceptors that express the pain receptor TRPV1. We will identify the microbial factors that control neuron-microglia interactions during bacterial colonization and post-inflammatory dysbiosis. Methods We have developed a germ-free TRPV1-GFP reporter mouse to be used for a combination of behavioural tests and phenotypic characterization of TRPV1+ nociceptors. RNA-sequencing of FACS isolated TRPV1+ neurons of germ-free mice will be used to identify genes that are under the control of the microbiota. We will restore discrepancies observed in germ-free mice by recolonization to assess the impact of the microbiota. Furthermore, we will investigate the regulation of Ahr in TRPV1+ neurons by the microbiota and the effect of its ligands on microglial activation and post-inflammatory visceral pain. Results Measuring somatic pain sensation in naive germ-free and SPF mice, we showed a 15% reduction in thermal pain threshold, as measured by the Hargreaves test, and a 50% reduction in mechanical pain threshold, as measured by the Von Frey test, in germ-free mice. When looking at the dorsal root ganglia of germ-free and SPF mice, we saw a 15% increase in the percentage of neurons that were TRPV1-GFP positive in germ-free mice. Conclusions Our results thus far highlight the importance of the microbiota in regulating the lineage of nociceptive neurons and the threshold of mechanical and thermal pain responses. These findings suggest a major contribution of the microbiota in shaping the neuro-immune axis, with major implications for visceral sensitization in the context of dysbiosis. My project will be looking further into the phenotype of nociceptors in germ-free mice and the effect of microbial-derived Ahr agonists on the maturation and function of colonic TRPV1+ nociceptors. My work will advance our understanding of mechanisms by which commensal bacteria regulate GI pain. Funding Agencies CIHR

The effect of gut microbiota on the intestinal lipidome of mice

Gut microbiota significantly influence the plasma and liver lipidome. An interconnecting metabolite is acetate generated after degradation and fermentation of dietary fiber by the gut microbiota, which is metabolized in the liver into longer chain fatty acids and complex lipids reaching the circulation. Whether these systemic changes are accompanied by alternations of the intestinal lipidome is unclear. Therefore, we quantified glycerophospholipids, sphingolipids and sterols in ileum and colon, the two segments containing the highest densities of microbes in the gastrointestinal tract, of germfree and specific pathogen free mice using mass spectrometry-based lipidomics. We found that the presence of gut microbes lowers the free cholesterol content in colon while elevating phosphatidylcholine levels. Further, PUFA-containing phosphatidylcholine and -ethanolamine fractions are increased in ileum and colon of germfree compared to SPF mice. A total fatty acid analysis by GC–MS revealed higher levels of arachidonic and docosahexaenoic acid in the ileum of germfree mice indicating that the gut microbiota inhibits PUFA metabolism in the small intestine.

Development of a reverse transcription loop mediated isothermal amplification assay for the detection of Mouse reovirus type 3 in laboratory mice

Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.

γδT Cells Are Essential for Orthodontic Tooth Movement

Sustained mechanical forces applied to tissue are known to shape local immunity. In the oral mucosa, mechanical stress, either naturally induced by masticatory forces or externally via mechanical loading during orthodontic tooth movement (OTM), is translated, in part, by T cells to alveolar bone resorption. Nevertheless, despite being considered critical for OTM, depletion of CD4+ and CD8+ T cells is reported to have no impact on tooth movement, thus questioning the function of αβT cells in OTM-associated bone resorption. To further address the role of T cells in OTM, we first characterized the leukocytes residing in the periodontal ligament (PDL), the tissue of interest during OTM, and compared it to the neighboring gingiva. Unlike the gingiva, monocytes and neutrophils represent the major leukocytes of the PDL. These myeloid cells were also the main leukocytes in the PDL of germ-free mice, although at lower levels than SPF mice. T lymphocytes were more enriched in the gingiva than the PDL, yet in both tissues, the relative fraction of the γδT cells was higher than the αβ T cells. We thus sought to examine the role of γδT cells in OTM. γδT cells residing in the PDL were mainly Vγ6+ and produced interleukin (IL)–17A but not interferon-γ. Using Tcrd-GDL mice allowing conditional ablation of γδT cells in vivo, we demonstrate that OTM was greatly diminished in the absence of γδT cells. Further analysis revealed that ablation of γδT cells decreased early IL-17A expression, monocyte and neutrophil recruitment, and the expression of the osteoclastogenic molecule receptor activator of nuclear factor–κβ ligand. This, eventually, resulted in reduced numbers of osteoclasts in the pressure site during OTM. Collectively, our data suggest that γδT cells are essential in OTM for translating orthodontic mechanical forces to bone resorption, required for relocating the tooth in the alveolar bone.

Evaluation of Propofol in Inhibiting Proliferation of Cardiac Fibroblasts in Angiotensin II-Induced Mouse

The present study was conducted to investigate the molecular mechanism of propofol in inhibiting the proliferation of mouse cardiac fibroblasts (CFs) induced by angiotensin II (Ag II). The ventricles of SPF mice from Kunming were cultured for the second to third generation of CFs under aseptic condition. On the basis of the different adding conditions, the mice were divided into five groups: (1) control group: no drug were added; (2) Ag II group: 100 nmol/L Ag II were added; (3) 10 μmol/L propofol + 100 nmol/L Ag II group; (4) 30 μmol/L propofol + 100 nmol/L Ag II group; (5) 50 μmol/L propofol + 100 nmol/L Ag II group. The effects of propofol on the proliferation of CFs induced by Ag II, the expression of CFs ET-1, the activity of NADPH oxidase and the formation of ROS were analyzed. In addition, our study also explored the potential role of Akt-eNOS-nitric oxide pathway regarding the inhibition of proliferation of Ag II induced CFs by propofol. We found that the proliferation of CFs, the secretion of ET-1, the activity of NADPH oxidase and the level of intracellular ROS and fibronectin expression were significantly increased after CFs exposure to Ag II for 24 h. The abovementioned indexes decreased significantly in CFs after treated with propofol for 24 h (10, 30, or 50 μmol/L) with significant statistical difference (P < 0.05). Akt and eNOS siRNA transfection significantly decreased the levels of Akt and eNOS protein, respectively. Blocking pathway of Akt-eNOS-nitric oxide decreased the inhibitory effect of propofol on Ag II-induced cell proliferation of CFs. Propofol exerts effect in inhibiting ET-1 and fibronectin expression and the formation of ROS induced by Ag II. Moreover, Akt-eNOS-nitric oxide signaling pathway may be involved in the effect of propofol on the proliferation of CFs induced by Ag II.

Evaluation of the Antiradiation Efficiency of Flagellin in Experiments on Mice

In experiments on SPF mice, the authors evaluated the radio-protective effectiveness of the recombinant Flagellin, developed at the Institute of High-Purity Preparations (St. Petersburg), administered at a wide range of doses of 0.004–2 mg/kg for 30 min prior to X-ray irradiation in the dose range of 7–10 Gy. The effectiveness of Flagellin was evaluated in terms of 30-day survival rates of the mice, the average life expectancy, and according to the DMF criterion for bone marrow death. The DMF values were 1.24–1.23–1.22 at the level of LD16, LD50, and LD84. ED50 approximately equals 25 μg/kg. The dose-response effect of Flagellin was evaluated to reach a plateau at a dose of 0.2 mg/kg.

The First Report to Evaluate Safety of Cyanobacterium Leptolyngbya sp. KIOST-1 for Use as a Food Ingredient: Oral Acute Toxicity and Genotoxicity Study.

Leptolyngbya sp. KIOST-1 (LK1) is a newly isolated cyanobacterium that shows no obvious cytotoxicity and contains high protein content for both human and animal diets. However, only limited information is available on its toxic effects. The purpose of this study was to validate the safety of LK1 powder. Following Organisation for Economic Co-operation and Development (OECD) guidelines, a single-dose oral toxicity test in Sprague Dawley rats was performed. Genotoxicity was assessed using a bacterial reverse mutation test with Salmonella typhimurium (strains TA98, TA100, TA1535, and TA1537) and Escherichia coli WP2 uvrA, an in vitro mammalian chromosome aberration test using Chinese hamster lung cells, and an in vivo mammalian erythrocyte micronucleus test using Hsd:ICR (CD-1) SPF mouse bone marrow. After LK1 administration (2,500 mg/kg), there were no LK1-related body weight changes or necropsy findings. The reverse mutation test showed no increased reverse mutation upon exposure to 5,000 μg/plate of the LK1 powder, the maximum tested amount. The chromosome aberration test and micronucleus assay demonstrated no chromosomal abnormalities and genotoxicity, respectively, in the presence of the LK1 powder. The absence of physiological findings and genetic abnormalities suggests that LK1 powder is appropriate as a candidate biomass to be used as a safe food ingredient.

Lactobacillus plantarum BSGP201683 Improves the Intestinal Barrier of Giant Panda Microbiota-Associated Mouse Infected by Enterotoxigenic Escherichia coli K88.

Giant pandas often suffered from gastrointestinal disease, especially the captive sub-adult one. Our study aims to investigate whether L. plantarum G83, a good panda-derived probiotic, can improve the intestinal barrier against the enterotoxigenic Escherichia coli K88 (E. coli K88) infection in giant panda microbiota-associated mice (GPAM). We treated SPF mice with antibiotics cocktail and transplanted the giant panda intestinal microbiota to set up a GPAM. Our results demonstrated that the microbiota of GPAM changed over time and was relatively stable in the short-term experiment (2-4weeks). Whereafter, the GPAM pretreated with L. plantarum G83 for 15days and infected with enterotoxigenic E. coli K88. The result indicated that the number of Bifidobacteria spp. increased in GPAM-G and GPAM-GE groups; the Lactobacillus spp. only increased in the GPAM-G group. Although the abundance of Enterobacteriaceae spp. only decreased in the GPAM-G group, the copy number of Escherichia coli in the GPAM-E group was significantly lower than that in the other groups. Meanwhile, the L. plantarum G83-induced alteration of microbiota could increase the mRNA expression of Claudin-1, Zo-1, and Occludin-1 in the GPAM-G group in the ileum; only Occludin-1 was increased in the GPAM-GE group. The sIgA in the ileum showed a positive response, also the result of body weight and histology in both the GPAM-G and GPAM-GE group. These results indicated that the L. plantarum G83 could improve the intestinal barrier to defense the enterotoxigenic E. coli K88 invasion.