Germ-free Mice Research Articles

A Passion for Small Things and Staying Primed: My Career in Virology and Immunology.

A love of science and animals, perseverance, and happenstance propelled my career in veterinary virology and immunology. I have focused on deadly enteric and respiratory viral infections in neonatal livestock and humans with an aim to understand their prevalence, pathogenesis, interspecies transmission, and immunity and develop vaccines. Research on animal coronaviruses (CoVs), including their broad interspecies transmission, provided a foundation to understand emerging zoonotic fatal human respiratory CoVs [severe acute respiratory syndrome, Middle East respiratory syndrome, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)] and reverse zoonosis of SARS-CoV-2 to animals. A highlight of my early research was the discovery of the gut-mammary gland-sIgA axis, documenting a common mucosal immune system. The latter remains pivotal to designing maternal vaccines for passive immunity in neonates. Our discovery and innovative cell propagation of fastidious human and animal rotaviruses and caliciviruses and their infectivity in germ-free animals has provided cell-adapted and animal disease models for ongoing virologic and immunologic investigations and vaccines. Nevertheless, besides the research discoveries, my lasting legacy remains the outstanding mentees who have enriched my science and my life.

Age-related loss of intestinal barrier integrity plays an integral role in thymic involution and T cell ageing.

The intestinal epithelium serves as a physical and functional barrier against harmful substances, preventing their entry into the circulation and subsequent induction of a systemic immune response. Gut barrier dysfunction has recently emerged as a feature of ageing linked to declining health, and increased intestinal membrane permeability has been shown to promote heightened systemic inflammation in aged hosts. Concurrent with age-related changes in the gut microbiome, the thymic microenvironment undergoes a series of morphological, phenotypical and architectural alterations with age, including disorganisation of the corticomedullary junction, increased fibrosis, increased thymic adiposity and the accumulation of senescent cells. However, a direct link between gut barrier dysbiosis and thymic involution leading to features of immune ageing has not been explored thus far. Herein, we reveal strong associations between enhanced microbial translocation and the peripheral accumulation of terminally differentiated, senescent and exhausted T cells and the compensatory expansion of regulatory T cells in older adults. Crucially, we demonstrate that aged germ-free mice are protected from age-related increases in intestinal permeability, highlighting the direct impact of mucosal permeability on thymic ageing. Together, these findings establish a novel mechanism by which gut barrier dysfunction drives systemic activation of the immune system during ageing through thymic involution. This enhances our understanding of drivers of T cell ageing and opens up the possibility for the use of microbiome-based interventions to restore immune homeostasis and promote healthy ageing in older adults.

Deciphering the Therapeutic Efficacy and Underlying Mechanisms of Dendrobium officinale Polysaccharides in the Intervention of Alzheimer's Disease Mice: Insights from Metabolomics and Microbiome.

As a traditional drug-food homologous plant, Dendrobium officinale is widely recognized for its nutritional and medicinal value. Specifically, D. officinale polysaccharide (DOP) has garnered attention as a potential prebiotic for its protective effects on gut microbiota and the nervous system. However, the underlying mechanism by which DOP improves cognitive dysfunction in Alzheimer's disease (AD) remains unclear. This study intends to elucidate the beneficial effects of DOP on AD mice from the perspectives of metabolomics and the intestinal microbiome. The results showed that DOP significantly ameliorated cognitive dysfunction, attenuated hippocampal neuronal damage and Aβ plaque deposition, and restored intestinal barrier integrity in AD mice. The antibiotic-cocktail-induced germ-free mouse model confirmed that the neuroprotective effect of DOP was dependent on gut microbiota. Further investigations demonstrated that DOP influenced the composition of gut microbiota and restored its diversity. Additionally, DOP reshaped metabolic profile disorders in AD mice and increased the short-chain fatty acids (SCFAs) content. Correlation analysis further highlighted that specific gut microbiota was associated with the metabolism of AD mice. In conclusion, this study sheds light on the positive impact of DOP in reshaping the gut microbiota and enhancing cognitive function, offering important perspectives for the possible advancement and utilization of DOP.

Bone loss with aging is independent of gut microbiome in mice

Emerging evidence suggests a significant role of gut microbiome in bone health. Aging is well recognized as a crucial factor influencing the gut microbiome. In this study, we investigated whether age-dependent microbial change contributes to age-related bone loss in CB6F1 mice. The bone phenotype of 24-month-old germ-free (GF) mice was indistinguishable compared to their littermates colonized by fecal transplant at 1-month-old. Moreover, bone loss from 3 to 24-month-old was comparable between GF and specific pathogen-free (SPF) mice. Thus, GF mice were not protected from age-related bone loss. 16S rRNA gene sequencing of fecal samples from 3-month and 24-month-old SPF males indicated an age-dependent microbial shift with an alteration in energy and nutrient metabolism potential. An integrative analysis of 16S predicted metagenome function and LC-MS fecal metabolome revealed an enrichment of protein and amino acid biosynthesis pathways in aged mice. Microbial S-adenosyl methionine metabolism was increased in the aged mice, which has previously been associated with the host aging process. Collectively, aging caused microbial taxonomic and functional alteration in mice. To demonstrate the functional importance of young and old microbiome to bone, we colonized GF mice with fecal microbiome from 3-month or 24-month-old SPF donor mice for 1 and 8 months. The effect of microbial colonization on bone phenotypes was independent of the microbiome donors’ age. In conclusion, our study indicates age-related bone loss occurs independent of gut microbiome.

TMIC-15. GUT MICROBIOTA CONTROLS NEUROFIBROMATOSIS-1 (NF1) OPTIC GLIOMA GROWTH THROUGH IMMUNE CIRCUIT MODULATION

Abstract Changes in the gut microbiome can have profound effects on the nervous system through modulation of T cell and microglia function. Since our prior studies demonstrated that T cells and microglia positively regulate low-grade glioma growth through the establishment of T cell-microglia immune circuit, we sought to explore the impact of changes in the gut microbiota on tumor biology. For these studies, genetically engineered Neurofibromatosis type 1 (NF1) optic glioma (Nf1-OPG) mice were raised in a germ-free environment or treated with antibiotics to deplete gut bacteria. First, we demonstrated that Nf1-OPG mice raised in a germ-free (gnotobiotic) environment or treated with specific antibiotics lacked optic gliomas and had improved OPG-induced retinal pathology (increased retinal nerve fiber layer thickness). Second, germ-free and antibiotic-treated Nf1-OPG mice gavaged with fecal microbiota from Nf1-OPG mice raised in a standard barrier facility restored optic glioma growth. Third, we showed that both germ-free and antibiotic-treated Nf1-OPG mice exhibited reduced intra-tumoral CD8+ T cell content resulting from decreased microglia chemokine production. Fourth, we discovered a gut-brain paracrine axis whose interruption reduced optic glioma growth and attenuated retinal nerve fiber layer thinning. Collectively, these findings establish a mechanistic relationship between the gut microbiota and brain tumor growth relevant to potential therapeutic interventions for pediatric low-grade gliomas.

Probiotics and their metabolite spermidine enhance IFN-γ+CD4+ Tcell immunity to inhibit hepatitis B virus.

The therapeutic potential of commensal microbes and their metabolites is promising in the functional cure of chronic hepatitis B virus (HBV) infection, which is defined as hepatitis B surface antigen (HBsAg) loss. Here, using both specific-pathogen-free and germ-free mice, we report that probiotics significantly promote the decline of HBsAg and inhibit HBV replication by enhancing intestinal homeostasis and provoking intrahepatic interferon (IFN)-γ+CD4+ Tcell immune response. Depletion of CD4+ Tcells or blockage of IFN-γ abolishes probiotics-mediated HBV inhibition. Specifically, probiotics-derived spermidine accumulates in the gut and transports to the liver, where it exhibits a similar anti-HBV effect. Mechanistically, spermidine enhances IFN-γ+CD4+ Tcell immunity by autophagy. Strikingly, administration of probiotics in HBV patients reveals a preliminary trend to accelerate the decline of serum HBsAg. In conclusion, probiotics and their derived spermidine promote HBV clearance via autophagy-enhanced IFN-γ+CD4+ Tcell immunity, highlighting the therapeutic potential of probiotics and spermidine for the functional cure of HBV patients.

Maternal consumption of yoghurt activating the aryl hydrocarbon receptor increases group 3 innate lymphoid cells in murine offspring

ABSTRACT Indole derivatives are microbial metabolites of the tryptophan pathway involved in gut immune homeostasis. They bind to the aryl hydrocarbon receptor (AhR), thereby modulating development of intestinal group 3 innate lymphoid cells (ILC3) and subsequent interleukin-22 production. In mice, indole derivatives of the maternal microbiota can reach the milk and drive early postnatal ILC3 development. Apart from the gut microbiota, lactic acid bacteria (LAB) also produce indole compounds during milk fermentation. Using germ-free mice, the aim of our study was to test if maternal intake of a dairy product enriched in AhR-activating indoles produced by fermentation could boost maturation of the intestinal innate immune system in the offspring. A set of 631 LAB strains were genetically screened for their potential to produce indole compounds. Among these, 125 strains were tested in combination with standard strains to produce yoghurts that were screened for their ability to activate AhR in vitro using the HepG2–AhR–Luc cell line. The most active yoghurt and a control yoghurt were formulated as pellets and fed to germ-free dams during pregnancy and lactation. Analysis of the offspring on postnatal day 14 using flow cytometry revealed an increase in the frequency of small intestinal lamina propria NKp46 +ILC3 s in the pups born to dams that had consumed the purified diet containing an AhR-active yoghurt (AhrY-diet) compared to control yoghurt (ConY-diet). Selection of LABs based on their ability to produce a fermented dairy able to activate AhR appears to be an effective approach to produce a yoghurt with immunomodulatory properties. IMPORTANCE Key progresses in the sequencing and functional annotation of microbial organisms have revolutionized research in the fields of human metabolism and food biotechnology. In particular, the gut microbiome is now recognized as an important mediator of the impact of nutrition on human metabolism. Annotated genomes of a large number of bacteria are now available worldwide, which selectively transform food through fermentation to produce specific bioactive compounds with the potential to modulate human health. A previous research has demonstrated that the maternal microbiota shapes the neonatal immune system. Similarly, this report shows that lactic acid bacteria can be selected to produce fermented food that can also modulate postnatal intestinal immunity.

Heat exposure promotes sarcopenia via gut microbiota-derived metabolites.

The unprecedented rise in global ambient temperatures in the last decade has significantly impacted human health, yet how heat exposure affects the development of sarcopenia remains enigmatic. Here, we demonstrate that chronic heat exposure induces skeletal muscle volume loss, leading to muscle strength and functional decline in mice. The microbiota composition of heat-exposed mice was analyzed using 16S ribosomal DNA analysis. Liquid chromatography-mass spectrometry (LC-MS) was used to explore the effects of heat exposure on the blood metabolome and to further analyze the correlation between blood metabolism and gut microbiota. Transplantation of microbiota from heat-exposed mice to germ-free mice was sufficient to increase adverse effects on skeletal muscle function in the host. Mechanistically, using an untargeted metabolomics strategy, we reveal that altered gut microbiota due to high temperatures is associated with elevated serum levels of homocitrulline. Homocitrulline causes mitochondrial dysfunction in myocytes by exacerbating ferroptosis levels. And Nrf2 activator (Oltipraz) supplementation alleviates muscle atrophy and dysfunction induced by heat exposure. Our findings reveal the detrimental effects of heat exposure on muscle function and provide new strategies for treating sarcopenia.

Fecal microbiota transplantation from protozoa-exposed donors downregulates immune response in a germ-free mouse model, its role in immune response and physiology of the intestine.

Intestinal parasites are part of the intestinal ecosystem and have been shown to establish close interactions with the intestinal microbiota. However, little is known about the influence of intestinal protozoa on the regulation of the immune response. In this study, we analyzed the regulation of the immune response of germ-free mice transplanted with fecal microbiota (FMT) from individuals with multiple parasitic protozoans (P) and non-parasitized individuals (NP). We determined the production of intestinal cytokines, the lymphocyte populations in both the colon and the spleen, and the genetic expression of markers of intestinal epithelial integrity. We observed a general downregulation of the intestinal immune response in mice receiving FMT-P. We found significantly lower intestinal production of the cytokines IL-6, TNF, IFN-γ, MCP-1, IL-10, and IL-12 in the FMT-P. Furthermore, a significant decrease in the proportion of CD3+, CD4+, and Foxp3+ T regulatory cells (Treg) was observed in both, the colon and spleen with FMT-P in contrast to FMT-NP. We also found that in FMT-P mice there was a significant decrease in tjp1 expression in all three regions of the small intestine; ocln in the ileum; reg3γ in the duodenum and relmβ in both the duodenum and ileum. We also found an increase in colonic mucus layer thickness in mice colonized with FMT-P in contrast with FMT-NP. Finally, our results suggest that gut protozoa, such as Blastocystis hominis, Entamoeba coli, Endolimax nana, Entamoeba histolytica/E. dispar, Iodamoeba bütschlii, and Chilomastix mesnili consortia affect the immunoinflammatory state and induce functional changes in the intestine via the gut microbiota. Likewise, it allows us to establish an FMT model in germ-free mice as a viable alternative to explore the effects that exposure to intestinal parasites could have on the immune response in humans.

Genetically Engineered Clostridium Prevents Perivascular Adipose Tissue and Endothelial Senescence and Dysfunction in Aging

Abstract Introduction (Peri)vascular senescence is a bona fide risk for several aging-associated diseases that threatens healthspan. Gut microbiome may be one of triggers that promotes vascular diseases as host ages. However, the interplay between vascular senescence and gut microbiome is largely unknown. Our recent studies revealed an increase in gut-derived phenylacetic acid (PAA) in the aging population (TwinsUK Aging Cohort, n=7,303) and aged mice. Our preliminary shotgun metagenomics also showed that ppfor-harboring Clostridium sp. ASF356 age-dependently generates PAA, which induces perivascular adipose tissue (PVAT) and endothelial cell (EC) senescence and dysfunction in old mice. Yet, it remains unclear whether and how genetic engineering of Clostridium sp. ASF356 (by depleting ppfor gene) can prevent aortic PVAT-EC senescence and restore vascular function in aging. Methods For bacterial genetic engineering, we depleted ppfor gene in Clostridium ASF356 by ClosTron technique and further mono-colonized aged mice (24-months old) and germ-free mice, as control, to reduce plasma PAA and thereby prevent aortic PVAT-EC senescence and improve endothelial dysfunction. Results As a proof of the concept, our studies exhibited that mono-colonization of germ-free mice with wild-type Clostridium sp. ASF356 markedly elevates plasma PAA levels. It was accompanied by hallmarks of aortic PVAT-EC senescence, including increased DNA damage (↑ γ-H2A.X phosphorylation), heightened SASP (↑ IL-6 and VCAM1), and proliferative arrest (↑ p16INK4a and p21WAF1/Cip1). Additionally, our findings revealed upreglation of Notch1 in PVAT, contributing to reduced energy supply (↓ UCP1) and augmented senescence-messaging secretome (↑ IL-6) towards ECs. Our further studies revealed that mono-colonization of aged mice (pre-treated with antibiotics) with Δppfor Clostridium ASF356 mutants intriguingly resulted in decreased plasma PAA levels. This intervention led to the rescues of cellular senescence in both aortic PVAT and ECs, consequently improving vascular function. Specifically, mice subjected to this treatment exhibited a significant reduction in SA-β-galactosidase+ cells, as well as decreased levels of γ-H2A.X phosphorylation, p16INK4a, p21WAF1/Cip1, and IL-6 in their aortic PVAT and ECs compared to wild-type aged mice. Conclusions The current study suggests that genetically engineered Δppfor Clostridium ASF356 mutants reverse cellular senescence and restore function in aortic PVAT and endothelial cells, highlighting the potential for targeted gut microbiome manipulation as an innovative senotherapy to combat vascular aging.

A human gut Faecalibacterium prausnitzii fatty acid amide hydrolase.

Undernutrition in Bangladeshi children is associated with disruption of postnatal gut microbiota assembly; compared with standard therapy, a microbiota-directed complementary food (MDCF) substantially improved their ponderal and linear growth. Here, we characterize a fatty acid amide hydrolase (FAAH) from a growth-associated intestinal strain of Faecalibacterium prausnitzii cultured from these children. This enzyme, expressed and purified from Escherichia coli, hydrolyzes a variety of N-acylamides, including oleoylethanolamide (OEA), neurotransmitters, and quorum sensing N-acyl homoserine lactones; it also synthesizes a range of N-acylamides, notably N-acyl amino acids. Treating germ-free mice with N-oleoylarginine and N-oleolyhistidine, major products of FAAH OEA metabolism, markedly affected expression of intestinal immune function pathways. Administering MDCF to Bangladeshi children considerably reduced fecal OEA, a satiety factor whose levels were negatively correlated with abundance and expression of their F. prausnitzii FAAH. This enzyme, structurally and catalytically distinct from mammalian FAAH, is positioned to regulate levels of a variety of bioactive molecules.

The microbiome shapes immunity in a sex-specific manner in mouse models of Alzheimer's disease.

INTRODUCTION : Preclinical studies reveal that the microbiome broadly affects immune responses and deposition and/or clearance of amyloid-beta (Aβ) in mouse models of Alzheimer's disease (AD). Whether the microbiome shapes central and peripheral immune profiles in AD models remains unknown. METHODS : We examined adaptive immune responses in two mouse models containing AD- related genetic predispositions (3xTg and 5xFAD) in the presence or absence of the microbiome. RESULTS : T and B cells were altered in brain-associated and systemic immune tissues between genetic models and wildtype mice, with earlier signs of immune activity in females. Systemic immune responses were modulated by the microbiome and differed by sex. Further, the absence of a microbiome in germ-free mice resulted in reduced cognitive deficits, primarily in females. DISCUSSION : These data reveal sexual dimorphism in early signs of immune activity and microbiome effects, and highlight an interesting interaction between sex and the microbiome in mouse models of AD.

Fetal programming by the parental microbiome of offspring behavior, and DNA methylation and gene expression within the hippocampus.

The microorganisms colonizing the gastrointestinal tract of animals, collectively referred to as the gut microbiome, affect numerous host behaviors dependent on the central nervous system (CNS). Studies comparing germ-free mice to normally colonized mice have demonstrated influences of the microbiome on anxiety-related behaviors, voluntary activity, and gene expression in the CNS. Additionally, there is epidemiologic evidence supporting an intergenerational influence of the maternal microbiome on neurodevelopment of offspring and behavior later in life. There is limited experimental evidence however directly linking the maternal microbiome to long-term neurodevelopmental outcomes, or knowledge regarding mechanisms responsible for such effects. Here we show that that the maternal microbiome has a dominant influence on several offspring phenotypes including anxiety-related behavior, voluntary activity, and body weight. Adverse outcomes in offspring were associated with features of the maternal microbiome including bile salt hydrolase activity gene expression ( bsh ), abundance of certain bile acids, and hepatic expression of Slc10a1 . In cross-foster experiments, offspring resembled their birth dam phenotypically, despite faithful colonization in the postnatal period with the surrogate dam microbiome. Genome-wide methylation analysis of hippocampal DNA identified microbiome- associated differences in methylation of 196 loci in total, 176 of which show conserved profiles between mother and offspring. Further, single-cell transcriptional analysis revealed accompanying differences in expression of several differentially methylated genes within certain hippocampal cell clusters, and vascular expression of genes associated with bile acid transport. Inferred cell-to-cell communication in the hippocampus based on coordinated ligand-receptor expression revealed differences in expression of neuropeptides associated with satiety. Collectively, these data provide proof-of-principle that the maternal gut microbiome has a dominant influence on the neurodevelopment underlying certain offspring behaviors and activities, and selectively affects genome methylation and gene expression in the offspring CNS in conjunction with that neurodevelopment.

The expression of intestinal Cyp2c55 is regulated by the microbiota and inflammation.

Although the mutualistic relationship between the intestinal microbiota and the human host is crucial for maintaining health, the underlying mechanisms of this relationship remain unclear. In the present study, aiming to elucidate the regulatory mechanisms governing the Cyp2c55 expression, which is predominantly observed in colonic tissues, germ-free, antibiotic-administered and colitic mice, as well as mouse colonoids, were used as experimental models. RNA sequencing showed comparable decreases in the colonic Cyp2c55 expression in germ-free and antibiotic-administered mice, when compared with that in specific pathogen-free mice. Furthermore, administration of dextran sulfate sodium decreased the Cyp2c55 expression in colitic mice. For these mice, a Pearson correlation analysis also showed a positive correlation between the Cyp2c55 expression and unconjugated bile acids (BAs), including chenodeoxycholic, muricholic, deoxycholic, lithocholic, and ursodeoxycholic acids, as well as taurine (T)-conjugated secondary BAs, including deoxycholic acid. Moreover, bacterial genera, such as Muribaculaceae and unclassified Lachnospiraceae, also exhibited a positive correlation with these BAs. While administration of an agonist of the pregnane X receptor (PXR) increased the Cyp2c55 expression in mouse colonoids, inflammatory cytokines decreased it. In conclusion, Cyp2c55 was highly expressed in the colonic epithelial cells of mice in a microbiota-dependent manner. The underlying mechanism seemed to involve a BA-mediated PXR activation. In addition, the colonic expression of Cyp2c55 was regulated by the inflammatory response. Although the physiological function of Cyp2c55 remains largely unidentified, our findings suggested that Cyp2c55 may play a role in the mutualistic interaction between the intestinal microbiota and the intestinal homeostasis.

From microbes to mind: germ-free models in neuropsychiatric research.

The gut-microbiota-brain axis refers to the bidirectional communication system between the gut, its microbial community, and the brain. This interaction involves a complex interplay of neural pathways, chemical transmitters, and immunological mechanisms. Germ-free animal models have been extensively employed to investigate gut-microbiota-brain interactions, significantly contributing to our current understanding of the role of intestinal microbes in brain function. However, despite the many benefits, this absence of microbiota is not futile. Germ-free animals present physiological and neurodevelopmental alterations that can persist even after reconstitution with normal microbiota. Therefore, the main goal of this minireview is to discuss how some of the inherent limitations of this model can interfere with the conclusion obtained when using these animals to study the complex nature of neuropsychiatric disorders. Furthermore, we examine the inclusion and use of antibiotic-based treatments as an alternative in the research of gut-brain interactions.

Expansion of a bacterial operon during cancer treatment ameliorates drug toxicity.

Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Herein, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal study of the human gut microbiome during colorectal cancer (CRC) treatment ( NCT04054908 ) coupled to cell culture and mouse experiments. 16S rRNA gene sequencing revealed significant shifts in gut microbial community structure during oral fluoropyrimidine treatment across multiple patient cohorts, in mouse small and large intestinal contents, and in patient-derived ex vivo communities. Metagenomic sequencing revealed marked shifts in pyrimidine-related gene abundance during oral fluoropyrimidine treatment, including enrichment of the preTA operon, which is sufficient for the inactivation of active metabolite 5-fluorouracil (5-FU). preTA + bacteria depleted 5-FU in gut microbiota grown ex vivo and the mouse distal gut. Germ-free and antibiotic-treated mice experienced increased fluoropyrimidine toxicity, which was rescued by colonization with the mouse gut microbiota, preTA + E. coli , or preTA -high CRC patient stool. Finally, preTA abundance was negatively associated with fluoropyrimidine toxicity in patients. Together, these data support a causal, clinically relevant interaction between a human gut bacterial operon and the dose-limiting side effects of cancer treatment. Our approach is generalizable to other drugs, including cancer immunotherapies, and provides valuable insights into host-microbiome interactions in the context of disease. One Sentence Summary: Gut microbial enzymes can be used to predict and prevent anticancer drug toxicity.

Clostridium sporogenes-derived metabolites protect mice against colonic inflammation

ABSTRACT Gut microbiota-derived metabolites play a pivotal role in the maintenance of intestinal immune homeostasis. Here, we demonstrate that the human commensal Clostridium sporogenes possesses a specific metabolic fingerprint, consisting predominantly of the tryptophan catabolite indole-3-propionic acid (IPA), the branched-chain acids (BCFAs) isobutyrate and isovalerate and the short-chain fatty acids (SCFAs) acetate and propionate. Mono-colonization of germ-free mice with C. sporogenes (CS mice) affected colonic mucosal immune cell phenotypes, including up-regulation of Il22 gene expression, and increased abundance of transcriptionally active colonic tuft cells and Foxp3+ regulatory T cells (Tregs). In DSS-induced colitis, conventional mice suffered severe inflammation accompanied by loss of colonic crypts. These symptoms were absent in CS mice. In conventional, but not CS mice, bulk RNAseq analysis of the colon revealed an increase in inflammatory and Th17-related gene signatures. C. sporogenes-derived IPA reduced IL-17A protein expression by suppressing mTOR activity and by altering ribosome-related pathways in Th17 cells. Additionally, BCFAs and SCFAs generated by C. sporogenes enhanced the activity of Tregs and increased the production of IL-22, which led to protection from colitis. Collectively, we identified C. sporogenes as a therapeutically relevant probiotic bacterium that might be employed in patients with inflammatory bowel disease (IBD).

Modelling a Western Lifestyle in Mice: A Novel Approach to Eradicating Aerobic Spore-Forming Bacteria from the Colonic Microbiome and Assessing Long-Term Clinical Outcomes.

The environmentally acquired aerobic spore-forming (EAS-Fs) bacteria that are ubiquitous in nature (e.g., soil) are transient colonisers of the mammalian gastro-intestinal tract. Without regular exposure, their numbers quickly diminish. These species of bacteria have been suggested to be essential to the normal functioning of metabolic and immunogenic health. The modern Western lifestyle restricts exposure to these EAS-Fs, possibly explaining part of the pathogenesis of many Western diseases. To date, the only animal studies that address specific microbiome modelling are based around germ-free animals. We have designed a new animal model that specifically restricts exposure to environmental sources of bacteria. A new protocol, termed Super Clean, which involves housing mice in autoclaved individually ventilated cages (IVCs), with autoclaved food/water and strict ascetic handling practice was first experimentally validated. The quantification of EAS-Fs was assessed by heat-treating faecal samples and measuring colony-forming units (CFUs). This was then compared to mice in standard conditions. Mice were housed in their respective groups from birth until 18 months. Stool samples were taken throughout the experiment to assess for abundance in transiently acquired environmental bacteria. Clinical, biochemical, histological, and gene expression markers were analysed for diabetes, hypercholesterolaemia, obesity, inflammatory bowel disease, and non-alcoholic fatty liver disease (the "diseases of the West"). Our results show that stringent adherence to the Super Clean protocol produces a significantly decreased abundance of aerobic spore-forming Bacillota after 21 days. This microbiomic shift was correlated with significantly increased levels of obesity and impaired glucose metabolism. There was no evidence of colitis, liver disease or hypercholesterolaemia. This new murine model successfully isolates EAS-Fs and has potential utility for future research, allowing for an investigation into the clinical impact of living in relative hygienic conditions.

8090 A Gut Commensal, Parabacteroides distasonis, and Molecular Mimicry in Type 1 Diabetes

Abstract Disclosure: A. Randall: None. Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic, insulin-producing beta cells by autoreactive T-cells. The trigger(s) of T1D are unclear and genetics alone cannot explain the recent increase in T1D incidence. The gut microbiome composition in T1D patients is altered and thought to play a key role in T1D pathogenesis. Most longitudinal studies have been descriptive. However, a direct cause-and-effect relationship hasn't been established yet. Insulin is a key autoantigen in T1D autoimmunity, and insulin B-chain amino acids 9-23 (insB:9-23) is an important, commonly recognized T-cell epitope. Molecular mimicry is an autoimmunity mechanism where host-like microbial proteins trigger autoimmune responses. We previously identified a peptide mimic of insB:9-23, amino acids 4-18 of hypoxanthine phosphoribosyl transferase (HPRT:4-18) of Parabacteroides distasonis (Girdhar et al., 2022). HPRT:4-18 stimulated insB:9-23-specific human and non-obese diabetic (NOD) mice T-cells. We hypothesize that P. distasonis HPRT:4-18 is required to accelerate diabetes via molecular mimicry of insB:9-23. By orally gavaging NOD mice (n=40 mice/group), we showed that P. distasonis accelerated diabetes onset. Next, we colonized NOD mice with another common gut commensal, Bacteroides fragilis, as a control, and did not observe any effect on islet infiltration. In addition, P. distasonis colonization increased infiltration of immune cells into the islets of germ-free NOD mice (n=6-8 mice/group), while the diabetes incidence experiment in mice is ongoing. P. distasonis-colonized mice had significantly more splenic CD8 and naïve T-cells, dendritic cells and macrophages but fewer FOXP3 cells (n=3-6 mice/group). To determine whether P. distasonis’s effect is specific to the pancreas, we checked intestinal barrier function and intestinal immune cell composition. Disruption of intestinal barrier function has been associated with diabetes. P. distasonis colonization did not alter gut permeability. Furthermore, P. distasonis did not increase intestinal inflammation. Among the intestinal intraepithelial lymphocytes (IELs), there were fewer naïve, central and effector T-cells and B-cells. The findings indicate that P. distasonis does not stimulate an unspecific immune response and its inflammatory effect is specific to the pancreas, supporting our initial hypothesis. Taken together, our results indicate that exposure to P. distasonis in the first years of life has the potential to stimulate molecular mimicry mechanism and cause T1D onset. Reference: Girdhar, K., Huang, Q., Chow, I. T., Vatanen, T., Brady, C., Raisingani, A., . . . Altindis, E. (2022). A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes. Proc Natl Acad Sci U S A, 119(31), e2120028119. doi:10.1073/pnas.2120028119 Presentation: 6/2/2024

Perturbed Microbial Ecology in Neuromyelitis Optica Spectrum Disorder: Evidence from the Gut Microbiome and Fecal Metabolome

BackgroundNeuromyelitis optica spectrum disorder (NMOSD) is a central nervous system inflammatory demyelinating immune-mediated ailment, which is influenced by genetic, epigenetic, and environmental elements. The escalating incidence of NMOSD in recent years implies alterations in environmental risk factors. Recent research has established a correlation between gut microbiomes and the development of NMOSD. MethodsMetagenomic shotgun sequencing and gas chromatography-mass spectrometry (GC-MS) were employed to assess alterations of the structure and function in the fecal microbiome, as well as levels of short-chain fatty acids (SCFAs) in fecal and blood samples, among individuals with neuromyelitis optica spectrum disorder (NMOSD) during the acute phase (n=25), the remission phase (n=11), and a group of healthy controls (HCs) (n = 24). We further explored the correlation between gut microbiota and the pathogenesis of NMOSD through fecal microbiota transplantation (FMT). The gut microbiome from human donors diagnosed with NMOSD or HCs was transplanted into germ-free mice, followed by an analysis of the alterations in the structure and functionality of the transplanted mice's gut microbiome. Additionally, the impact of microbiome transfer on the immunity and spinal cord of germ-free mice was assessed through various techniques, including ELISA, flow cytometry, western blot, histopathology, and transcriptome sequencing. Results(1) At the taxonomic levels of genus and species, there were significant differences in the α-diversity of the microbiome between HCs and NMOSD patients in the acute phase, with NMOSD patients having higher species diversity. (2) In the acute phase, the gut microbiota of NMOSD patients was characterized by Ruminococcaceae_unclassified, Campylobacter, Parabacteroides, Lactobacillus, Akkermansia, Streptococcus oralis, Clostridium leptum, Clostridium asparagiforme, Firmicutes bacterium CAG 238, and Lactobacillus fermentum. (3) The relative abundances of Coprobacter, Turicimonas, Gemmiger, Enterobacter, Roseburia sp.CAG 471, Veillonella tobetsuensis, Proteobacteria bacterium CAG 139, Ruminococcus bicirculans, Lactococcus lactis, Flavonifractor plautii, and Streptococcus cristatus were notably lower in patients experiencing remission compared to NMOSD patients in the acute phase, On the other hand, the relative abundances of Flavonifractor (P = 0.049) and Clostridium aldenense (P = 0.049) were significantly higher. Following medication, the gut microbiome distribution in NMOSD patients during remission closely resembled that of healthy controls (HCs). (4) Compared with HCs, acetate levels in the feces of patients with NMOSD in the acute phase were significantly lower. (5) In addition, we transplanted feces from NMOSD patients into germ-free mice and revealed a significant increase in the levels of IL-6, IL-17A, and IL-23 in the blood of mice belonging to the NMOSD fecal transplantation (NFMT) group. Additionally, the IL-10 level exhibited a significant reduction. Moreover, the proportion of Th17 cells displayed a significant increase, while the proportion of Treg cells exhibited a significant decrease in the spleens of NFMT mice. ConclusionPatients in the acute phase of neuromyelitis optica spectrum disorder (NMOSD) exhibited imbalances in their gut microbiota and a deficiency in short-chain fatty acids (SCFAs). Following drug treatment, the composition of intestinal microbes in NMOSD patients during the remission phase closely resembled that of the healthy control population. The FMT experiment provided evidence of the significant association between intestinal flora and the pathogenesis of NMOSD. Consequently, investigating gut microbiota and identifying novel microbial markers hold promise for the diagnosis and treatment of NMOSD patients.