Temporally Distinct Functions of the Cytokines IL-12 and IL-23 Drive Chronic Colon Inflammation in Response to Intestinal Barrier Impairment.

Epithelial barrier defects are implicated in the pathogenesis of inflammatory bowel disease (IBD); however, the role of microbiome dysbiosis and the cytokine networks orchestrating chronic intestinal inflammation in response to barrier impairment remain poorly understood. Here, we showed that altered Schaedler flora (ASF), a benign minimal microbiota, was sufficient to trigger colitis in a mouse model of intestinal barrier impairment. Colitis development required myeloid-cell-specific adaptor protein MyD88 signaling and was orchestrated by the cytokines IL-12, IL-23, and IFN-γ. Colon inflammation was driven by IL-12 during the early stages of the disease, but as the mice aged, the pathology shifted toward an IL-23-dependent inflammatory response driving disease chronicity. These findings reveal that IL-12 and IL-23 act in a temporally distinct, biphasic manner to induce microbiota-driven chronic intestinal inflammation. Similar mechanisms might contribute to the pathogenesis of IBD particularly in patients with underlying intestinal barrier defects.

Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes.

The community of microorganisms in the mammalian gastrointestinal tract, referred to as the gut microbiota, influences host physiology and immunity. The last decade of microbiome research has provided significant advancements for the field and highlighted the importance of gut microbes to states of both health and disease. Novel molecular techniques have unraveled the tremendous diversity of intestinal symbionts that potentially influence the host, many proof-of-concept studies have demonstrated causative roles of gut microbial communities in various pathologies, and microbiome-based approaches are beginning to be implemented in the clinic for diagnostic purposes or for personalized treatments. However, several challenges for the field remain: purely descriptive reports outnumbering mechanistic studies and slow translation of experimental results obtained in animal models into the clinics. Moreover, there is a dearth of knowledge regarding how gut microbes, including novel species that have yet to be identified, impact host immune responses. The sheer complexity of the gut microbial ecosystem makes it difficult, in part, to fully understand the microbiota-host networks that regulate immunity. In the present manuscript, we review key findings on the interactions between gut microbiota members and the immune system. Because culturing microbes allows performing functional studies, we have emphasized the impact of specific taxa or communities thereof. We also highlight underlying molecular mechanisms and discuss opportunities to implement minimal microbiome-based strategies.

Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.

In advanced chronic liver disease (CLD), the translocation of intestinal bacteria and the resultant increase of proinflammatory cytokines in the splanchnic and systemic circulation may contribute to the progression of fibrosis. We therefore speculated that fibrosis and portal hypertension (PHT) would be attenuated in a mouse model of limited intestinal colonization with altered Schaedler flora (ASF) compared to a more complex colonization with specific pathogen-free (SPF) flora. We induced liver fibrosis in ASF and SPF mice by common bile duct ligation (BDL) or by carbon tetrachloride (CCl4) treatment. We then measured portal pressure (PP), portosystemic shunts (PSSs), and harvested tissues for further analyses. There were no differences in PP between sham-treated ASF or SPF mice. After BDL or CCl4 treatment, PP, PSSs, and hepatic collagen deposition increased in both groups. However, the increase in PP and the degree of fibrosis was significantly higher in ASF than SPF mice. Expression of fibrotic markers α-smooth muscle actin, desmin, and platelet-derived growth factor receptor ß were significantly higher in ASF than SPF mice. This was associated with higher activation of hepatic immune cells (macrophages, neutrophils) and decreased expression of the intestinal epithelial tight junction proteins (claudin-1, occludin-1). In 2 models of advanced CLD, SPF mice presented significantly attenuated liver injury, fibrosis, and PHT compared to ASF mice. In contrast to our hypothesis, these findings suggest that a complex intestinal microbiota may play a "hepato-protective" role.-Moghadamrad, S., Hassan, M., McCoy, K. D., Kirundi, J., Kellmann, P., De Gottardi, A. Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.

Specific Localization and Quantification of the Oligo-Mouse-Microbiota (OMM12 ) by Fluorescence In Situ Hybridization (FISH).

The oligo-mouse-microbiota (OMM12 ) is a widely used syncom that colonizes gnotobiotic mice in a stable manner. It provides several fundamental functions to its murine host, including colonization resistance against enteric pathogens. Here, we designed and validated specific fluorescence in situ hybridization (FISH) probes to detect and quantify OMM12 strains on intestinal tissue cross sections. 16S rRNA-specific probes were designed, and specificity was validated on fixed pure cultures. A hybridization protocol was optimized for sensitive detection of the individual bacterial cells in cryosections. Using this method, we showed that the intestinal mucosal niche of Akkermansia muciniphila can be influenced by global gut microbial community context. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Localization and quantification of OMM12 single strains in mouse cecum cross section Support Protocol: Establishment of specific FISH probe set for OMM12 syncom.

Fusobacterium nucleatum drives a pro-inflammatory intestinal microenvironment through metabolite receptor-dependent modulation of IL-17 expression.

The colorectal cancer (CRC)-associated microbiota creates a pro-tumorigenic intestinal milieu and shapes immune responses within the tumor microenvironment. However, how oncomicrobes - like Fusobacterium nucleatum, found in the oral cavity and associated with CRC tissues- affect these distinct aspects of tumorigenesis is difficult to parse. Herein, we found that neonatal inoculation of ApcMin/+ mice with F. nucleatum strain Fn7-1 circumvents technical barriers preventing its intestinal colonization, drives colonic Il17a expression prior to tumor formation, and potentiates intestinal tumorigenesis. Using gnotobiotic mice colonized with a minimal complexity microbiota (the altered Schaedler's flora), we observed that intestinal Fn7-1 colonization increases colonic Th17 cell frequency and their IL-17A and IL-17F expression, along with a concurrent increase in colonic lamina propria Il23p19 expression. As Fn7-1 stably colonizes the intestinal tract in our models, we posited that microbial metabolites, specifically short-chain fatty acids (SCFA) that F. nucleatum abundantly produces in culture and, as we demonstrate, in the intestinal tract, might mediate part of its immunomodulatory effects in vivo. Supporting this hypothesis, we found that Fn7-1 did not alter RORγt+ CD4+T cell frequency in the absence of the SCFA receptor FFAR2. Taken together, our work suggests that F. nucleatum influences intestinal immunity by shaping Th17 responses in an FFAR2-dependent manner, although further studies are necessary to clarify the precise and multifaceted roles of FFAR2. The potential to increase intestinal Th17 responses is shared by another oncomicrobe, enterotoxigenic Bacteroides fragilis, highlighting a conserved pathway that could potentially be targeted to slow oncomicrobe-mediated CRC.

Colonization with Altered Schaedler Flora Impacts Leukocyte Adhesion in Mesenteric Ischemia-Reperfusion Injury.

The microbiota impacts mesenteric ischemia-reperfusion injury, aggravating the interaction of leukocytes with endothelial cells in mesenteric venules. The role of defined gut microbiomes in this life-threatening pathology is unknown. To investigate how a defined model microbiome affects the adhesion of leukocytes in mesenteric ischemia-reperfusion, we took advantage of gnotobiotic isolator technology and transferred altered Schaedler flora (ASF) from C3H/HeNTac to germ-free C57BL/6J mice. We were able to detect all eight bacterial taxa of ASF in fecal samples of colonized C57BL/6J mice by PCR. Applying qRT-PCR for quantification of species-specific 16S rDNA sequences of ASF bacteria, we found a major shift in the abundance of ASF 500, which was greater in C57BL/6J mice relative to the C3H/HeNTac founder breeding pair. Using high-speed epifluorescence intravital microscopy to visualize the venules of the small bowel mesentery, we found that gnotobiotic ASF-colonized mice showed reduced leukocyte adherence, both pre- and post-ischemia. Relative to germ-free mice, the counts of adhering leukocytes were increased pre-ischemia but did not significantly increase in ASF-colonized mice in the post-ischemic state. Collectively, our results suggest a protective role of the minimal microbial consortium ASF in mesenteric ischemia-reperfusion injury.

Campylobacter jejuni persistently colonizes gnotobiotic altered Schaedler flora C3H/HeN mice and induces mild colitis.

Campylobacter jejuni is a major cause of food-borne human bacterial gastroenteritis but animal models for C. jejuni mediated disease remain limited because C. jejuni poorly colonizes immunocompetent, conventionally-reared (Conv-R) mice. Thus, a reliable rodent model (i.e. persistent colonization) is desirable in order to evaluate C. jejuni-mediated gastrointestinal disease and mechanisms of pathogenicity. As the nature and complexity of the microbiota likely impacts colonization resistance for C. jejuni, Conv-R and gnotobiotic C3H/HeN mice were used to evaluate the persistence of C. jejuni colonization and development of disease. A total of four C. jejuni isolates readily and persistently colonized ASF mice and induced mild mucosal inflammation in the proximal colon, but C. jejuni did not stably colonize nor induce lesions in Conv-R mice. This suggests that the pathogenesis of C. jejuni is influenced by the microbiota, and that ASF mice offer a reproducible model to study the influence of the microbiota on the ability of C. jejuni to colonize the gut and to mediate gastroenteritis.

"Western Diet"-Induced Adipose Inflammation Requires a Complex Gut Microbiota.

BACKGROUND & AIMS: Consumption of a low-fiber, high-fat, Western-style diet (WSD) induces adiposity and adipose inflammation characterized by increases in the M1:M2 macrophage ratio and proinflammatory cytokine expression, both of which contribute to WSD-induced metabolic syndrome. WSD-induced adipose inflammation might result from endoplasmic reticulum stress in lipid-overloaded adipocytes and/or dissemination of gut bacterial products, resulting in activation of innate immune signaling. Hence, we aimed to investigate the role of the gut microbiota, and its detection by innate immune signaling pathways, in WSD-induced adipose inflammation. METHODS: Mice were fed grain-based chow or a WSD for 8 weeks, assessed metabolically, and intestinal and adipose tissue were analyzed by flow cytometry and quantitative reverse transcription polymerase chain reaction. Microbiota was ablated via antibiotics and use of gnotobiotic mice that completely lacked microbiota (germ-free mice) or had a low-complexity microbiota (altered Schaedler flora). Innate immune signaling was ablated by genetic deletion of Toll-like receptor signaling adaptor myeloid differentiation primary response 88. RESULTS: Ablation of microbiota via antibiotic, germ-free, or altered Schaedler flora approaches did not significantly impact WSD-induced adiposity, yet dramatically reduced WSD-induced adipose inflammation as assessed by macrophage populations and cytokine expression. Microbiota ablation also prevented colonic neutrophil and CD103- dendritic cell infiltration. Such reduced indices of inflammation correlated with protection against WSD-induced dysglycemia, hypercholesterolemia, and liver dysfunction. Genetic deletion of myeloid differentiation primary response 88 also prevented WSD-induced adipose inflammation. CONCLUSIONS: These results indicate that adipose inflammation, and some aspects of metabolic syndrome, are not purely a consequence of diet-induced adiposity per se but, rather, may require disturbance of intestine-microbiota interactions and subsequent activation of innate immunity.

Mouse Genetic Background Affects Transfer of an Antibiotic Resistance Plasmid in the Gastrointestinal Tract.

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.

Altered Schaedler flora mice: A defined microbiota animal model to study the microbiota-gut-brain axis.

Despite considerable attention, the mechanisms by which the microbiota affect brain function and host behaviour via the gut-brain axis remain undefined. Identifying microbe-specific pathways that influence neuronal function and bi-directional communication between the gut microbiota and the host central nervous system is challenging due to the extreme microbial diversity in the gut of conventionally-reared mice. Herein, we describe the use of the altered Schaedler flora (ASF) mouse model as an alternative to conventionally-reared and germ-free animals. Colonized with only 8 bacterial species, use of ASF mice greatly simplifies the examination of microbiota-host interactions. We assessed the extent to which behaviour differed between mice with a limited consortium of bacteria compared with a complex, conventional microbiota. The elevated plus maze and open-field assays were utilized to assess murine behaviour. Histological analysis of ileum and colon was performed to evaluate intestinal morphology, and 16 s rRNA gene taxonomic profiling was performed to determine host-stress induced changes in fecal microbial communities. Behavioural and serum corticosterone differences were observed between ASF and conventionally-reared mice, while no differences were found between the intestinal morphology of these two groups. The stress of the behavioural tests induced significant changes in the ASF fecal microbial community but not in that of the conventionally-reared mice. In contrast to the conventionally-reared mice, the results indicated that the ASF mice displayed a marked anxiogenic-like behaviour. These data indicate that ASF mice represent a unique model to elucidate mechanisms governing microbiota-gut-brain communication affecting behaviour.

Mucispirillum schaedleri Antagonizes Salmonella Virulence to Protect Mice against Colitis.

The microbiota and the gastrointestinal mucus layer play a pivotal role in protection against non-typhoidal Salmonella enterica serovar Typhimurium (S. Tm) colitis. Here, we analyzed the course of Salmonella colitis in mice lacking a functional mucus layer in the gut. Unexpectedly, in contrast to mucus-proficient littermates, genetically deficient mice were protected against Salmonella-induced gut inflammation in the streptomycin colitis model. This correlated with microbiota alterations and enrichment of the bacterial phylum Deferribacteres. Using gnotobiotic mice associated with defined bacterial consortia, we causally linked Mucispirillum schaedleri, currently the sole known representative of Deferribacteres present in the mammalian microbiota, to host protection against S. Tm colitis. Inhibition by M. schaedleri involves interference with S. Tm invasion gene expression, partly by competing for anaerobic electron acceptors. In conclusion, this study establishes M. schaedleri, a core member of the murine gut microbiota, as a key antagonist of S. Tm virulence in the gut.

Mechanisms of Salmonella pathogenesis in animal models.

Animal models play an important role in understanding the mechanisms of bacterial pathogenesis. Here we review recent studies of Salmonella infection in various animal models. Although mice are a classic animal model for Salmonella, mice do not normally get diarrhea, raising the question of how well the model represents normal human infection. However, pretreatment of mice with oral streptomycin, which apparently reduces the normal microbiota, leads to an inflammatory diarrheal response upon oral infection with Salmonella. This has led to a re-evaluation of the role of various Salmonella virulence factors in colonization of the intestine and induction of diarrhea. Indeed, it is now clear that Salmonella purposefully induces inflammation, which leads to the production of both carbon sources and terminal electron acceptors by the host that allow Salmonella to outgrow the normal intestinal microbiota. Overall use of this modified mouse model provides a more nuanced understanding of Salmonella intestinal infection in the context of the microbiota with implications for the ability to predict human risk.

A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.

Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.

A fast and robust protocol for metataxonomic analysis using RNAseq data.

BACKGROUND: Metagenomics is a rapidly emerging field aimed to analyze microbial diversity and dynamics by studying the genomic content of the microbiota. Metataxonomics tools analyze high-throughput sequencing data, primarily from 16S rRNA gene sequencing and DNAseq, to identify microorganisms and viruses within a complex mixture. With the growing demand for analysis of the functional microbiome, metatranscriptome studies attract more interest. To make metatranscriptomic data sufficient for metataxonomics, new analytical workflows are needed to deal with sparse and taxonomically less informative sequencing data. RESULTS: We present a new protocol, IMSA+A, for accurate taxonomy classification based on metatranscriptome data of any read length that can efficiently and robustly identify bacteria, fungi, and viruses in the same sample. The new protocol improves accuracy by using a conservative reference database, employing a new counting scheme, and by assembling shotgun reads. Assembly also reduces analysis runtime. Simulated data were utilized to evaluate the protocol by permuting common experimental variables. When applied to the real metatranscriptome data for mouse intestines colonized by ASF, the protocol showed superior performance in detection of the microorganisms compared to the existing metataxonomics tools. IMSA+A is available at https://github.com/JeremyCoxBMI/IMSA-A . CONCLUSIONS: The developed protocol addresses the need for taxonomy classification from RNAseq data. Previously not utilized, i.e., unmapped to a reference genome, RNAseq reads can now be used to gather taxonomic information about the microbiota present in a biological sample without conducting additional sequencing. Any metatranscriptome pipeline that includes assembly of reads can add this analysis with minimal additional cost of compute time. The new protocol also creates an opportunity to revisit old metatranscriptome data, where taxonomic content may be important but was not analyzed.

Helminth co-infection in Helicobacter pylori infected INS-GAS mice attenuates gastric premalignant lesions of epithelial dysplasia and glandular atrophy and preserves colonization resistance of the stomach to lower bowel microbiota.

Higher prevalence of helminth infections in Helicobacter pylori infected children was suggested to potentially lower the life-time risk for gastric adenocarcinoma. In rodent models, helminth co-infection does not reduce Helicobacter-induced inflammation but delays progression of pre-malignant gastric lesions. Because gastric cancer in INS-GAS mice is promoted by intestinal microflora, the impact of Heligmosomoides polygyrus co-infection on H. pylori-associated gastric lesions and microflora were evaluated. Male INS-GAS mice co-infected with H. pylori and H. polygyrus for 5 months were assessed for gastrointestinal lesions, inflammation-related mRNA expression, FoxP3(+) cells, epithelial proliferation, and gastric colonization with H. pylori and Altered Schaedler Flora. Despite similar gastric inflammation and high levels of proinflammatory mRNA, helminth co-infection increased FoxP3(+) cells in the corpus and reduced H. pylori-associated gastric atrophy (p < 0.04), dysplasia (p < 0.02) and prevented H. pylori-induced changes in the gastric flora (p < 0.05). This is the first evidence of helminth infection reducing H. pylori-induced gastric lesions while inhibiting changes in gastric flora, consistent with prior observations that gastric colonization with enteric microbiota accelerated gastric lesions in INS-GAS mice. Identifying how helminths reduce gastric premalignant lesions and impact bacterial colonization of the H. pylori infected stomach could lead to new treatment strategies to inhibit progression from chronic gastritis to cancer in humans.