Bacteroides ovatus Promotes IL-22 Production and Reduces Trinitrobenzene Sulfonic Acid-Driven Colonic Inflammation.

The intestinal microbiota influences the development and function of the mucosal immune system. However, the exact mechanisms by which commensal microbes modulate immunity is not clear. We previously demonstrated that commensal Bacteroides ovatus ATCC 8384 reduces mucosal inflammation. Herein, we aimed to identify immunomodulatory pathways employed by B. ovatus. In germ-free mice, mono-association with B. ovatus shifted the CD11b+/CD11c+ and CD103+/CD11c+ dendritic cell populations. Because indole compounds are known to modulate dendritic cells, B. ovatus cell-free supernatant was screened for tryptophan metabolites by liquid chromatography-tandem mass spectrometry and larger quantities of indole-3-acetic acid were detected. Analysis of cecal and fecal samples from germ-free and B. ovatus mono-associated mice confirmed that B. ovatus could elevate indole-3-acetic acid concentrations in vivo. Indole metabolites have previously been shown to stimulate immune cells to secrete the reparative cytokine IL-22. Addition of B. ovatus cell-free supernatant to immature bone marrow-derived dendritic cells stimulated IL-22 secretion. The ability of IL-22 to drive repair in the intestinal epithelium was confirmed using a physiologically relevant human intestinal enteroid model. Finally, B. ovatus shifted the immune cell populations in trinitrobenzene sulfonic acid-treated mice and up-regulated colonic IL-22 expression, effects that correlated with decreased inflammation. Our data suggest that B. ovatus-produced indole-3-acetic acid promotes IL-22 production by immune cells, yielding beneficial effects on colitis.

Select Streptococci Can Degrade Candida Mannan To Facilitate Growth.

Multiple studies have found that streptococci have a synergistic relationship with Candida species, but the details of these interactions are still being discovered. Candida species are covered by mannan, a polymer of mannose, which could serve as a carbon source for certain microbes. We hypothesized that streptococci that possess mannan-degrading glycosyl hydrolases would be able to enzymatically cleave mannose residues, which could serve as a primary carbohydrate source to support growth. We analyzed 90 streptococcus genomes to predict the capability of streptococci to transport and utilize mannose and to degrade diverse mannose linkages found on mannan. The genome analysis revealed mannose transporters and downstream pathways in most streptococci, but only <50% of streptococci harbored the glycosyl hydrolases required for mannan degradation. To confirm the ability of streptococci to use mannose or mannan, we grew 6 representative streptococci in a chemically defined medium lacking glucose supplemented with mannose, yeast extract, or purified mannan isolated from Candida and Saccharomyces strains. Although all tested Streptococcus strains could use mannose, Streptococcus salivarius and Streptococcus agalactiae, which did not possess mannan-degrading glycosyl hydrolases, could not use yeast extract or mannan to enhance their growth. In contrast, we found that Streptococcus mitis, Streptococcus parasanguinis, Streptococcus sanguinis, and Streptococcus pyogenes possessed the necessary glycosyl hydrolases to use yeast extract and isolated mannan, which promoted robust growth. Our data indicate that several streptococci are capable of degrading fungal mannans and harvesting mannose for energy. IMPORTANCE This work highlights a previously undescribed aspect of streptococcal Candida interactions. Our work identifies that certain streptococci possess the enzymes required to degrade mannan, and through this mechanism, they can release mannose residues from the cell wall of fungal species and use them as a nutrient source. We speculate that streptococci that can degrade fungal mannan may have a competitive advantage for colonization. This finding has broad implications for human health, as streptococci and Candida are found at multiple body sites.

Distinct roles of histamine H1- and H2-receptor signaling pathways in inflammation-associated colonic tumorigenesis.

Inflammatory bowel disease (IBD) is a well-known risk factor for the development of colorectal cancer. Prior studies have demonstrated that microbial histamine can ameliorate intestinal inflammation in mice. We tested the hypothesis whether microbe-derived luminal histamine suppresses inflammation-associated colon cancer in Apcmin/+ mice. Mice were colonized with the human-derived Lactobacillus reuteri. Chronic inflammation was induced by repeated cycles of low-dose dextran sulfate sodium (DSS). Mice that were given histamine-producing L. reuteri via oral gavage developed fewer colonic tumors, despite the presence of a complex mouse gut microbiome. We further demonstrated that administration of a histamine H1-receptor (H1R) antagonist suppressed tumorigenesis, while administration of histamine H2-receptor (H2R) antagonist significantly increased both tumor number and size. The bimodal functions of histamine include protumorigenic effects through H1R and antitumorigenic effects via H2R, and these results were supported by gene expression profiling studies on tumor specimens of patients with colorectal cancer. Greater ratios of gene expression of H2R ( HRH2) vs. H1R ( HRH1) were correlated with improved overall survival outcomes in patients with colorectal cancer. Additionally, activation of H2R suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibited chemokine gene expression induced by H1R activation in colorectal cancer cells. Moreover, the combination of a H1R antagonist and a H2R agonist yielded potent suppression of lipopolysaccharide-induced MAPK signaling in macrophages. Given the impact on intestinal epithelial and immune cells, simultaneous modulation of H1R and H2R signaling pathways may be a promising therapeutic target for the prevention and treatment of inflammation-associated colorectal cancer. NEW & NOTEWORTHY Histamine-producing Lactobacillus reuteri can suppress development of inflammation-associated colon cancer in an established mouse model. The net effects of histamine may depend on the relative activity of H1R and H2R signaling pathways in the intestinal mucosa. Our findings suggest that treatment with H1R or H2R antagonists could yield opposite effects. However, by harnessing the ability to block H1R signaling while stimulating H2R signaling, novel strategies for suppression of intestinal inflammation and colorectal neoplasia could be developed.

Gut Microbe-Mediated Suppression of Inflammation-Associated Colon Carcinogenesis by Luminal Histamine Production.

Microbiome-mediated suppression of carcinogenesis may open new avenues for identification of therapeutic targets and prevention strategies in oncology. Histidine decarboxylase (HDC) deficiency has been shown to promote inflammation-associated colorectal cancer by accumulation of CD11b+Gr-1+ immature myeloid cells, indicating a potential antitumorigenic effect of histamine. Here, we demonstrate that administration of hdc+Lactobacillus reuteri in the gut resulted in luminal hdc gene expression and histamine production in the intestines of Hdc-/- mice. This histamine-producing probiotic decreased the number and size of colon tumors and colonic uptake of [18F]-fluorodeoxyglucose by positron emission tomography in Hdc-/- mice. Administration of L. reuteri suppressed keratinocyte chemoattractant (KC), Il22, Il6, Tnf, and IL1α gene expression in the colonic mucosa and reduced the amounts of proinflammatory, cancer-associated cytokines, keratinocyte chemoattractant, IL-22, and IL-6, in plasma. Histamine-generating L. reuteri also decreased the relative numbers of splenic CD11b+Gr-1+ immature myeloid cells. Furthermore, an isogenic HDC-deficient L. reuteri mutant that was unable to generate histamine did not suppress carcinogenesis, indicating a significant role of the cometabolite, histamine, in suppression of chronic intestinal inflammation and colorectal tumorigenesis. These findings link luminal conversion of amino acids to biogenic amines by gut microbes and probiotic-mediated suppression of colorectal neoplasia.

Monospecies probiotic preparation and administration with downstream analysis of sex-specific effects on gut microbiome composition in mice.

Dysbiosis of the gut microbiome is implicated in the growing burden of non-communicable chronic diseases, including neurodevelopmental disorders, and both preclinical and clinical studies highlight the potential for precision probiotic therapies in their prevention and treatment. Here, we present an optimized protocol for the preparation and administration of Limosilactobacillus reuteri MM4-1A (ATCC-PTA-6475) to adolescent mice. We also describe steps for performing downstream analysis of metataxonomic sequencing data with careful assessment of sex-specific effects on microbiome composition and structure. For complete details on the use and execution of this protocol, please refer to Di Gesù et al.1.

A Lesson from the Pandemic: Utilizing Digital Tools To Support Student Engagement during Instructional Assistant-Led Sessions.

Student instructional assistants (IAs) are an integral part of most students' college experience in higher education. When properly trained, IAs can improve students' grades, engagement with course content, persistence, and retention. Recently, the COVID-19 pandemic forced the transition of nearly all instructional practices online. At the University of Alabama at Birmingham, IAs, including Biology Learning Assistants (BLAs), began hosting their instructional sessions virtually, outside of class time. The goals of these sessions were to reinforce fundamental concepts using active learning strategies and to address student questions by building a supportive learning community. In this article, we summarize the training and guidance we provided to the BLAs regarding how best to adapt digital educational tools to engage students during their virtual sessions. We recommend that institutions of higher education recognize the expansion of digital educational tools as an opportunity to increase the technological literacy and competence of their IAs to best serve their student body in this increasingly digital age of education.

Loss of H2R Signaling Disrupts Neutrophil Homeostasis and Promotes Inflammation-Associated Colonic Tumorigenesis in Mice.

BACKGROUND & AIMS: We previously showed that histamine suppressed inflammation-associated colonic tumorigenesis through histamine type 2 receptor (H2R) signaling in mice. This study aimed to precisely elucidate the downstream effects of H2R activation in innate immune cells. METHODS: Analyses using online databases of single-cell RNA sequencing of intestinal epithelial cells in mice and RNA sequencing of mouse immune cells were performed to determine the relative abundances of 4 histamine receptors among different cell types. Mouse neutrophils, which expressed greater amounts of H2R, were collected from the peritoneum of wild-type and H2R-deficient mice, of which low-density and high-density neutrophils were extracted by centrifugation and were subjected to RNA sequencing. The effects of H2R activation on neutrophil differentiation and its functions in colitis and inflammation-associated colon tumors were investigated in a mouse model of dextran sulfate sodium-induced colitis. RESULTS: Data analysis of RNA sequencing and quantitative reverse-transcription polymerase chain reaction showed that Hrh2 is highly expressed in neutrophils, but barely detectable in intestinal epithelial cells. In mice, the absence of H2R activation promoted infiltration of neutrophils into both sites of inflammation and colonic tumors. H2R-deficient high-density neutrophils yielded proinflammatory features via nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and suppressed T-cell proliferation. On the other hand, low-density neutrophils, which totally lack H2R activation, showed an immature phenotype compared with wild-type low-density neutrophils, with enhanced MYC pathway signaling and reduced expression of the maturation marker Toll-like receptor 4. CONCLUSIONS: Blocking H2R signaling enhanced proinflammatory responses of mature neutrophils and suppressed neutrophil maturation, leading to accelerated progression of inflammation-associated colonic tumorigenesis.

Bacteroides ovatus colonization influences the abundance of intestinal short chain fatty acids and neurotransmitters.

Gut microbes can synthesize multiple neuro-active metabolites. We profiled neuro-active compounds produced by the gut commensal Bacteroides ovatus in vitro and in vivo by LC-MS/MS. We found that B. ovatus generates acetic acid, propionic acid, isobutyric acid, and isovaleric acid. In vitro, B. ovatus consumed tryptophan and glutamate and synthesized the neuro-active compounds glutamine and GABA. Consistent with our LC-MS/MS-based in vitro data, we observed elevated levels of acetic acid, propionic acid, isobutyric acid, and isovaleric acid in the intestines of B. ovatus mono-associated mice compared with germ-free controls. B. ovatus mono-association also increased the concentrations of intestinal GABA and decreased the concentrations of tryptophan and glutamine compared with germ-free controls. Computational network analysis revealed unique links between SCFAs, neuro-active compounds, and colonization status. These results highlight connections between microbial colonization and intestinal neurotransmitter concentrations, suggesting that B. ovatus selectively influences the presence of intestinal neurotransmitters.

Maternal gut microbiota mediate intergenerational effects of high-fat diet on descendant social behavior.

Dysbiosis of the maternal gut microbiome during pregnancy is associated with adverse neurodevelopmental outcomes. We previously showed that maternal high-fat diet (MHFD) in mice induces gut dysbiosis, social dysfunction, and underlying synaptic plasticity deficits in male offspring (F1). Here, we reason that, if HFD-mediated changes in maternal gut microbiota drive offspring social deficits, then MHFD-induced dysbiosis in F1 female MHFD offspring would likewise impair F2 social behavior. Metataxonomic sequencing reveals reduced microbial richness among female F1 MHFD offspring. Despite recovery of microbial richness among MHFD-descendant F2 mice, they display social dysfunction. Post-weaning Limosilactobacillus reuteri treatment increases the abundance of short-chain fatty acid-producing taxa and rescues MHFD-descendant F2 social deficits. L. reuteri exerts a sexually dimorphic impact on gut microbiota configuration, increasing discriminant taxa between female cohorts. Collectively, these results show multigenerational impacts of HFD-induced dysbiosis in the maternal lineage and highlight the potential of maternal microbiome-targeted interventions for neurodevelopmental disorders.

Immunomodulation of dendritic cells by Lactobacillus reuteri surface components and metabolites.

BACKGROUND: Lactic acid bacteria are commensal members of the gut microbiota and are postulated to promote host health. Secreted factors and cell surface components from Lactobacillus species have been shown to modulate the host immune system. However, the precise role of L. reuteri secreted factors and surface proteins in influencing dendritic cells (DCs) remains uncharacterized. HYPOTHESIS: We hypothesize that L. reuteri secreted factors will promote DC maturation, skewing cells toward an anti-inflammatory phenotype. In acute colitis, we speculate that L. reuteri promotes IL-10 and dampens pro-inflammatory cytokine production, thereby improving colitis. METHODS & RESULTS: Mouse bone marrow-derived DCs were differentiated into immature dendritic cells (iDCs) via IL-4 and GM-CSF stimulation. iDCs exposed to L. reuteri secreted factors or UV-irradiated bacteria exhibited greater expression of DC maturation markers CD83 and CD86 by flow cytometry. Additionally, L. reuteri stimulated DCs exhibited phenotypic maturation as denoted by cytokine production, including anti-inflammatory IL-10. Using mouse colonic organoids, we found that the microinjection of L. reuteri secreted metabolites and UV-irradiated bacteria was able to promote IL-10 production by DCs, indicating potential epithelial-immune cross-talk. In a TNBS-model of acute colitis, L. reuteri administration significantly improved histological scoring, colonic cytokine mRNA, serum cytokines, and bolstered IL-10 production. CONCLUSIONS: Overall these data demonstrate that both L. reuteri secreted factors and its bacterial components are able to promote DC maturation. This work points to the specific role of L. reuteri in modulating intestinal DCs. NEW & NOTEWORTHY: Lactobacillus reuteri colonizes the mammalian gastrointestinal tract and exerts beneficial effects on host health. However, the mechanisms behind these effects have not been fully explored. In this article, we identified that L. reuteri ATTC PTA 6475 metabolites and surface components promote dendritic cell maturation and IL-10 production. In acute colitis, we also demonstrate that L. reuteri can promote IL-10 and suppress inflammation. These findings may represent a crucial mechanism for maintaining intestinal immune homeostasis.

Unraveling the Metabolic Requirements of the Gut Commensal Bacteroides ovatus.

Background: Bacteroidetes are the most common bacterial phylum in the mammalian intestine and the effects of several Bacteroides spp. on multiple facets of host physiology have been previously described. Of the Bacteroides spp., Bacteroides ovatus has recently garnered attention due to its beneficial effects in the context of intestinal inflammation. In this study, we aimed to examine model host intestinal physiological conditions and dietary modifications to characterize their effects on B. ovatus growth. Methods and Results: Using Biolog phenotypic microarrays, we evaluated 62 primary carbon sources and determined that B. ovatus ATCC 8384 can use the following carbohydrates as primary carbon sources: 10 disaccharides, 4 trisaccharides, 4 polysaccharides, 4 polymers, 3 L-linked sugars, 6 D-linked sugars, 5 amino-sugars, 6 alcohol sugars, and 15 organic acids. Proteomic profiling of B. ovatus bacteria revealed that a significant portion of the B. ovatus proteome contains proteins important for metabolism. Among the proteins, we found glycosyl hydrolase (GH) familes GH2, GH5, GH20, GH 43, GH88, GH92, and GH95. We also identified multiple proteins with antioxidant properties and reasoned that these proteins may support B. ovatus growth in the GI tract. Upon further testing, we showed that B. ovatus grew robustly in various pH, osmolarity, bile, ethanol, and H2O2 concentrations; indicating that B. ovatus is a well-adapted gut microbe. Conclusion: Taken together, we have demonstrated that key host and diet-derived changes in the intestinal environment influence B. ovatus growth. These data provide the framework for future work toward understanding how diet and lifestyle interventions may promote a beneficial environment for B. ovatus growth.

Phagocytosis by macrophages depends on histamine H2 receptor signaling and scavenger receptor 1.

The histamine H2 receptor (H2R) is a G protein-coupled receptor that mediates cyclic AMP production, protein kinase A activation, and MAP kinase signaling. In order to explore the multifaceted effects of histamine signaling on immune cells, phagocytosis was evaluated using primary mouse-derived macrophages. Phagocytosis is initiated by signaling via surface-bound scavenger receptors and can be regulated by autophagy. Absence of H2R signaling resulted in diminished phagocytosis of live bacteria and synthetic microspheres by primary macrophages from histamine H2 receptor gene (Hrh2)-deficient mice. Flow cytometry and immunofluorescence microscopy were used to quantify phagocytosis of phylogenetically diverse bacteria as well as microspheres of defined chemical composition. Autophagy and scavenger receptor gene expression were quantified in macrophages after exposure to Escherichia coli. Expression of the autophagy genes, Becn1 and Atg12, was increased in Hrh2-/- macrophages, indicating upregulation of autophagy pathways. Expression of the Macrophage Scavenger Receptor 1 gene (Msr1) was diminished in Hrh2-deficient macrophages, supporting the possible importance of histamine signaling in scavenger receptor abundance and macrophage function. Flow cytometry confirmed diminished MSR1 surface abundance in Hrh2-/- macrophages. These data suggest that H2R signaling is required for effective phagocytosis by regulating the process of autophagy and scavenger receptor MSR1 abundance in macrophages.

Microbial interactions with the intestinal epithelium and beyond: Focusing on immune cell maturation and homeostasis.

Microbial metabolites influence the function of epithelial, endothelial and immune cells in the intestinal mucosa. Microbial metabolites like SCFAs and B complex vitamins direct macrophage polarization whereas microbial derived biogenic amines modulate intestinal epithelium and immune response. Aberrant bacterial lipopolysaccharide-mediated signaling may be involved in the pathogenesis of chronic intestinal inflammation and colorectal carcinogenesis. Our perception of human microbes has changed from that of opportunistic pathogens to active participants maintaining intestinal and whole body homeostasis. This review attempts to explain the dynamic and enriched interactions between the intestinal epithelial mucosa and commensal bacteria in homeostasis maintenance.