Jack Arnold’s research while affiliated with University of Chicago and other places

The increasing prevalence of food allergies has been linked to reduced commensal microbial diversity. In this article, we describe two features of allergy-protective Clostridia that contribute to their beneficial effects. Some Clostridial taxa bear flagella (a ligand for TLR5) and produce indole (a ligand for the aryl hydrocarbon receptor [AhR]). Lysates and flagella from a Clostridia consortium induced interleukin-22 (IL-22) secretion from ileal explants. IL-22 production is abrogated in explants from mice in which TLR5 or MyD88 signaling is deficient either globally or conditionally in CD11c⁺ antigen-presenting cells. AhR signaling in RORγt⁺ cells is necessary for the induction of IL-22. Mice deficient in AhR in RORγt⁺ cells exhibit increased intestinal permeability and are more susceptible to an anaphylactic response to food. Our findings implicate TLR5 and AhR signaling in a molecular mechanism by which commensal Clostridia protect against allergic responses to food.

Techniques by which to genetically manipulate members of the microbiota enable both the evaluation of host-microbe interactions and an avenue by which to monitor and modulate human physiology. Genetic engineering applications have traditionally focused on model gut residents, such as Escherichia coli and lactic acid bacteria. However, emerging efforts by which to develop synthetic biology toolsets for "nonmodel" resident gut microbes could provide an improved foundation for microbiome engineering. As genome engineering tools come online, so too have novel applications for engineered gut microbes. Engineered resident gut bacteria facilitate investigations of the roles of microbes and their metabolites on host health and allow for potential live microbial biotherapeutics. Due to the rapid pace of discovery in this burgeoning field, this minireview highlights advancements in the genetic engineering of all resident gut microbes.

Perturbations to the commensal gut microbiota are associated with the increasing prevalence of food allergies. Our lab has reported that colonization of germ-free mice with feces from healthy, but not cow’s milk allergic, infants protects against anaphylaxis to a cow’s milk allergen. Protection was associated with higher levels of the secondary bile acid (SBA) isodeoxycholic acid (isoDCA) in the feces of healthy-colonized mice. IsoDCA has been reported to induce regulatory T cells (Tregs) which suppress inflammation. We hypothesize that isoDCA-driven induction of mucosal Tregs promotes non-responsiveness and protects against food allergy. To study this, we identified two Clostridiaspecies, Peptacetobacter hiranonisand Ruminococcus gnavus, that together produce isoDCA from taurocholic acid (TCA), an abundant host bile acid. Using a Group II intron-based system, we engineered R. gnavus Δrumgna_00694, a functional knock-out of a 3β-hydroxysteroid dehydrogenase required to produce isoDCA. In our system, P. hiranonisconverts TCA to deoxycholic acid (DCA) and R. gnavusWT, but not Δrumgna_00694, converts DCA to isoDCA. Ongoing experiments will compare isoDCA-dependent Treg induction in gut-associated lymphoid tissues of mice co-colonized with P. hiranonisand R. gnavusWT or Δrumgna_00694. Once optimized, this system can be used in our allergic sensitization model to investigate the role of SBA in protection against food allergy and maintenance of intestinal homeostasis.

It is well known that accumulated changes in microbiota composition over several decades have contributed to an increase in non-communicable chronic diseases. While much effort has been devoted toward achieving a consensus on the microbes, and their products, driving this increase, inconsistent results suggest more research is required to understand the disparity in observed outcomes. Previous work from our lab identified a consortium of commensal Clostridia that alone was sufficient to preserve intestinal barrier integrity and prevent sensitization to food allergens in mice by inducing IL-22. Further investigation determined that members of this consortium display flagella. When isolated, these commensal flagella (Fla-C) exhibited TLR5-dependent IL-22 induction at a magnitude comparable to flagellin from SalmonellaTyphimurium (Fla-ST). However, mice treated with Fla-C maintained intestinal barrier function significantly more effectively than Fla-ST or negative control PBS-treated mice as measured by a serum FITC-dextran permeability assay. This was accompanied by a striking difference in the expression of Reg3 antimicrobial peptides and IL-17; all of which were significantly increased by treatment with Fla-ST when compared to Fla-C. Sequencing of Clostridia consortium-derived flagellated isolates revealed flagellin genes with unique hypervariable regions when compared to fliC from S. Typhimurium. These results highlight the distinct impacts of commensal and pathogenic flagella and suggest an important role for flagellated commensal bacteria in the maintenance of intestinal epithelial barrier function. Supported by a grant from National Institutes of Health (RO1 AI06302)

Lifestyle-induced changes to the diversity of the commensal microbiota have been causally linked to the increasing prevalence of food allergies and other non-communicable chronic diseases. We have shown that bacteria from the Clostridia class prevent an allergic response to food by eliciting an IL-22 dependent barrier protective response that limits allergen access to the systemic circulation. We have now examined the mechanisms by which commensal Clostridia induce this allergy-protective effect. We identified taxa in a consortium of Clostridia that possess flagella and produce indole, which are ligands for TLR5 and AhR, respectively. Lysates and flagella isolated from this consortium induced IL-22 in mouse intestinal explants. IL-22 was not induced in explants from mice in which TLR5 or MyD88 was knocked out globally or conditionally in CD11c+ cells. Treatment with the commensal flagellar isolate also reduced detection of intragastrically administered FITC dextran in the serum of antibiotic-treated mice. Similarly, indole exposure induced IL-22 in intestinal explants and reduced intestinal permeability to FITC dextran. Importantly, AhR signaling in RORγt+ cells was necessary for IL-22 induction by flagella. These results suggest that flagella and indole act synergistically to prevent an allergic response. Finally, we have isolated and characterized two Clostridial taxa which bear flagella and produce indole. We hypothesize that germ-free mice colonized with these two taxa will exhibit improved IL-22 dependent barrier function and be protected against an allergic response. Our work reveals novel features of Clostridia key to their allergy-protective capability which may be further exploited to develop therapeutics. Supported by NIH AI106302 and the Bunning Professorship Endowment Fund

The human microbiome has been inextricably linked to multiple facets of human physiology. From an engineering standpoint, the ability to precisely control the composition and activity of the microbiome holds great promise for furthering our understanding of disease etiology and for new avenues of therapeutic and diagnostic agents. While the field of microbiome research is still in its infancy, growing engineering efforts are emerging to enable new studies in the microbiome and to rapidly translate these findings to microbiome‐based interventions. At the 3rd International Conference on Microbiome Engineering, leading experts in the field presented state‐of‐the‐art work in microbiome engineering, discussing probiotics, prebiotics, engineered microbes, microbially derived biomolecules, and bacteriophage.

We previously reported that a consortium of bacteria from the Clostridia class modulates intestinal barrier function through induction of IL-22 and prevents allergic sensitization to food in mice. We further investigated the properties of these bacteria and the mechanisms that underly their protective impact. Shotgun metagenomic sequencing of the anaerobically cultured Clostridia consortium identified genes which suggest the presence of flagellated bacteria. Motility was then confirmed using agar-based assays. Lysates generated from the consortium stimulated ileal tissue to produce IL-23 and IL-22 in vitro in a MyD88 and TLR5-dependent manner, specifically MyD88 signaling in CD11c+ cells. Together, these experiments support the presence of flagellin in our consortium. Flagellin is a TLR5 ligand that induces IL-23 production by CD11c+ dendritic cells, which stimulates IL-22 production in RORγt+ innate lymphoid cells (ILC3). In addition, we found that the Clostridia consortium produced indoles, known ligands for the aryl hydrocarbon receptor (AhR). In vitro stimulation of ileal tissue with indoles induced IL-22 production, and oral administration to antibiotic treated mice improved intestinal barrier function. Lastly, we observed impaired intestinal IL-22 production and barrier function, and exacerbated food allergy in mice that lack AhR signaling in RORγt+ cells such as ILC3, confirming the relevance of AhR signaling in our food allergy model. Production of flagellin and indoles are two potentially beneficial aspects of allergy-protective commensal bacteria and our knowledge of these pathways and metabolites will allow us to develop targeted therapeutics for the prevention or treatment of food allergy.