Aryl Hydrocarbon Receptor Signaling Cell Intrinsically Inhibits Intestinal Group 2 Innate Lymphoid Cell Function.

Innate lymphoid cells (ILCs) are important for mucosal immunity. The intestine harbors all ILC subsets, but how these cells are balanced to achieve immune homeostasis and mount appropriate responses during infection remains elusive. Here, we show that aryl hydrocarbon receptor (Ahr) expression in the gut regulates ILC balance. Among ILCs, Ahr is most highly expressed by gut ILC2s and controls chromatin accessibility at the Ahr locus via positive feedback. Ahr signaling suppresses Gfi1 transcription-factor-mediated expression of the interleukin-33 (IL-33) receptor ST2 in ILC2s and expression of ILC2 effector molecules IL-5, IL-13, and amphiregulin in a cell-intrinsic manner. Ablation of Ahr enhances anti-helminth immunity in the gut, whereas genetic or pharmacological activation of Ahr suppresses ILC2 function but enhances ILC3 maintenance to protect the host from Citrobacter rodentium infection. Thus, the host regulates the gut ILC2-ILC3 balance by engaging the Ahr pathway to mount appropriate immunity against various pathogens.

Ikaros Inhibits Group 3 Innate Lymphoid Cell Development and Function by Suppressing the Aryl Hydrocarbon Receptor Pathway.

Group 3 innate lymphoid cells (ILC3s) expressing the transcription factor (TF) RORγt are important for the defense and homeostasis of host intestinal tissues. The zinc finger TF Ikaros, encoded by Ikzf1, is essential for the development of RORγt(+) fetal lymphoid tissue inducer (LTi) cells and lymphoid organogenesis, but its role in postnatal ILC3s is unknown. Here, we show that small-intestinal ILC3s had lower Ikaros expression than ILC precursors and other ILC subsets. Ikaros inhibited ILC3s in a cell-intrinsic manner through zinc-finger-dependent inhibition of transcriptional activity of the aryl hydrocarbon receptor, a key regulator of ILC3 maintenance and function. Ablation of Ikzf1 in RORγt(+) ILC3s resulted in increased expansion and cytokine production of intestinal ILC3s and protection against infection and colitis. Therefore, in contrast to being required for LTi development, Ikaros inhibits postnatal ILC3 development and function to regulate gut immune responses at steady state and in disease.

Integrated Multi-Cohort Analysis of the Parkinson's Disease Gut Metagenome.

BACKGROUND: The gut microbiome is altered in several neurologic disorders, including Parkinson's disease (PD). OBJECTIVES: The aim is to profile the fecal gut metagenome in PD for alterations in microbial composition, taxon abundance, metabolic pathways, and microbial gene products, and their relationship with disease progression. METHODS: Shotgun metagenomic sequencing was conducted on 244 stool donors from two independent cohorts in the United States, including individuals with PD (n = 48, n = 47, respectively), environmental household controls (HC, n = 29, n = 30), and community population controls (PC, n = 41, n = 49). Microbial features consistently altered in PD compared to HC and PC subjects were identified. Data were cross-referenced to public metagenomic data sets from two previous studies in Germany and China to determine generalizable microbiome features. RESULTS: We find several significantly altered taxa between PD and controls within the two cohorts sequenced in this study. Analysis across global cohorts returns consistent changes only in Intestinimonas butyriciproducens. Pathway enrichment analysis reveals disruptions in microbial carbohydrate and lipid metabolism and increased amino acid and nucleotide metabolism in PD. Global gene-level signatures indicate an increased response to oxidative stress, decreased cellular growth and microbial motility, and disrupted intercommunity signaling. CONCLUSIONS: A metagenomic meta-analysis of PD shows consistent and novel alterations in functional metabolic potential and microbial gene abundance across four independent studies from three continents. These data reveal that stereotypic changes in the functional potential of the gut microbiome are a consistent feature of PD, highlighting potential diagnostic and therapeutic avenues for future research. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Dichotomous regulation of group 3 innate lymphoid cells by nongastric Helicobacter species.

Intestinal innate lymphoid cells (ILCs) contribute to the protective immunity and homeostasis of the gut, and the microbiota are critically involved in shaping ILC function. However, the role of the gut microbiota in regulating ILC development and maintenance still remains elusive. Here, we identified opposing effects on ILCs by two Helicobacter species, Helicobacter apodemus and Helicobacter typhlonius, isolated from immunocompromised mice. We demonstrated that the introduction of both Helicobacter species activated ILCs and induced gut inflammation; however, these Helicobacter species negatively regulated RORγt+ group 3 ILCs (ILC3s), especially T-bet+ ILC3s, and diminished their proliferative capacity. Thus, these findings underscore a previously unknown dichotomous regulation of ILC3s by Helicobacter species, and may serve as a model for further investigations to elucidate the host-microbe interactions that critically sustain the maintenance of intestinal ILC3s.

Innate lymphoid cells in intestinal immunity and inflammation.

Innate lymphoid cells (ILCs) are a new and distinct family of innate immune cells that play an important role in immunity and inflammation. In this review, we focus on the role of ILCs in mucosal tissues, especially in the gut, in health and disease. ILCs support intestinal homeostasis by protecting the intestine from pathogens, contributing to the development of gut lymphoid tissue, and helping to repair injuries. By cooperating with epithelial cells and other innate and adaptive immune cells, ILCs participate in the control of pathogens and tolerance of commensal bacteria. The development and maintenance of ILCs are influenced by nutrients and metabolites sourced from diet and/or gut bacteria. ILCs have been shown to be involved in host metabolism and to participate in various diseases of the intestine including infectious and chronic inflammatory diseases, and cancer. Thus, the elucidation of ILC biology provides an exciting potential for development of novel therapeutic means to modulate immune responses in various disease settings.

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 the deposition and/or clearance of amyloid-beta (Aß) 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 if inflammation in female mice. 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 female mice. DISCUSSION: These data reveal sexual dimorphism in early signs of inflammation and the effects of the microbiome, and highlight a previously unrecognized interaction between sex and the microbiome in mouse models of AD. Research in Context: Systemic review: We reviewed the literature related to Alzheimer's disease (AD), inflammation, and the microbiome using PubMed. We cite several studies that demonstrate the influence of the microbiome on inflammation and cognitive performance in both animal models and humans. However, the mechanisms linking immunity to AD are not well understood. Interpretation: Using two well-established mouse models of AD, we found that the microbiome does not strongly influence the onset of inflammation in brain-draining lymph nodes; rather, it largely modulates systemic immune responses, local cytokine production, and cognitive performance. Notably, the inflammatory state in mice was affected by sex, and this sex effect differed between local and systemic tissues and mice with or without a microbiome. Future directions: Our work identified a sex- and microbiome-mediated effect on inflammation and cognitive performance. Future studies may focus on microbiome-dependent mechanisms that intersect with sex hormone and immune responses to determine peripheral effects on AD outcomes. Highlights: Adaptive immunity is activated at early ages and differentially by sex in mouse models of AD.Inflammation in 5xFAD mice is characterized by increased IL-17A-producing T cells.Inflammation in 3xTg mice is characterized by increased cytokine responses in males, but attenuated cytokine responses in female mice.Longitudinal immune responses differ between 3xTg mice and 5xFAD mice.Both 3xTg and 5xFAD female mice show improved learning and cognition in the absence of a microbiome.

Gut microbiome-mediated regulation of neuroinflammation.

The intestinal microbiome influences neuroinflammatory disease in animal models, and recent studies have identified multiple pathways of communication between the gut and brain. Microbes are able to produce metabolites that enter circulation, can alter inflammatory tone in the intestines, periphery, and central nervous system (CNS), and affect trafficking of immune cells into the brain. Additionally, the vagus nerve that connects the enteric nervous system to the CNS is implicated in modulation of brain immune responses. As preclinical research findings and concepts are applied to humans, the potential impacts of the gut microbiome-brain axis on neuroinflammation represent exciting frontiers for further investigation.

A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice.

Parkinson's disease (PD) is a movement disorder characterized by neuroinflammation, α-synuclein pathology, and neurodegeneration. Most cases of PD are non-hereditary, suggesting a strong role for environmental factors, and it has been speculated that disease may originate in peripheral tissues such as the gastrointestinal (GI) tract before affecting the brain. The gut microbiome is altered in PD and may impact motor and GI symptoms as indicated by animal studies, although mechanisms of gut-brain interactions remain incompletely defined. Intestinal bacteria ferment dietary fibers into short-chain fatty acids, with fecal levels of these molecules differing between PD and healthy controls and in mouse models. Among other effects, dietary microbial metabolites can modulate activation of microglia, brain-resident immune cells implicated in PD. We therefore investigated whether a fiber-rich diet influences microglial function in α-synuclein overexpressing (ASO) mice, a preclinical model with PD-like symptoms and pathology. Feeding a prebiotic high-fiber diet attenuates motor deficits and reduces α-synuclein aggregation in the substantia nigra of mice. Concomitantly, the gut microbiome of ASO mice adopts a profile correlated with health upon prebiotic treatment, which also reduces microglial activation. Single-cell RNA-seq analysis of microglia from the substantia nigra and striatum uncovers increased pro-inflammatory signaling and reduced homeostatic responses in ASO mice compared to wild-type counterparts on standard diets. However, prebiotic feeding reverses pathogenic microglial states in ASO mice and promotes expansion of protective disease-associated macrophage (DAM) subsets of microglia. Notably, depletion of microglia using a CSF1R inhibitor eliminates the beneficial effects of prebiotics by restoring motor deficits to ASO mice despite feeding a prebiotic diet. These studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.

Ahr-Foxp3-RORγt axis controls gut homing of CD4+ T cells by regulating GPR15.

The orphan chemoattractant receptor GPR15 is important for homing T lymphocytes to the large intestine, thereby maintaining intestinal immune homeostasis. However, the molecular mechanisms underlying the regulation of GPR15 expression remain elusive. Here, we show a central role of the aryl hydrocarbon receptor (Ahr) in promoting GPR15 expression in both mice and human, thus gut homing of T lymphocytes. Mechanistically, Ahr directly binds to open chromatin regions of the Gpr15 locus to enhance its expression. Ahr transcriptional activity in directing GPR15 expression was modulated by two transcription factors, Foxp3 and RORγt, both of which are expressed preferentially by gut regulatory T cells (Tregs) in vivo. Specifically, Foxp3 interacted with Ahr and enhanced Ahr DNA binding at the Gpr15 locus, thereby promoting GPR15 expression. In contrast, RORγt plays an inhibitory role, at least in part, by competing with Ahr binding to the Gpr15 locus. Our findings thus demonstrate a key role for Ahr in regulating Treg intestinal homing under the steady state and during inflammation and the importance of Ahr-RORγt-Foxp3 axis in regulating gut homing receptor GPR15 expression by lymphocytes.

Requirement of Mitochondrial Transcription Factor A in Tissue-Resident Regulatory T Cell Maintenance and Function.

Regulatory T cells (Tregs) are pivotal for immune suppression. Cellular metabolism is important for Treg homeostasis and function. However, the exact role of mitochondrial respiration in Tregs remains elusive. Mitochondrial transcription factor A (Tfam) is essential for mitochondrial respiration and controls mitochondrial DNA replication, transcription, and packaging. Here, we show that genetic ablation of Tfam in Tregs impairs Treg maintenance in non-lymphoid tissues in the steady state and in tumors. Tfam-deficient Tregs have reduced proliferation and Foxp3 expression upon glucose deprivation in vitro. Tfam deficiency preferentially affects gene activation in Tregs through regulation of DNA methylation, with enhanced methylation in the TSDR of the Foxp3 locus. Deletion of Tfam in Tregs affects Treg homing and stability, resulting in tissue inflammation in colitis, but enhances tumor rejection. Thus, our work reveals a critical role of Tfam-mediated mitochondrial respiration in Tregs to regulate inflammation and anti-tumor immunity.

Detection of a Peptide Biomarker by Engineered Yeast Receptors.

Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.

Regulation of Innate Lymphoid Cells by Aryl Hydrocarbon Receptor.

With striking similarity to their adaptive T helper cell counterparts, innate lymphoid cells (ILCs) represent an emerging family of cell types that express signature transcription factors, including T-bet+ Eomes+ natural killer cells, T-bet+ Eomes- group 1 ILCs, GATA3+ group 2 ILCs, RORγt+ group 3 ILCs, and newly identified Id3+ regulatory ILC. ILCs are abundantly present in barrier tissues of the host (e.g., the lung, gut, and skin) at the interface of host-environment interactions. Active research has been conducted to elucidate molecular mechanisms underlying the development and function of ILCs. The aryl hydrocarbon receptor (Ahr) is a ligand-dependent transcription factor, best known to mediate the effects of xenobiotic environmental toxins and endogenous microbial and dietary metabolites. Here, we review recent progresses regarding Ahr function in ILCs. We focus on the Ahr-mediated cross talk between ILCs and other immune/non-immune cells in host tissues especially in the gut. We discuss the molecular mechanisms of the action of Ahr expression and activity in regulation of ILCs in immunity and inflammation, and the interaction between Ahr and other pathways/transcription factors in ILC development and function with their implication in disease.

The Aryl Hydrocarbon Receptor Preferentially Marks and Promotes Gut Regulatory T Cells.

The local environment may affect the development and function of tissue-resident T regulatory cells (Tregs), which are crucial for controlling inflammation. Although the aryl hydrocarbon receptor (Ahr), an environmental sensor, is expressed by Tregs, its role in Treg cell development and/or function remains elusive. Here, we generated mouse genetic models to ablate or activate Ahr expression specifically in Tregs. We showed that Ahr was expressed more abundantly by peripherally induced Tregs (pTregs) in the gut and that its expression was independent of microbiota. Ahr was important for Treg gut homing and function. Ahr inhibited pro-inflammatory cytokines produced by Tregs but was dispensable for Treg stability. Furthermore, Ahr-expressing Tregs had enhanced in vivo suppressive activity compared with Tregs lacking Ahr expression in a T cell transfer model of colitis. Our data suggest that Ahr signaling in Tregs may be important for gut immune homeostasis.

N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli.

Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits.