Rapid detection and online analysis of microbial changes through flow cytometry.

Short-read 16 S rRNA gene sequencing is the dominating technology to profile microbial communities in different habitats. Its uncontested taxonomic resolution paved the way for major contributions to the field. Sample measurement and analysis, that is, sequencing, is rather slow-in order of days. Alternatively, flow cytometry can be used to profile the microbiota of various sources within a few minutes per sample. To keep up with high measurement speed, we developed the open source-analyzing tool FlowSoFine. To validate the ability to distinguish microbial profiles, we examined human skin samples of three body sites (N = 3 × 54) with flow cytometry and 16 S rRNA gene amplicon sequencing. Confirmed by sequencing of the very same samples, body site was found to be significantly different by flow cytometry. For a proof-of-principle multidimensional approach, using stool samples of patients (N = 40) with/without inflammatory bowel diseases, we could discriminate the health status by their bacterial patterns. In conclusion, FlowSoFine enables the generation and comparison of cytometric fingerprints of microbial communities from different sources. The implemented interface supports the user through all analytical steps to work out the biological relevant signals from raw measurements to publication ready figures. Furthermore, we present flow cytometry as a valid method for skin microbiota analysis.

The Ah Receptor from Toxicity to Therapeutics: Report from the 5th AHR Meeting at Penn State University, USA, June 2022.

The aryl hydrocarbon receptor (AHR) is a sensor of low-molecular-weight molecule signals that originate from environmental exposures, the microbiome, and host metabolism. Building upon initial studies examining anthropogenic chemical exposures, the list of AHR ligands of microbial, diet, and host metabolism origin continues to grow and has provided important clues as to the function of this enigmatic receptor. The AHR has now been shown to be directly involved in numerous biochemical pathways that influence host homeostasis, chronic disease development, and responses to toxic insults. As this field of study has continued to grow, it has become apparent that the AHR is an important novel target for cancer, metabolic diseases, skin conditions, and autoimmune disease. This meeting attempted to cover the scope of basic and applied research being performed to address possible applications of our basic knowledge of this receptor on therapeutic outcomes.

Smad7 in intestinal CD4+ T cells determines autoimmunity in a spontaneous model of multiple sclerosis.

Environmental triggers acting at the intestinal barrier are thought to contribute to the initiation of autoimmune disorders. The transforming growth factor beta inhibitor Smad7 determines the phenotype of CD4+ T cells. We hypothesized that Smad7 in intestinal CD4+ T cells controls initiation of opticospinal encephalomyelitis (OSE), a murine model of multiple sclerosis (MS), depending on the presence of gut microbiota. Smad7 was overexpressed or deleted in OSE CD4+ T cells to determine the effect on clinical progression, T cell differentiation, and T cell migration from the intestine to the central nervous system (CNS). Smad7 overexpression worsened the clinical course of OSE and increased CNS inflammation and demyelination. It favored expansion of intestinal CD4+ T cells toward an inflammatory phenotype and migration of intestinal CD4+ T cells to the CNS. Intestinal biopsies from MS patients revealed decreased transforming growth factor beta signaling with a shift toward inflammatory T cell subtypes. Smad7 in intestinal T cells might represent a valuable therapeutic target for MS to achieve immunologic tolerance in the intestine and suppress CNS inflammation.

The aryl hydrocarbon receptor at the forefront of host-microbe interactions in the skin: A perspective on current knowledge gaps and directions for future research and therapeutic applications.

The skin is home to a community of skin microbiota including bacteria, viruses and fungi, which are widely accepted to be of importance for skin homeostasis but also associated with skin diseases. Detailed knowledge on the skin microbiota composition and its changes in a number of skin diseases is available. Yet, specific interactions between microbes and the host skin cells or how they communicate with each other are less well understood. To identify, understand and eventually therapeutically exploit causal relationships of microbial dysbiosis with disease, studies are required that address the receptors and mediators involved in host-microbe interactions. In this perspective article, we provide an outlook on one of such receptors, namely the aryl hydrocarbon receptor (AHR). The AHR is well known for being a ligand-activated transcription factor regulating the proliferation, differentiation and function of many cell types present in the skin. Its targeting by anti-inflammatory therapeutics such as coal tar and Tapinarof is effective in atopic dermatitis and psoriasis. AHR signalling is activated upon binding of wide variety of small chemicals or ligands, including microbiota-derived metabolites. New evidence has emerged pointing towards a key role for epidermal AHR signalling through skin microbiota-derived metabolites. In response, AHR-driven expression of antimicrobial peptides and stratum corneum formation may alter the skin microbiota composition. This a self-perpetuating feedback loop calls for novel therapeutic intervention strategies for which we herein discuss the requirements in future mechanistic studies.

Proximal Lck Promoter-Driven Cre Function Is Limited in Neonatal and Ineffective in Adult γδ T Cell Development.

During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αß and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αß T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αß T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.

Trajectory Shifts in Interdisciplinary Research of the Aryl Hydrocarbon Receptor-A Personal Perspective on Thymus and Skin.

Identifying historical trajectories is a useful exercise in research, as it helps clarify important, perhaps even "paradigmatic", shifts in thinking and moving forward in science. In this review, the development of research regarding the role of the transcription factor "aryl hydrocarbon receptor" (AHR) as a mediator of the toxicity of environmental pollution towards a link between the environment and a healthy adaptive response of the immune system and the skin is discussed. From this fascinating development, the opportunities for targeting the AHR in the therapy of many diseases become clear.

AHR Signaling Dampens Inflammatory Signature in Neonatal Skin γδ T Cells.

Background Aryl hydrocarbon receptor (AHR)-deficient mice do not support the expansion of dendritic epidermal T cells (DETC), a resident immune cell population in the murine epidermis, which immigrates from the fetal thymus to the skin around birth. Material and Methods In order to identify the gene expression changes underlying the DETC disappearance in AHR-deficient mice, we analyzed microarray RNA-profiles of DETC, sorted from the skin of two-week-old AHR-deficient mice and their heterozygous littermates. In vitro studies were done for verification, and IL-10, AHR repressor (AHRR), and c-Kit deficient mice analyzed for DETC frequency. Results We identified 434 annotated differentially expressed genes. Gene set enrichment analysis demonstrated that the expression of genes related to proliferation, ion homeostasis and morphology differed between the two mouse genotypes. Importantly, with 1767 pathways the cluster-group "inflammation" contained the majority of AHR-dependently regulated pathways. The most abundant cluster of differentially expressed genes was "inflammation." DETC of AHR-deficient mice were inflammatory active and had altered calcium and F-actin levels. Extending the study to the AHRR, an enigmatic modulator of AHR-activity, we found approximately 50% less DETC in AHRR-deficient mice than in wild-type-littermates. Conclusion AHR-signaling in DETC dampens their inflammatory default potential and supports their homeostasis in the skin.

Role of the Aryl Hydrocarbon Receptor in Environmentally Induced Skin Aging and Skin Carcinogenesis.

The skin is constantly exposed to a variety of environmental threats, including solar electromagnetic radiation, microbes, airborne particulate matter, and chemicals. Acute exposure to these environmental factors results in the activation of different signaling pathways that orchestrate adaptive stress responses to maintain cell and tissue homeostasis. Chronic exposure of skin to these factors, however, may lead to the accumulation of damaged macromolecules and loss of cell and tissue integrity, which, over time, may facilitate aging processes and the development of aging-related malignancies. One transcription factor that is expressed in all cutaneous cells and activated by various environmental stressors, including dioxins, polycyclic aromatic hydrocarbons, and ultraviolet radiation, is the aryl hydrocarbon receptor (AHR). By regulating keratinocyte proliferation and differentiation, epidermal barrier function, melanogenesis, and immunity, a certain degree of AHR activity is critical to maintain skin integrity and to adapt to acute stress situations. In contrast, a chronic activation of cutaneous AHR signaling critically contributes to premature aging and the development of neoplasms by affecting metabolism, extracellular matrix remodeling, inflammation, pigmentation, DNA repair, and apoptosis. This article provides an overview of the detrimental effects associated with sustained AHR activity in chronically stressed skin and pinpoints AHR as a promising target for chemoprevention.

The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology.

The aryl hydrocarbon receptor (AhR) is an evolutionarily old transcription factor belonging to the Per-ARNT-Sim-basic helix-loop-helix protein family. AhR translocates into the nucleus upon binding of various small molecules into the pocket of its single-ligand binding domain. AhR binding to both xenobiotic and endogenous ligands results in highly cell-specific transcriptome changes and in changes in cellular functions. We discuss here the role of AhR for immune cells of the barrier organs: skin, gut, and lung. Both adaptive and innate immune cells require AhR signaling at critical checkpoints. We also discuss the current two prevailing views-namely, 1) AhR as a promiscuous sensor for small chemicals and 2) a role for AhR as a balancing factor for cell differentiation and function, which is controlled by levels of endogenous high-affinity ligands. AhR signaling is considered a promising drug and preventive target, particularly for cancer, inflammatory, and autoimmune diseases. Therefore, understanding its biology is of great importance.

Old Receptor, New Tricks-The Ever-Expanding Universe of Aryl Hydrocarbon Receptor Functions. Report from the 4th AHR Meeting, 29⁻31 August 2018 in Paris, France.

In a time where "translational" science has become a mantra in the biomedical field, it is reassuring when years of research into a biological phenomenon suddenly points towards novel prevention or therapeutic approaches to disease, thereby demonstrating once again that basic science and translational science are intimately linked. The studies on the aryl hydrocarbon receptor (AHR) discussed here provide a perfect example of how years of basic toxicological research on a molecule, whose normal physiological function remained a mystery for so long, has now yielded a treasure trove of actionable information on the development of targeted therapeutics. Examples are autoimmunity, metabolic imbalance, inflammatory skin and gastro-intestinal diseases, cancer, development and perhaps ageing. Indeed, the AHR field no longer asks, "What does this receptor do in the absence of xenobiotics?" It now asks, "What doesn't this receptor do?".

Normal mast cell numbers in the tissues of AhR-deficient mice.

The transcription factor aryl hydrocarbon receptor (AhR) acts as an immunomodulatory molecule in several immune cell lineages. Recently, it has been implicated in development and maintenance of immune cells in barrier tissues such as skin and mucosa. To investigate its role on mast cell development and maintenance in skin, peritoneal exudate cells (PECs) and lymph nodes, we studied in depth their phenotype in AhR-deficient mice. Our findings do not provide any evidence for a suspected role of the AhR in mast cell homeostasis.

Differential cell type-specific function of the aryl hydrocarbon receptor and its repressor in diet-induced obesity and fibrosis.

OBJECTIVE: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating xenobiotic responses as well as physiological metabolism. Dietary AhR ligands activate the AhR signaling axis, whereas AhR activation is negatively regulated by the AhR repressor (AhRR). While AhR-deficient mice are known to be resistant to diet-induced obesity (DIO), the influence of the AhRR on DIO has not been assessed so far. METHODS: In this study, we analyzed AhRR-/- mice and mice with a conditional deletion of either AhRR or AhR in myeloid cells under conditions of DIO and after supplementation of dietary AhR ligands. Moreover, macrophage metabolism was assessed using Seahorse Mito Stress Test and ROS assays as well as transcriptomic analysis. RESULTS: We demonstrate that global AhRR deficiency leads to a robust, but not as profound protection from DIO and hepatosteatosis as AhR deficiency. Under conditions of DIO, AhRR-/- mice did not accumulate TCA cycle intermediates in the circulation in contrast to wild-type (WT) mice, indicating protection from metabolic dysfunction. This effect could be mimicked by dietary supplementation of AhR ligands in WT mice. Because of the predominant expression of the AhRR in myeloid cells, AhRR-deficient macrophages were analyzed for changes in metabolism and showed major metabolic alterations regarding oxidative phosphorylation and mitochondrial activity. Unbiased transcriptomic analysis revealed increased expression of genes involved in de novo lipogenesis and mitochondrial biogenesis. Mice with a genetic deficiency of the AhRR in myeloid cells did not show alterations in weight gain after high fat diet (HFD) but demonstrated ameliorated liver damage compared to control mice. Further, deficiency of the AhR in myeloid cells also did not affect weight gain but led to enhanced liver damage and adipose tissue fibrosis compared to controls. CONCLUSIONS: AhRR-deficient mice are resistant to diet-induced metabolic syndrome. Although conditional ablation of either the AhR or AhRR in myeloid cells did not recapitulate the phenotype of the global knockout, our findings suggest that enhanced AhR signaling in myeloid cells deficient for AhRR protects from diet-induced liver damage and fibrosis, whereas myeloid cell-specific AhR deficiency is detrimental.

Aryl hydrocarbon receptor is critical for homeostasis of invariant gammadelta T cells in the murine epidermis.

An immunoregulatory role of aryl hydrocarbon receptor (AhR) has been shown in conventional αß and γδ T cells, but its function in skin γδ T cells (dendritic epidermal T cells [DETC]) is unknown. In this study, we demonstrate that DETC express AhR in wild-type mice, and are specifically absent in the epidermis of AhR-deficient mice (AhR-KO). We show that DETC precursors are generated in the thymus and home to the skin. Proliferation of DETC in the skin was impaired in AhR-KO mice, resulting in a >90% loss compared with wild type. Surprisingly, DETC were not replaced by αß T cells or conventional γδ T cells, suggesting a limited time frame for seeding this niche. We found that DETC from AhR-KO mice failed to express the receptor tyrosine kinase c-Kit, a known growth factor for γδ T cells in the gut. Moreover, we found that c-kit is a direct target of AhR, and propose that AhR-dependent c-Kit expression is potentially involved in DETC homeostasis. DETC are a major source of GM-CSF in the skin. Recently, we had shown that impaired Langerhans cell maturation in AhR-KO is related to low GM-CSF levels. Our findings suggest that the DETCs are necessary for LC maturation, and provide insights into a novel role for AhR in the maintenance of skin-specific γδ T cells, and its consequences for the skin immune network.

The aryl hydrocarbon receptor controls mesenchymal stromal cell-mediated immunomodulation via ubiquitination of eukaryotic elongation factor-2 kinase.

Mesenchymal stem cells (MSCs) have great therapeutic advantages due to their immunosuppressive properties. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose signaling plays an important role in the immune system. AHR may be involved in the regulation of MSC-associated immunomodulatory functions. However, the mechanisms by which AHR controls the immunosuppressive functions of MSCs are not well understood. Here, we report that Ahr-deficient MSCs show decreased therapeutic efficacy against graft-versus-host disease (GVHD) compared to wild-type (WT)-MSCs. This was probably due to decreased iNOS protein expression, which is a key regulatory enzyme in MSC immunomodulation. The expression of eukaryotic elongation factor 2 kinase (eEF2K), which inhibits the elongation stage of protein synthesis, is significantly increased in the Ahr-deficient MSCs. Inhibition of eEF2K restored iNOS protein expression. AHR is known to act as an E3 ligase together with CUL4B. We observed constitutive binding of AHR to eEF2K. Consequently, ubiquitination and degradation of eEF2K were inhibited in Ahr-deficient MSCs and by the AHR antagonist CH223191 in WT-MSCs. In summary, AHR regulates the immunomodulatory functions of MSCs through ubiquitination of eEF2K, thereby controlling iNOS protein synthesis and its product, nitric oxide levels.

The aryl hydrocarbon receptor in immunity.

Low-molecular-weight chemicals or xenobiotics might contribute to the increasing prevalence of allergies and autoimmunity. Certain chemicals can alter immune responses via their action on the cytosolic transcription factor aryl hydrocarbon receptor (AhR). AhR recognizes numerous small xenobiotic and natural molecules, such as dioxin and the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole. Although AhR is best known for mediating dioxin toxicity, knockout studies have indicated that AhR also plays a role in normal physiology, including certain immune responses. In particular, Th17 cells and dendritic cells express high levels of AhR. We review here current evidence for the physiological role of AhR in the immune system, focussing in particular on T-cell biology.

Biology and function of the aryl hydrocarbon receptor: report of an international and interdisciplinary conference.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor present in many cells. The AhR links environmental chemical stimuli with adaptive responses, such as detoxification, cellular homoeostasis or immune responses. Furthermore, novel roles of AhR in physiological and genetic functions are being discovered. This is a report of a recent meeting in Düsseldorf. The meeting highlighted that AhR research has moved from its focus on toxic effects of dioxins and other environmental pollutants to its biological roles. For instance, it was recently discovered that AhR-responsive elements in retrotransposons contribute to the functional structure of the genome. Other exciting new reports concerned the way plant-derived compounds in our diet are necessary for a fully functioning immune system of the gut. Also, human brain tumours use the AhR system to gain growth advantages. Other aspects covered were neurotoxicology, the circadian rhythm, or the breadth of the adaptive and innate immune system (hematopoietic stem cells, dendritic cells, T cells, mast cells). Finally, the meeting dealt with the discovery of new xenobiotic and natural ligands and their use in translational medicine, or cancer biology and AhR.