The atypical IκB family member Bcl3 determines differentiation and fate of intestinal RORγt+ regulatory T-cell subsets.

Peripherally-induced regulatory T cells (pTregs) expressing the retinoic acid receptor-related orphan-receptor gamma t (RORγt) are indispensable for intestinal immune homeostasis. Nuclear factor kappa family members regulate the differentiation of thymic Tregs and promote their survival in the periphery. However, the Treg intrinsic molecular mechanisms controlling the size of the pTregs in the intestine and associated lymphoid organs remain unclear. Here, we provide direct evidence that B-cell lymphoma 3 (Bcl3) limits the development of pTregs in a T cell-intrinsic manner. Moreover, the absence of Bcl3 allowed for the formation of an unusual intestinal Treg population co-expressing the transcription factors Helios and RORγt. The expanded RORγt+ Treg populations in the absence of Bcl3 displayed an activated phenotype and secreted high levels of the anti-inflammatory cytokines interleukin (IL)-10 and transforming growth factor beta. They were fully capable of suppressing effector T cells in a transfer colitis model despite an intrinsic bias to trans-differentiate toward T helper 17-like cells. Finally, we provide a Bcl3-dependent gene signature in pTregs including altered responsiveness to the cytokines IL-2, IL-6, and tumor necrosis factor alpha. Our results demonstrate that Bcl3 acts as a molecular switch to limit the expansion of different intestinal Treg subsets and may thus serve as a novel therapeutic target for inflammatory bowel disease by restoring intestinal immune tolerance.

IgE and anaphylaxis specific to the carbohydrate alpha-gal depend on IL-4.

BACKGROUND: Alpha-gal (Galα1-3Galß1-4GlcNAc) is a carbohydrate with the potential to elicit fatal allergic reactions to mammalian meat and drugs of mammalian origin. This type of allergy is induced by tick bites, and therapeutic options for this skin-driven food allergy are limited to the avoidance of the allergen and treatment of symptoms. Thus, a better understanding of the immune mechanisms resulting in sensitization through the skin is crucial, especially in the case of a carbohydrate allergen for which underlying immune responses are poorly understood. OBJECTIVE: We aimed to establish a mouse model of alpha-gal allergy for in-depth immunologic analyses. METHODS: Alpha-galactosyltransferase 1-deficient mice devoid of alpha-gal glycosylations were sensitized with the alpha-gal-carrying self-protein mouse serum albumin by repetitive intracutaneous injections in combination with the adjuvant aluminum hydroxide. The role of basophils and IL-4 in sensitization was investigated by antibody-mediated depletion. RESULTS: Alpha-gal-sensitized mice displayed increased levels of alpha-gal-specific IgE and IgG1 and developed systemic anaphylaxis on challenge with both alpha-gal-containing glycoproteins and glycolipids. In accordance with alpha-gal-allergic patients, we detected elevated numbers of basophils at the site of sensitization as well as increased numbers of alpha-gal-specific B cells, germinal center B cells, and B cells of IgE and IgG1 isotypes in skin-draining lymph nodes. By depleting IL-4 during sensitization, we demonstrated for the first time that sensitization and elicitation of allergy to alpha-gal and correspondingly to a carbohydrate allergen is dependent on IL-4. CONCLUSION: These findings establish IL-4 as a potential target to interfere with alpha-gal allergy elicited by tick bites.

A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria.

The alpha-Gal epitope (α-Gal) with the determining element galactose-α1,3-galactose can lead to clinically relevant allergic reactions and rejections in xenotransplantation. These immune reactions can develop because humans are devoid of this carbohydrate due to evolutionary loss of the enzyme α1,3-galactosyltransferase (GGTA1). In addition, up to 1% of human IgG antibodies are directed against α-Gal, but the stimulus for the induction of anti-α-Gal antibodies is still unclear. Commensal bacteria have been suggested as a causal factor for this induction as α-Gal binding tools such as lectins were found to stain cultivated bacteria isolated from the intestinal tract. Currently available tools for the detection of the definite α-Gal epitope, however, are cross-reactive, or have limited affinity and, hence, offer restricted possibilities for application. In this study, we describe a novel monoclonal IgG1 antibody (27H8) specific for the α-Gal epitope. The 27H8 antibody was generated by immunization of Ggta1 knockout mice and displays a high affinity towards synthetic and naturally occurring α-Gal in various applications. Using this novel tool, we found that intestinal bacteria reported to be α-Gal positive cannot be stained with 27H8 questioning whether commensal bacteria express the native α-Gal epitope at all.

Therapeutic Potential of the Intestinal Microbiota for Immunomodulation of Food Allergies.

Food allergy is an atopic disease that is caused by the immune system targeting harmless food antigens that can result in life-threatening anaphylaxis. As humans and microbes have co-evolved, inevitably commensal microbes have a tremendous impact on our health. As such, the gut with its enormous microbial richness reflects a highly tolerogenic environment at steady state, in which immune cells are educated to react in a well-calibrated manner to food and microbial antigens. Recent evidence indicates that the susceptibility to food allergy is critically linked to microbial dysbiosis and can be transmitted by microbial transfer from humans to mice. Experimental work and epidemiological studies further point toward a critical time window in early childhood during which the immune system is imprinted by microbial colonization. Particularly, Foxp3-expressing regulatory T cells turn out to be key players, acting as rheostats for controlling the magnitude of food allergic reactions. An increasing number of bacterial metabolites has recently been shown to regulate directly or indirectly the differentiation of peripherally induced Tregs, most of which co-express the RAR-related orphan receptor gamma t (RORγt). Genetic ablation provided additional direct evidence for the importance of RORγt+ Tregs in food allergy. Future strategies for the stratification of food allergic patients with the aim to manipulate the intestinal microbiota by means of fecal transplantation efforts, pre- or probiotic regimens or for boosting oral immunotherapy may improve diagnosis and therapy. In this review some of the key underlying mechanisms are summarized and future directions for potential microbial therapy are explored.

Preclinical evaluation of the anti-tumor effects of the natural isoflavone genistein in two xenograft mouse models monitored by [18F]FDG, [18F]FLT, and [64Cu]NODAGA-cetuximab small animal PET.

UNLABELLED: The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting. PROCEDURES: A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 3'-deoxy-3'-[18F]fluorothymidine ([18F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [18F]FDG, [18F]FLT and [64Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data. RESULTS: Genistein reduced tumor growth significantly in both xenografts. [18F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [18F]FDG and [64Cu]NODAGA-cetuximab were not suitable for therapy monitoring. CONCLUSIONS: As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [18F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting.

Signal integration by the CYP1A1 promoter--a quantitative study.

Genes involved in detoxification of foreign compounds exhibit complex spatiotemporal expression patterns in liver. Cytochrome P450 1A1 (CYP1A1), for example, is restricted to the pericentral region of liver lobules in response to the interplay between aryl hydrocarbon receptor (AhR) and Wnt/ß-catenin signaling pathways. However, the mechanisms by which the two pathways orchestrate gene expression are still poorly understood. With the help of 29 mutant constructs of the human CYP1A1 promoter and a mathematical model that combines Wnt/ß-catenin and AhR signaling with the statistical mechanics of the promoter, we systematically quantified the regulatory influence of different transcription factor binding sites on gene induction within the promoter. The model unveils how different binding sites cooperate and how they establish the promoter logic; it quantitatively predicts two-dimensional stimulus-response curves. Furthermore, it shows that crosstalk between Wnt/ß-catenin and AhR signaling is crucial to understand the complex zonated expression patterns found in liver lobules. This study exemplifies how statistical mechanical modeling together with combinatorial reporter assays has the capacity to disentangle the promoter logic that establishes physiological gene expression patterns.

Cooperation of structurally different aryl hydrocarbon receptor agonists and ß-catenin in the regulation of CYP1A expression.

The ligand-activated nuclear receptor AhR (aryl hydrocarbon receptor) mediates the response of hepatocytes to various exogenous compounds. AhR is classically activated by planar, aromatic hydrocarbons, but also by other, structurally rather unrelated compounds. Recent data show that the canonical Wnt/ß-catenin signaling pathway is also involved in the regulation of hepatic zonal gene expression and drug metabolism in mammalian liver. Previous studies indicate that the loss of ß-catenin in hepatocytes diminishes the response to the AhR agonists 3-methylcholanthrene (3MC) in vivo and to 2,3,7,8-tetrachlorodibenzo-[p]-dioxin in vitro. The knockout of ß-catenin also impairs the zonal pattern of AhR target gene induction by 3MC. However, it is presently unknown whether the chemical nature of the AhR agonist influences the AhR/ß-catenin interaction. Moreover, no information is available about the dose-response curves of AhR activation in the absence or presence of Wnt/ß-catenin signaling. In the present study, we have analyzed AhR-dependent responses to different concentrations of structurally unrelated AhR agonists in vivo and in vitro. The results demonstrate that ß-catenin is essential to obtain the maximum AhR response. Moreover, using transgenic mouse models which allow for the ablation of ß-catenin at different age of mice, we demonstrate that the presence of ß-catenin, not postnatal developmental effects in ß-catenin-deficient livers, is responsible for the observed interplay of ß-catenin and the AhR.