New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Effects of exercise during chemo- or radiotherapy on immune markers - a systematic review.

Introduction Patients with cancer receiving radio- or chemotherapy undergo many immunological stressors. Chronic regular exercise was shown to positively influence the immune system in several populations, while exercise overload may have negative effects. Exercise is currently recommended for all patients with cancer. However, knowledge regarding the effects of exercise on immune markers in patients undergoing chemo- or radiotherapy is limited. The aim of this study is to systematically review the effects of moderate and high intensity exercise interventions in patients with cancer during chemotherapy or radiotherapy, on immune markers. Methods For this review, a search was performed in PubMed and EMBASE, until March 2023. Methodological quality was assessed with the Pedro tool and best-evidence syntheses were performed both per immune marker and for the inflammatory profile. Results Methodological quality of the 15 included articles was rated fair to good. The majority of markers was unaltered, but observed effects included a suppressive effect of exercise during radiotherapy on some pro-inflammatory markers, a preserving effect of exercise during chemotherapy on NK cell degranulation and cytotoxicity, a protective effect on the decrease in thrombocytes during chemotherapy, and a positive effect of exercise during chemotherapy on IgA. Discussion/conclusion Although exercise only influenced a few markers, the results are promising. Exercise did not negatively influence immune markers, and some were positively affected since suppressed inflammation might have positive clinical implications. For future research, consensus is needed regarding a set of markers that are most responsive to exercise. Next, differential effects of training types and intensities on these markers should be further investigated, as well as their clinical implications.

Environmentally weathered polystyrene particles induce phenotypical and functional maturation of human monocyte-derived dendritic cells.

Micro- and nanoplastics (MNP) are ubiquitously present in the environment due to their high persistence and bioaccumulative properties. Humans get exposed to MNP via various routes and consequently, they will encounter dendritic cells (DC) which are antigen-presenting cells involved in regulating immune responses. The consequences of DC exposure to MNP are an important, yet understudied, cause of concern. Therefore, this study aimed to assess the uptake and effect of MNP in vitro by exposing human monocyte-derived dendritic cells (MoDC) to virgin and environmentally weathered polystyrene (PS) particles of different sizes (0.2, 1, and 10 µm), at different concentrations ranging from 1 to 100 µg/ml. The effects of these particles were examined by measuring co-stimulatory surface marker (i.e. CD83 and CD86) expression. In addition, T-cell proliferation was measured via a mixed-leukocyte reaction (MLR) assay. The results showed that MoDC were capable of absorbing PS particles, and this was facilitated by pre-incubation in heat-inactivated (HI) plasma. Furthermore, depending on their size, weathered PS particles in particular caused increased expression of CD83 and CD86 on MoDC. Lastly, weathered 0.2 µm PS particles were able to functionally activate MoDC, leading to an increase in T-cell activation. These in vitro data suggest that, depending on their size, weathered PS particles might act as an immunostimulating adjuvant, possibly leading to T-cell sensitization.

Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures.

Intestinal organoids are advanced cellular models, which are widely used in mammalian studies to mimic and study in vivo intestinal function and host-pathogen interactions. Growth factors WNT3 and RSPO1 are crucial for the growth of intestinal organoids. Chicken intestinal organoids are currently cultured with mammalian Wnt3a and Rspo1, however, maintaining their longevity has shown to be challenging. Based on the limited homology between mammalian and avian RSPO1, we expect that chicken-derived factors are required for the organoid cultures. Isolated crypts from embryonic tissue of laying hens were growing in the presence of chicken WNT3 and RSPO1, whereas growth in the presence of mammalian Wnt3a and Rspo1 was limited. Moreover, the growth was increased by using Prostaglandin E2 (PGE2) and a Forkhead box O1-inhibitor (FOXO1-inhibitor), allowing to culture these organoids for 15 passages. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes and enteroendocrine cells in 2D structures. Overall, we show that chicken intestinal organoids can be cultured for multiple passages using chicken-derived WNT3 and RSPO1, PGE2, and FOXO1-inhibitor.

Natural language processing in toxicology: Delineating adverse outcome pathways and guiding the application of new approach methodologies.

Adverse Outcome Pathways (AOPs) are conceptual frameworks that tie an initial perturbation (molecular initiating event) to a phenotypic toxicological manifestation (adverse outcome), through a series of steps (key events). They provide therefore a standardized way to map and organize toxicological mechanistic information. As such, AOPs inform on key events underlying toxicity, thus supporting the development of New Approach Methodologies (NAMs), which aim to reduce the use of animal testing for toxicology purposes. However, the establishment of a novel AOP relies on the gathering of multiple streams of evidence and information, from available literature to knowledge databases. Often, this information is in the form of free text, also called unstructured text, which is not immediately digestible by a computer. This information is thus both tedious and increasingly time-consuming to process manually with the growing volume of data available. The advancement of machine learning provides alternative solutions to this challenge. To extract and organize information from relevant sources, it seems valuable to employ deep learning Natural Language Processing techniques. We review here some of the recent progress in the NLP field, and show how these techniques have already demonstrated value in the biomedical and toxicology areas. We also propose an approach to efficiently and reliably extract and combine relevant toxicological information from text. This data can be used to map underlying mechanisms that lead to toxicological effects and start building quantitative models, in particular AOPs, ultimately allowing animal-free human-based hazard and risk assessment.

Short-term associations between barbecue fumes and respiratory health in young adults.

BACKGROUND: Epidemiological studies have associated biomass combustion with (respiratory) morbidity and mortality, primarily in indoor settings. Barbecuing results in high outdoor air pollution exposures, but the health effects are unknown. OBJECTIVE: The objective was to investigate short-term changes in respiratory health in healthy adults, associated with exposure to barbecue fumes. METHODS: 16 healthy, adult volunteers were exposed to barbecue smoke in outdoor air in rest during 1.5 h, using a repeated-measures design. Major air pollutants were monitored on-site, including particulate matter <2.5 µm (PM2.5), particle number concentrations (PNC) and black- and brown carbon. At the same place and time-of-day, subjects participated in a control session, during which they were not exposed to barbecue smoke. Before and immediately after all sessions lung function was measured. Before, immediately after, 4- and 18 h post-sessions nasal expression levels of interleukin (IL)-8, IL6 and Tumor Necrosis Factor alpha (TNFα) were determined in nasal swabs, using quantitative polymerase chain reaction. Associations between major air pollutants, lung function and inflammatory markers were assessed using mixed linear regression models. RESULTS: High PM2.5 levels and PNCs were observed during barbecue sessions, with averages ranging from 553 to 1062 µg/m3 and 109,000-463,000 pt/cm3, respectively. Average black- and brown carbon levels ranged between 4.1-13.0 and 5.0-16.2 µg/m3. A 1000 µg/m3 increase in PM2.5 was associated with 2.37 (0.97, 4.67) and 2.21 (0.98, 5.00) times higher expression of IL8, immediately- and 18 h after exposure. No associations were found between air pollutants and lung function, or the expression of IL6 or TNFα. DISCUSSION: Short-term exposure to air pollutants emitted from barbecuing was associated with a mild respiratory response in healthy young adults, including prolonged increase in nasal IL8 without a change in lung function and other measured inflammatory markers. The results might indicate prolonged respiratory inflammation, due to short-term exposure to barbecue fumes.

Nanomaterials and the Serosal Immune System in the Thoracic and Peritoneal Cavities.

The thoracic and peritoneal cavities are lined by serous membranes and are home of the serosal immune system. This immune system fuses innate and adaptive immunity, to maintain local homeostasis and repair local tissue damage, and to cooperate closely with the mucosal immune system. Innate lymphoid cells (ILCs) are found abundantly in the thoracic and peritoneal cavities, and they are crucial in first defense against pathogenic viruses and bacteria. Nanomaterials (NMs) can enter the cavities intentionally for medical purposes, or unintentionally following environmental exposure; subsequent serosal inflammation and cancer (mesothelioma) has gained significant interest. However, reports on adverse effects of NM on ILCs and other components of the serosal immune system are scarce or even lacking. As ILCs are crucial in the first defense against pathogenic viruses and bacteria, it is possible that serosal exposure to NM may lead to a reduced resistance against pathogens. Additionally, affected serosal lymphoid tissues and cells may disturb adipose tissue homeostasis. This review aims to provide insight into key effects of NM on the serosal immune system.

The hepatotoxic fluoroquinolone trovafloxacin disturbs TNF- and LPS-induced p65 nuclear translocation in vivo and in vitro.

Idiosyncratic drug-induced liver injury (IDILI) is a severe disease that cannot be detected during drug development. It has been shown that hepatotoxicity of some compounds associated with IDILI becomes apparent when these are combined in vivo and in vitro with LPS or TNF. Among these compounds trovafloxacin (TVX) induced apoptosis in the liver and increased pro-inflammatory cytokines in mice exposed to LPS/TNF. The hepatocyte survival and the cytokine release after TNF/LPS stimulation relies on a pulsatile activation of NF-κB. We set out to evaluate the dynamic activation of NF-κB in response to TVX + TNF or LPS models, both in mouse and human cells. Remarkably, TVX prolonged the first translocation of NF-κB induced by TNF both in vivo and in vitro. The prolonged p65 translocation caused by TVX was associated with an increased phosphorylation of IKK and MAPKs and accumulation of inhibitors of NF-κB such as IκBα and A20 in HepG2. Coherently, TVX suppressed further TNF-induced NF-κB translocations in HepG2 leading to decreased transcription of ICAM-1 and inhibitors of apoptosis. TVX prolonged LPS-induced NF-κB translocation in RAW264.7 macrophages increasing the secretion of TNF. In summary, this study presents new, relevant insights into the mechanism of TVX-induced liver injury underlining the resemblance between mouse and human models. In this study we convincingly show that regularly used toxicity models provide a coherent view of relevant pathways for IDILI. We propose that assessment of the kinetics of activation of NF-κB and MAPKs is an appropriate tool for the identification of hepatotoxic compounds during drug development.

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models.

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI.

Deamidation and Enzymatic Hydrolysis of Gliadins Alter Their Processing by Dendritic Cells in Vitro.

Gliadins are major wheat allergens. Their treatment by acid or enzymatic hydrolysis has been shown to modify their allergenic potential. As the interaction of food proteins with dendritic cells (DCs) is a key event in allergic sensitization, we wished to investigate whether deamidation and enzymatic hydrolysis influence gliadin processing by DC and to examine the capacity of gliadins to activate DCs. We compared the uptake and degradation of native and modified gliadins by DCs using mouse bone marrow-derived DCs. We also analyzed the effects of these interactions on the phenotypes of DCs and T helper (Th) lymphocytes. Modifying gliadins induced a change in physicochemical properties (molecular weight, hydrophobicity, and sequence) and also in the peptide size. These alterations in turn led to increased uptake and intracellular degradation of the proteins by DCs. Native gliadins (NGs) (100 µg/mL), but not modified gliadins, increased the frequency of DC expressing CD80 (15.41 ± 2.36% vs 6.81 ± 1.10%, p < 0.001), CCR7 (28.53 ± 8.17% vs 17.88 ± 2.53%, p < 0.001), CXCR4 (70.14 ± 4.63% vs 42.82 ± 1.96%, p < 0.001), and CCR7-dependent migration (2.46 ± 1.45 vs 1.00 ± 0.22, p < 0.01) compared with NGs. This was accompanied by Th lymphocyte activation (30.37 ± 3.87% vs 21.53 ± 3.14%, p < 0.1) and proliferation (16.39 ± 3.97% vs 9.31 ± 2.80%, p > 0.1). Moreover, hydrolysis decreases the peptide size and induces an increase in gliadin uptake and degradation. Deamidation and extensive enzymatic hydrolysis of gliadins modify their interaction with DCs, leading to alteration of their immunostimulatory capacity. These findings demonstrate the strong relationship between the biochemical characteristics of proteins and immune cell interactions.

A network-based approach for identifying suitable biomarkers for oral immunotherapy of food allergy.

BACKGROUND: Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, concerns with regards to safety and long-term efficacy of OIT remain. There is a need to identify biomarkers that predict, monitor and/or evaluate the effects of OIT. Here we present a method to select candidate biomarkers for efficacy and safety assessment of OIT using the computational approaches Bayesian networks (BN) and Topological Data Analysis (TDA). RESULTS: Data were used from fructo-oligosaccharide diet-supported OIT experiments performed in 3 independent cow's milk allergy (CMA) and 2 independent peanut allergy (PNA) experiments in mice. Bioinformatical approaches were used to understand the data structure. The BN predicted the efficacy of OIT in the CMA with 86% and indicated a clear effect of scFOS/lcFOS on allergy parameters. For the PNA model, this BN (trained on CMA data) predicted an efficacy of OIT with 76% accuracy and shows similar effects of the allergen, treatment and diet as compared to the CMA model. The TDA identified clusters of biomarkers closely linked to biologically relevant clinical symptoms and also unrelated and redundant parameters within the network. CONCLUSIONS: Here we provide a promising application of computational approaches to a) compare mechanistic features of two different food allergies during OIT b) determine the biological relevance of candidate biomarkers c) generate new hypotheses to explain why CMA has a different disease pattern than PNA and d) select relevant biomarkers for future studies.

Non-digestible oligosaccharides scFOS/lcFOS facilitate safe subcutaneous immunotherapy for peanut allergy.

BACKGROUND: Improving the safety of subcutaneous immunotherapy (SCIT) for food allergy is necessary to reduce side effects and achieve long-term tolerance. We determined the effect of dietary supplementation with 1% non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) on safety and efficacy of SCIT using a peanut allergy mouse model. METHODS: After sensitization, mice received a scFOS/lcFOS or control diet for the rest of the study. To study safety of SCIT, mice were dosed with a single subcutaneous injection of peanut extract (PE) or PBS. To study efficacy, mice were dosed subcutaneously (SCIT, 3 times/week) with PE or PBS for 3 weeks. Hereafter, acute allergic skin responses, anaphylactic shock symptoms and body temperature were assessed. To study the mechanism in vitro, the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) line was sensitized with an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation. RESULTS: scFOS/lcFOS reduced anaphylaxis caused by a single PE SCIT dose. scFOS/lcFOS alone also reduced the acute allergic skin response. Moreover, scFOS/lcFOS supplementation resulted in lower MMCP-1 levels in serum after PE SCIT dose compared to control diet, while antibody levels were not affected by the diet. In vitro incubation with scFOS/lcFOS at 0.5% suppressed the degranulation of IgE-sensitized RBL cells. However, dietary supplementation with scFOS/lcFOS did not improve the efficacy of SCIT. CONCLUSIONS: We show that scFOS/lcFOS diet improves the safety of SCIT, as evidenced by lower anaphylactic responses without compromising the efficacy in a mouse model for peanut allergy. This effect is likely to result from the suppression of mast cell effector function.

Mouse strain differences in response to oral immunotherapy for peanut allergy.

BACKGROUND: Promising therapies for food allergy are emerging, mostly based on animal experimentation. However, different mouse strains are used, which may make it hard to compare experiments. The current study investigated whether the immunological differences between C3H/HeOuJ (C3H) and BALB/c mice lead to differences in efficacy of peanut-specific immunotherapy. METHODS: After sensitization using peanut extract (PE), C3H and BALB/c mice received oral immunotherapy (OIT) by intragastric dosing for three weeks. Hereafter, mice were exposed to PE via the intradermal, intragastric and intraperitoneal route, to determine allergic outcomes. Furthermore, PE-specific antibody and cytokine production were determined and the number of various immune cells at different time points during the study were measured. RESULTS: OIT protected C3H mice against anaphylaxis, whereas no anaphylaxis was seen in BALB/c mice. In contrast, OIT induced an increase in MMCP-1 levels in BALB/c mice but not in C3H mice. No effect of OIT on the acute allergic skin response was observed in either strain. Specific antibody responses showed similar patterns in both strains for IgA and IgG1. IgE levels were a tenfold higher in BALB/c mice and after the intragastric challenge (day 70) OIT-treated BALB/c mice showed induced IgE levels. Moreover, in C3H mice IgG2a levels were higher and increased in response to OIT and challenges. After the final challenge, but not at other timepoints MLN-derived lymphocytes from OIT-treated BALB/c mice produced less IL-13 and IL-5 compared to control-treated mice, whereas no differences were seen in case of C3H mice. CONCLUSIONS: Taken together, these results show that the C3H strain is more suitable to study clinical outcomes of OIT, whereas the BALB/c strain is more optimal to study T cell responses.

Butyrate Enhances Desensitization Induced by Oral Immunotherapy in Cow's Milk Allergic Mice.

BACKGROUND: In previous studies, we showed that a fructo-oligosaccharide- (FOS-) supplemented diet enhanced oral immunotherapy (OIT) efficacy in a mouse model for cow's milk allergy. Fermentation of FOS by intestinal bacteria leads to production of short-chain fatty acids (SCFA) including butyrate. AIM: To investigate the contribution of butyrate in the enhanced efficacy of OIT + FOS. METHODS: C3H/HeOuJ mice were sensitized and received OIT with or without FOS or butyrate supplementation. After treatment, whole blood was collected to conduct a basophil activation test (BAT) and allergen challenges were performed to measure acute allergic symptoms. CD4 + CD25 + regulatory T cells (Tregs) were isolated from treated mice or differentiated in vitro and used in a bone marrow-derived mast cell (BMMC) suppression assay. Cecum content was collected to analyze SCFA concentrations. RESULTS: Allergen-induced basophil activation was reduced in OIT + butyrate samples compared to OIT. Accordingly, the acute allergic skin response and mast cell degranulation upon challenge were reduced in OIT + butyrate and OIT + FOS mice compared to sensitized controls. Butyrate was increased in the cecum content of OIT + FOS mice compared to OIT mice and sensitized controls. Treg-mediated BMMC suppression was enhanced after in vivo butyrate and FOS exposure in combination with OIT but with a more pronounced effect for butyrate. CONCLUSION: Butyrate supplementation enhanced OIT-induced desensitization of basophils and mast cells and Treg functionality. Only OIT + FOS treatment induced potential microbial alterations, shown by increased butyrate levels in cecum content. Both butyrate and FOS are promising candidates to improve OIT efficacy in human studies to treat food allergies.

Chemically modified peanut extract shows increased safety while maintaining immunogenicity.

BACKGROUND: Peanuts are most responsible for food-induced anaphylaxis in adults in developed countries. An effective and safe immunotherapy is urgently needed. The aim of this study was to investigate the immunogenicity, allergenicity, and immunotherapeutic efficacy of a well-characterized chemically modified peanut extract (MPE) adsorbed to Al(OH)3 . METHODS: Peanut extract (PE) was modified by reduction and alkylation. Using sera of peanut-allergic patients, competitive IgE-binding assays and mediator release assays were performed. The immunogenicity of MPE was evaluated by measuring activation of human PE-specific T-cell lines and the induction of PE-specific IgG in mice. The safety and efficacy of MPE adsorbed to Al(OH)3 was tested in two mouse models by measuring allergic manifestations upon peanut challenge in peanut-allergic mice. RESULTS: Compared to PE, the IgE-binding and capacity to induce allergic symptoms of MPE were lower in all patients. PE and MPE displayed similar immunogenicity in vivo and in vitro. In mice sensitized to PE, the threshold for anaphylaxis (drop in BT) upon subcutaneous challenge with PE was 0.01 mg, while at 0.3 mg MPE no allergic reaction occurred. Anaphylaxis was not observed when PE and MPE were fully adsorbed to Al(OH)3 . Both PE and MPE + Al(OH)3 showed to be efficacious in a model for immunotherapy. CONCLUSION: In our studies, an Al(OH)3 adsorbed MPE showed reduced allergenicity compared to unmodified PE, while the efficacy of immunotherapy is maintained. The preclinical data presented in this study supports further development of modified peanut allergens for IT.

Dietary Supplementation with Nondigestible Oligosaccharides Reduces Allergic Symptoms and Supports Low Dose Oral Immunotherapy in a Peanut Allergy Mouse Model.

SCOPE: A major downside of oral immunotherapy (OIT) for food allergy is the risk of severe side effects. Non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) reduce allergy development in murine models. Therefore, it is hypothesized that scFOS/lcFOS can also support the efficacy of OIT in a peanut allergy model. METHODS AND RESULTS: After sensitization to peanut extract (PE) using cholera toxin, C3H/HeOuJ mice are fed a 1% scFOS/lcFOS or control diet and receive OIT (1.5 or 15 mg PE). Hereafter, mice are exposed to PE via different routes to determine the safety and efficacy of treatment in clinical outcomes, PE-specific antibody production, and numbers of various immune cells. scFOS/lcFOS increases short-chain fatty acid levels in the caecum and reduce the acute allergic skin response and drop in body temperature after PE exposure. Interestingly, 15 mg and 1.5 mg OIT with scFOS/lcFOS induce protection against anaphylaxis, whereas 1.5 mg OIT alone does not. OIT, with or without scFOS/lcFOS, induces PE-specific immunoglobulin (Ig) IgG and IgA levels and increases CD103+ dendritic cells in the mesenteric lymph nodes. CONCLUSIONS: scFOS/lcFOS and scFOS/lcFOS combined with low dose OIT are able to protect against a peanut-allergic anaphylactic response.

Glycation of the Major Milk Allergen ß-Lactoglobulin Changes Its Allergenicity by Alterations in Cellular Uptake and Degradation.

SCOPE: During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen ß-lactoglobulin (BLG), in their interactions with cells crucially involved in allergy. METHODS AND RESULTS: BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T-cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco-2 monolayer. Uptake of glycated BLG by bone marrow-derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor-mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4+ T-cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG-specific IgE sensitized basophil cells. CONCLUSIONS: This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy.

The efficacy of oral and subcutaneous antigen-specific immunotherapy in murine cow's milk- and peanut allergy models.

BACKGROUND: Antigen-specific immunotherapy (AIT) is a promising therapeutic approach for both cow's milk allergy (CMA) and peanut allergy (PNA), but needs optimization in terms of efficacy and safety. AIM: Compare oral immunotherapy (OIT) and subcutaneous immunotherapy (SCIT) in murine models for CMA and PNA and determine the dose of allergen needed to effectively modify parameters of allergy. METHODS: Female C3H/HeOuJ mice were sensitized intragastrically (i.g.) to whey or peanut extract with cholera toxin. Mice were treated orally (5 times/week) or subcutaneously (3 times/week) for three consecutive weeks. Hereafter, the acute allergic skin response, anaphylactic shock symptoms and body temperature were measured upon intradermal (i.d.) and intraperitoneal (i.p.) challenge, and mast cell degranulation was measured upon i.g. challenge. Allergen-specific IgE, IgG1 and IgG2a were measured in serum at different time points. Single cell suspensions derived from lymph organs were stimulated with allergen to induce cytokine production and T cell phenotypes were assessed using flow cytometry. RESULTS: Both OIT and SCIT decreased clinically related signs upon challenge in the CMA and PNA model. Interestingly, a rise in allergen-specific IgE was observed during immunotherapy, hereafter, treated mice were protected against the increase in IgE caused by allergen challenge. Allergen-specific IgG1 and IgG2a increased due to both types of AIT. In the CMA model, SCIT and OIT reduced the percentage of activated Th2 cells and increased the percentage of activated Th1 cells in the spleen. OIT increased the percentage of regulatory T cells (Tregs) and activated Th2 cells in the MLN. Th2 cytokines IL-5, IL-13 and IL-10 were reduced after OIT, but not after SCIT. In the PNA model, no differences were observed in percentages of T cell subsets. SCIT induced Th2 cytokines IL-5 and IL-10, whereas OIT had no effect. CONCLUSION: We have shown clinical protection against allergic manifestations after OIT and SCIT in a CMA and PNA model. Although similar allergen-specific antibody patterns were observed, differences in T cell and cytokine responses were shown. Whether these findings are related to a different mechanism of AIT in CMA and PNA needs to be elucidated.

Improved Efficacy of Oral Immunotherapy Using Non-Digestible Oligosaccharides in a Murine Cow's Milk Allergy Model: A Potential Role for Foxp3+ Regulatory T Cells.

BACKGROUND: Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, there are some concerns regarding its safety and long-term efficacy. The use of non-digestible oligosaccharides might improve OIT efficacy since they are known to directly modulate intestinal epithelial and immune cells in addition to acting as prebiotics. AIM: To investigate whether a diet supplemented with plant-derived fructo-oligosaccharides (FOS) supports the efficacy of OIT in a murine cow's milk allergy model and to elucidate the potential mechanisms involved. METHODS: After oral sensitization to the cow's milk protein whey, female C3H/HeOuJ mice were fed either a control diet or a diet supplemented with FOS (1% w/w) and received OIT (10 mg whey) 5 days a week for 3 weeks by gavage. Intradermal (i.d.) and intragastric (i.g.) challenges were performed to measure acute allergic symptoms and mast cell degranulation. Blood and organs were collected to measure antibody levels and T cell and dendritic cell populations. Spleen-derived T cell fractions (whole spleen- and CD25-depleted) were transferred to naïve recipient mice to confirm the involvement of regulatory T cells (Tregs) in allergy protection induced by OIT + FOS. RESULTS: OIT + FOS decreased acute allergic symptoms and mast cell degranulation upon challenge and prevented the challenge-induced increase in whey-specific IgE as observed in sensitized mice. Early induction of Tregs in the mesenteric lymph nodes (MLN) of OIT + FOS mice coincided with reduced T cell responsiveness in splenocyte cultures. CD25 depletion in OIT + FOS-derived splenocyte suspensions prior to transfer abolished protection against signs of anaphylaxis in recipients. OIT + FOS increased serum galectin-9 levels. No differences in short-chain fatty acid (SCFA) levels in the cecum were observed between the treatment groups. Concisely, FOS supplementation significantly improved OIT in the acute allergic skin response, %Foxp3+ Tregs and %LAP+ Th3 cells in MLN, and serum galectin-9 levels. CONCLUSION: FOS supplementation improved the efficacy of OIT in cow's milk allergic mice. Increased levels of Tregs in the MLN and abolished protection against signs of anaphylaxis upon transfer of CD25-depleted cell fractions, suggest a role for Foxp3+ Tregs in the protective effect of OIT + FOS.

Indole-3-carbinol, a plant nutrient and AhR-Ligand precursor, supports oral tolerance against OVA and improves peanut allergy symptoms in mice.

In general, dietary antigens are tolerated by the gut associated immune system. Impairment of this so-called oral tolerance is a serious health risk. We have previously shown that activation of the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) by the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects both oral tolerance and food allergy. In this study, we determine whether a common plant-derived, dietary AhR-ligand modulates oral tolerance as well. We therefore fed mice with indole-3-carbinole (I3C), an AhR ligand that is abundant in cruciferous plants. We show that several I3C metabolites were detectable in the serum after feeding, including the high-affinity ligand 3,3´-diindolylmethane (DIM). I3C feeding robustly induced the AhR-target gene CYP4501A1 in the intestine; I3C feeding also induced the aldh1 gene, whose product catalyzes the formation of retinoic acid (RA), an inducer of regulatory T cells. We then measured parameters indicating oral tolerance and severity of peanut-induced food allergy. In contrast to the tolerance-breaking effect of TCDD, feeding mice with chow containing 2 g/kg I3C lowered the serum anti-ovalbumin IgG1 response in an experimental oral tolerance protocol. Moreover, I3C feeding attenuated symptoms of peanut allergy. In conclusion, the dietary compound I3C can positively influence a vital immune function of the gut.