Iron is essential for oxygen transport, energy metabolism, and immune regulation. Yet iron deficiency is the most common micronutrient disorder across all age groups, affecting nearly one quarter of the global population. Iron deficiency triggers nutritional immunity, a host defense mechanism that withholds and redistributes iron, contributing to increased morbidity and mortality. This review outlines normal iron physiology, distribution and absorption pathways and on the consequences of deficiency across body compartments, with particular attention to type 2-driven diseases. Beyond anemia, insufficient iron availability disrupts immune homeostasis by promoting type 2 inflammation, elevating IgE, and activating mast cells and eosinophils. Regulatory macrophages, the central hub of iron cycling, adopt an inflammatory, iron-sequestering state that reinforces malabsorption and redistribution. Epidemiology studies show higher iron-deficiency risk in allergic individuals; low maternal iron or early-life iron predisposes to eczema, wheeze, and asthma, while food-allergen elimination (notably cow's milk) further worsens anemia risk. Clinical evidence indicates that restoring iron status through diet, supplementation, or fortification lowers IgE levels, improves lung function, and alleviates symptoms of rhinitis, urticaria, and asthma. Iron may therefore represent a modifiable determinant of allergic disease development and severity. Integrating iron assessment and nutritional care into allergy management may reduce disease burden and slow the progression of allergic march.

Toll-like receptor (TLRs) activation in multiple myeloma (MM) cells induces heterogeneous functional responses including cell growth and proliferation, survival or apoptosis. These effects have been suggested to be partly due to increase in secretion of cytokines such as IL-6 or IFNα among others from MM cells following TLR activation. However, whether triggering of these receptors also modulates production of immunoglobulin free light chains (FLCs), which largely contribute to MM pathology, has not been investigated in MM cells before. This study explored the effect of TLR1/2 ligand (Pam3CSK4) alone or combined with bortezomib (BTZ) on production of FLCs in human myeloma cell lines, L363, OPM-2, U266 and NCI-H929. It also investigated the above effect when MM cells were exposed to bone marrow stromal cells (BMSCs) or fibronectin (FN). Adhesion to BMSCs or FN increased secretion of FLC in MM cells. Pam3CSK4 decreased FLC production, and this effect was enhanced in combination with BTZ but attenuated when MM cells adhered to BMSCs or FN. The findings of this study imply that activation of TLR1/2 downregulates FLC production in MM cells even in the context of bone marrow microenvironment components and suggest that targeting some TLRs such as TLR1/2 might have therapeutic potential.

Autism Spectrum Disorder (ASD) comprises a group of neurodevelopmental disorders characterized by alterations in communication, repetitive behaviors and impaired socialization. The precise etiology and pathogenesis remain unclear, and there is currently no effective treatment for this condition. Emerging research highlights the role of immune dysregulation in ASD pathophysiology. Mast cells (MCs) are immune cells traditionally associated with allergic diseases but also play a crucial role in other inflammatory and immune processes. Increased MC activity may be linked to the development of ASD in certain patients. This review explores the potential mechanisms involving MCs in ASD, including neuroinflammation, autoimmunity, and disruptions in the gut-brain axis. Evidence suggests that MC mediators such as histamine, serotonin, and cytokines influence neuroinflammatory pathways that are altered in ASD, and MCs can interact with other immune and neuronal cells contributing to ASD symptoms. Autoimmunity is frequently present in both ASD patients and their mothers, and MCs could originate these processes by modulation of lymphocyte populations or secretion of self-antigens. Finally, MC involvement in gut permeability and microbiota dysbiosis underscores their role in gastrointestinal comorbidities frequent in ASD. Targeting MC activity through pharmacological agents offers promising therapeutic avenues. This comprehensive review sheds light on immune-mediated processes underlying ASD and discusses potential future strategies for intervention.

The basophil activation test (BAT) is gaining increasing relevance as an ex vivo functional assay in allergy to evaluate IgE-mediated hypersensitivity reactions to food allergens, venoms, and drugs and to monitor tolerance induction. Establishing universal standard operating protocols has been difficult, due to several challenges including variable activation markers, positive control selection, the need for processing fresh blood samples, and the existence of non-releasing individuals. In oncology, BAT is also an emerging promising diagnostic and management tool to assess hypersensitivity reactions to biologics and chemotherapy agents, monitor drug tolerance in desensitisation, and predict and address the safety of novel anti-cancer IgE-based therapeutics. This position paper highlights the emerging significance of BAT in AllergoOncology, in facilitating therapy monitoring, biomarker discovery, and risk stratification. Capitalising on long-acquired expertise in the development of BAT for allergy, we propose research directions and routes to clinical applications of this highly promising tool in AllergoOncology. We advocate the need for enhanced focus on addressing standardisation challenges and leveraging outputs for precision medicine. By linking allergy and oncology, the key remaining limitations can be addressed, with the aim of realising the significant promise of BAT as a robust tool to enhance personalised care in allergy and AllergoOncology.

The gut mucosa serves as an essential interface between the internal and external environment, providing a continuous barrier against possible harmful luminal content. The regulation of this protective function is controlled by immune-mediated and non-immune mechanisms, wherein mast cells (MCs) play a key role. These versatile immune cells are strategically located in the lining of the gastrointestinal (GI) tract, where they help maintain the integrity of the intestinal barrier, regulate blood flow, control the entry of immune cells into tissues, and participate in various physiological processes, such as wound healing and intestinal peristalsis. However, excessive MC activation may disturb the gut balance, which could cause a "leaky gut", where increased permeability of the intestinal lining allows substances to pass into the bloodstream, causing various health problems. Studies have confirmed an increased presence of MCs in the intestinal lining of individuals with compromised barriers, as seen in conditions like gastrointestinal diseases (GIDs). Hence, precise regulation of MC activity is essential for maintaining intestinal health and limiting disease progression. In this review, we aim to offer a comprehensive and current overview of the role of MCs in GIDs by delving into their origins, functions, and interactions in the GI environment. We explore the "leaky gut" concept, examining how MCs influence the intestinal barrier and its association with GIDs. Additionally, we describe the latest advancements in MC research, including targeted therapies and potential future directions.

Background: We previously proposed the whey protein beta-lactoglobulin (BLG) loaded with iron-siderophore complexes as the active principle in the farm protective effect against allergies. A lozenge as food for specific medical purposes (FSMP) was formulated to assess its therapeutical efficacy in BALB/c mice and in-vitro experiments. Methods: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic to milk, or tolerating milk, was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the lozenge or placebo, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with holoBLG in conjugation with catechin-iron complexes as ligands in a dietary supplement or with the apoBLG. Results: One major IgE- and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk allergic children and degranulation of mast cells. In mice, only supplementation with the lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. Conclusion: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen nonspecific manner, thereby conferring immune resilience against allergic symptoms.

Derived from the myeloid lineage, granulocytes, including basophils, eosinophils, and neutrophils, along with mast cells, play important, often disparate, roles across the allergic disease spectrum. While these cells and their mediators are commonly associated with allergic inflammation, they also exhibit several functions either promoting or restricting tumor growth. In this Position Paper we discuss common granulocyte and mast cell features relating to immunomodulatory functions in allergy and in cancer. We highlight key mechanisms which may inform cancer treatment and propose pertinent areas for future research. We suggest areas where understanding the communication between granulocytes, mast cells, and the tumor microenvironment, will be crucial for identifying immune mechanisms that may be harnessed to counteract tumor development. For example, a comprehensive understanding of allergic and immune factors driving distinct neutrophil states and those mechanisms that link mast cells with immunotherapy resistance, might enable targeted manipulation of specific subpopulations, leading to precision immunotherapy in cancer. We recommend specific areas of investigation in AllergoOncology and knowledge exchange across disease contexts to uncover pertinent reciprocal functions in allergy and cancer and allow therapeutic manipulation of these powerful cell populations. These will help address the unmet needs in stratifying and managing patients with allergic diseases and cancer.

Studies over the past years have provided evidence that Toll-like receptor (TLRs) activation in multiple myeloma (MM) cells induces heterogeneous functional responses including cell growth and proliferation, survival or apoptosis. These effects have been suggested to be partly due to increase in secretion of cytokines such as IL-6 or IFNα among others from MM cells following TLR activation. However, whether triggering of these receptors also modulates production of immunoglobulin free light chains (FLCs) in MM cells has never been investigated. FLCs contribute largely to MM pathology. Here we explored the effect of TLR1 ligand (Pam3CSK4) alone or combined with bortezomib (BTZ) on production of FLCs in human myeloma cell lines, L363, OPM-2, U266 and NCI-H929 in the absence or presence of bone marrow stromal cells (BMSCs) or fibronectin (FN) to examine the influence of bone marrow microenvironment. Adhesion to BMSCs or FN increased secretion of FLC in MM cells. Pam3CSK4 decreased FLC production in the presence or absence of BMSCs or FN and this effect was enhanced in combination with BTZ. However, the level of reduction was lower in the presence of BMSCs or FN. Our findings imply that activation of TLR1 downregulates FLC production in MM cells even in the context of bone marrow microenvironment components and suggest that some TLRs such as TLR1 might be considered a therapeutic target especially in combined treatment protocols in MM ### Competing Interest Statement The authors have declared no competing interest.

Background: Exposure of the esophageal mucosa to food allergens can cause acute mucosal responses in patients with eosinophilic esophagitis (EoE), but the underlying local immune mechanisms driving these acute responses are not well understood. Objective: We sought to gain insight into the early transcriptomic changes that occur during an acute mucosal response to food allergens in EoE. Methods: Bulk RNA sequencing was performed on esophageal biopsy specimens from adult patients with EoE (n = 5) collected before and 20 minutes after intramucosal injection of various food extracts in the esophagus. Baseline biopsy specimens from control subjects without EoE (n = 5) were also included. Results: At baseline, the transcriptome of the patients with EoE showed increased expression of genes related to an EoE signature. After local food injection, we identified 40 genes with a potential role in the early immune response to food allergens (most notably CEBPB, IL1B, TNFSF18, PHLDA2, and SLC15A3). These 40 genes were enriched in processes related to immune activation, such as the acute -phase response, cellular responses to external stimuli, and cell population proliferation. TNFSF18 (also called GITRL), a member of the TNF superfamily that is best studied for its costimulatory effect on T cells, was the most dysregulated early EoE gene, showing a 12 -fold increase compared with baseline and an 18 -fold increase compared with a negative visual response. Further experiments showed that the esophageal epithelium may be an important source of TNFSF18 in EoE, which was rapidly induced by costimulating esophageal epithelial cells with the EoE-relevant cytokines IL -13 and TNF-a. Conclusions: Our data provide unprecedented insight into the transcriptomic changes that mediate the acute mucosal immune response to food allergens in EoE and suggest that TNFSF18 may be an important effector molecule in this response. (J Allergy Clin Immunol 2024;153:780-92.)

Mustard seeds belong to the food category of mandatory labelling due to the severe reactions they can trigger in allergic patients. However, the mechanisms underlying allergic sensitization to mustard seeds are poorly understood. The aim of this work is to study type 2 immune activation induced by the mustard seed major allergen Sin a1 via the intestinal mucosa, employing an in vitro model mimicking allergen exposure via the intestinal epithelial cells (IECs). Sin a1 was isolated from the total protein extract and exposed to IEC, monocyte derived dendritic cells (DCs) or IEC/DC co-cultures. A system of consecutive co-cultures was employed to study the generic capacity of Sin a1 to induce type 2 activation leading to sensitization: IEC/DC, DC/T-cell, T/B-cell and stem cell derived mast cells (MCs) derived from healthy donors. Immune profiles were determined by ELISA and flow cytometry. Sin a1 activated IEC and induced type-2 cytokine secretion in IEC/DC co-culture or DC alone (IL-15, IL-25 and TSLP), and primed DC induced type 2 T-cell skewing. IgG secretion in the T-cell/B-cell phase was enhanced in the presence of Sin a1 in the first stages of the co-culture. Anti-IgE did not induce degranulation but promoted IL-13 and IL-4 release by MC primed with the supernatant from B-cells co-cultured with Sin a1-IEC/DC or -DC primed T-cells. Sin a1 enhanced the release of type-2 inflammatory mediators by epithelial and dendritic cells; the latter instructed generic type-2 responses in T-cells that resulted in B-cell activation, and finally MC activation upon anti-IgE exposure. This indicates that via activation of IEC and/or DC, mustard seed allergen Sin a1 is capable of driving type 2 immunity which may lead to allergic sensitization. Mustard seed allergen Sin a1 may activate mucosal barrier human intestinal epithelial cells (A) and/or dendritic cells (B), to initiate allergic sensitization by allowing consecutive type 2 activation of DC/T-cells, T/B-cells, and/or mast cells.

BackgroundThe global demand of sustainable food sources leads to introduction of novel foods on the market, which may pose a risk of inducing allergic sensitization. Currently there are no validated in vitro assays mimicking the human mucosal immune system to study sensitizing allergenicity risk of novel food proteins. The aim of this study was to introduce a series of sequential human epithelial and immune cell cocultures mimicking key immune events after exposure to the common food allergen ovalbumin from intestinal epithelial cell (IEC) activation up to mast cell degranulation.MethodsThis in vitro human mucosal food sensitizing allergenicity model combines crosstalk between IEC and monocyte-derived dendritic cells (moDC), followed by coculture of the primed moDCs with allogenic naïve CD4+ T cells. During subsequent coculture of primed CD4+ T cells with naïve B cells, IgE isotype-switching was monitored and supernatants were added to primary human mast cells to investigate degranulation upon IgE crosslinking. Mediator secretion and surface marker expression of immune cells were determined.ResultsOvalbumin activates IEC and underlying moDCs, both resulting in downstream IgE isotype-switching. However, only direct exposure of moDCs to ovalbumin drives Th2 polarization and a humoral B cell response allowing for IgE mediated mast cell degranulation, IL13 and IL4 release in this sequential DC-T cell-B cell-mast cell model, indicating also an immunomodulatory role for IEC.ConclusionThis in vitro coculture model combines multiple key events involved in allergic sensitization from epithelial cell to mast cell, which can be applied to study the allergic mechanism and sensitizing capacity of proteins.

To the Editor: Since the early nineties several approaches have been tested to develop safe and effective allergen immunotherapy (AIT) for the treatment of peanut allergy, starting with subcutaneous AIT with aqueous extract, followed by hypo-allergenic major allergens produced in bacteria that were administered rectally. These approaches were abandoned, mainly because of too many severe side-effects. Other administration routes that are considered to be safer, like sublingual and epicutaneous, have not yet reached the market. The only treatment that did receive market authorization is oral immunotherapy (OIT). It is effective for desensitization, disappointing for sustained efficacy, and side-effects occur frequently.1 Furthermore, the most sensitive patients being at risk of severe life-threatening allergic reactions are unlikely to be helped with this approach, as side-effects may include severe anaphylaxis.2 Therefore, there is an urgent need to provide all patients, including the severest ones, with a safe and effective treatment. Recently, nano- and micro-particulate strategies, such as virus-like particles, have been proposed as potentially safe and effective approaches. For example, a virus-based particle expressing the peanut allergens Ara h 1 or 2, based on the Cucumber Mosaic Virus, was shown to be hypo-allergenic in a mouse model.3 Here we report the ground-breaking hypo-allergenicity of a novel non-virus-based microparticle produced in plants, that is, enveloped bioparticles (eBPs) expressing ~3000 copies of Ara h 2 on the surface of each particle. Ara h 2 eBPs were generated by transfection of Nicotiana benthamiana with Agrobacterium tumefaciens carrying Ara h 2 cDNA constructs followed by oligomerization and membrane sequences, as described before for Der p 2 eBPs.4 Quantification of Ara h 2 was performed by immuno-slot blot using an in-house polyclonal rabbit anti-nAra h 2 serum and by dot-blot using serum from an Ara h 2-sensitized patient (Figure 1A, B). Dilutions of Ara h 2-BPs were compared by densitometric scanning to a standard curve of titrated natural purified Ara h 2 (nAra h 2) that had been quantified by a protein assay (BCA). The number of Ara h 2 molecules per bioparticle was calculated based on the number of particles, determined by tunable resistive pulse sensing (TRSP), and the concentration of Ara h 2, determined by SLOT-BLOT.4 The eBP platform, first tested with house dust mite (Der p 2) and cat dander (Fel d 1) allergens, demonstrated significantly stronger immunogenicity than alum-adsorbed allergen, and close to a 1000-fold reduction in allergenicity shown by the basophil activation test (BAT) and the rat basophilic leukaemia (RBL) cell test.4, 5 In the present study, hypo-allergenicity of Ara h 2 eBPs was first evaluated by reduction of IgE binding using ImmunoCAP inhibition. A pool of sera from eight Ara h 2-sensitized patients was incubated with dilution series of either nAra h 2 or the Ara h 2 eBP, followed by quantification of IgE binding to the recombinant Ara h 2 ImmunoCAP (f423; Thermo Fisher Scientific, Uppsala, Sweden). The Ara h 2 concentration of the bioparticles on the X-axis in Figure 1C was directly based on the quantitative SLOT-BLOT (Figure 1A). Collection of sera was approved by the Institutional Review Board of the University of Colorado, Denver, all subjects or their guardians signed informed consent and, for minors, assent. The Ara h 2 eBP showed a 10,000-fold reduction in IgE binding potency compared to nAra h 2 (Figure 1C). Additionally, the degree of functional hypo-allergenicity was assessed using either CD34+ stem-cell-derived human mast cells or RBL cells which were loaded with human IgE from nine Ara h 2-sensitized patients, and subsequently incubated with soluble nAra h 2 or with Ara h 2 eBPs. Both assays demonstrated around a 10,000-fold reduction of β-hexosaminidase release, a measure for degranulation, for Ara h 2 eBPs compared to nAra h 2 (Figure 2A, B). In the case of the RBL assay, concentrations of Ara h 2 required to induce half-maximal release could be calculated, showing that the eBPs are >14,000-fold less allergenic than nAra h 2 (Figure 2C). To take patient-associated effector-cell properties into account, a BAT with basophils from five peanut allergic patients was performed, demonstrating a similar degree of hypo-allergenicity, using the established activation markers CD203c and CD63 (Figure 2D). Altogether, these data demonstrate a very substantial reduction in the capacity of Ara h 2 eBPs to induce effector cell driven allergic responses. Another aspect of hypo-allergenicity, rarely taken into account, is IgE-facilitated allergen presentation by B cells to allergen-specific Th2 cells, contributing to sustained allergen-specific Th2 responses.6 Here, we evaluated the capacity of Ara h 2 eBPs to form immune complexes with Ara h 2-specific IgE that can bind to the low-affinity IgE receptor (CD23) on CD23+ EBV-transformed B cells, serving as a surrogate read-out for the capacity of B cells to present allergens to allergen-specific T cells.7 Either nAra h 2 or Ara h 2 eBPs were pre-incubated with patient serum at 37°C, followed by addition of the EBV-transformed B cells at 4°C (to avoid internalization). When IgE forms a complex with Ara h 2, accessible IgE Fc regions present in the complex can be detected by flow cytometry on the B cells (CD23+IgE+ B cells). Soluble nAra h 2 pre-incubated with patient sera resulted in the presence of CD23+IgE+ B cells already at much lower concentrations of allergen than observed with the Ara h 2 eBPs, either after incubation with a patient serum pool of nine Ara h 2-sensitized patients or with individual sera from two different patients (Figure 2E). These results indicate that the eBPs have a > 10,000-fold reduced capacity to form IgE-Ara h 2 complexes and subsequently activate Th2 cell responses via IgE-facilitated allergen presentation by B cells. Together, these data demonstrate an impressive degree of hypo-allergenicity of Ara h 2 eBPs, at the level of IgE binding, allergic effector cell activation, and IgE-facilitated allergen-presentation. Interestingly, the IgE-binding capacity during ImmunoCAP inhibition assays was enhanced for Der p 2 eBPs and unaffected for Fel d 1 eBPs (unpublished data). These differences compared to Ara h 2 eBPs may be explained by the localization of dominant IgE epitopes, which either remain available or become shielded off in the one-directional positioning of the allergens on the surface of the particles. Nevertheless, all three allergen eBPs are exceptionally hypo-allergenic in functional assays with allergenic effector cells (mast cells, RBL, BAT).4, 5 This may be caused by insufficient FcεR cross-linking due to the restricted freedom of movement of the allergens on the eBPs, while soluble allergen can freely move around. Moreover, there is limited space for the 150–200 nm eBPs to bind to the surface of a ~ 1.5 μm large cell, decreasing effective FcεR cross-linking by steric hindrance.8 In conclusion, plant-produced Ara h 2 eBPs have a very promising safety profile that has great potential to be used as a safe subcutaneous treatment, possibly even in patients with the highest sensitivity. As a next step, a skin prick test study will have to establish whether the same degree of hypo-allergenicity is indeed confirmed in vivo. C.Castenmiller and R. van Ree conceptualized the study and wrote, revised, and edited the manuscript. C.Castenmiller, J.H. Akkerdaas, S. Versteeg, B.R. Blokhuis, M.E. Kirpas and M. Stigler designed experiments and/or acquired, interpreted, and analysed the data. L. Auger, R. Desgagnés, C. Martel, L. Mirande, B. Morel, J. Roberge, V. Stordeur, G. Tropper, L.P. Vézina, and V. Gomord contributed to the concept design, development, or manufacturing of the plant-derived bioparticle platform. S.C. Dreskin provided peanut allergic patient serum. E.C. de Jong, W.G. Shreffler, F. Redegeld, L. Aglas and R. van Ree supervised experiments. R. van Ree supervised the study. All authors critically reviewed the manuscript. This reported studies were embedded in the Siallergen consortium, which was supported by Health Holland. The work of the authors has further been supported by Angany Inc. (Quebec, Canada), the Austrian Science Funds (FWF Project P32189), the University of Salzburg priority program "Allergy-Cancer-BioNano Research Centre", and a grant from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, Bethesda, MD, USA (RO1AI165866 (SCD)). Charlotte Castenmiller, Lorenz Aglas, and Maria Stigler received contract research funding and study material from Angany Inc. Louis-P Vézina is a cofounder board member, and CEO of Angany Inc. Réjean Desgagnés, Caroline Martel, Lydia Auger, Joanie Roberge, Bertrand Morel, Virginie Stordeur, Lucie Mirande, Guy Tropper, and Véronique Gomord are employees of Angany Inc. Ronald van Ree received contract research funding and research material form Angany Inc. and besides received consulting fees and/or speaker's fees from Angany Inc. HAL Allergy BV, Citeq BV, ThermoFisher Scientific, Reacta Healthcare Ltd., Mission MightyMe, and AB Enzymes, and has stock options from Angany. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Background Human milk extracellular vesicles (EVs) affect various cell types in the gastrointestinal tract, including T cells, and play a role in the development of the newborn’s immune system by delivering specific molecular cargo to target cells. Although maternal allergic sensitization alters the composition of milk, it is unknown whether this impacts the function of milk EVs. Therefore, we analyzed the T cell modulatory capacity and compared the protein and miRNA cargoes of EVs from milk of allergic and non-allergic mothers. Methods EVs were isolated from human milk from allergic and non-allergic donors by differential centrifugation, density gradient floatation and size exclusion chromatography. Functional modulation of primary human CD4+ T cells by EVs was assessed in vitro . Proteomic analysis and small RNA sequencing was performed on milk EVs to evaluate protein and miRNA abundance and to identify cellular targets of this EV cargo in relevant T cell signaling pathways. Results T cell proliferation, activation and cytokine production were suppressed in the presence of milk EVs. Remarkably, milk EVs from allergic mothers inhibited T cell activation to a lesser extent than EVs from non-allergic mothers. Integrative multi-omics analysis identified EV cargo of which the cellular targets could be linked to differential modulation of T cell activation-associated processes . Conclusions Milk EVs from non-allergic mothers are stronger inhibitors of T cell activation compared to milk EVs from allergic mothers. This altered functionality might be linked to changes in miRNA and protein cargo that modulate T cell signaling pathways in an integrative manner.

Mast cells (MC) are hematopoietic immune cells that play a major role during allergic reactions in adults by releasing a myriad of vasoactive and inflammatory mediators. MC seed all vascularized tissues and are most prominent in organs with a barrier function such as skin, lungs, and intestines. These secreted molecules cause mild symptoms such as localized itchiness and sneezing to life-threatening symptoms (i.e., anaphylactic shock). Presently, despite the extensive research on Th2-mediated immune responses in allergic diseases in adults, we are still unable to determine the mechanisms of the role of MC in developing pediatric allergic (PA) disorders. In this review, we will summarize the most recent findings on the origin of MC and discuss the underappreciated contribution of MC in the sensitization phase to maternal antibodies during pregnancy in allergic reactions and other diseases such as infectious diseases. Then, we will lay out potential MC-dependent therapeutic strategies to be considered in future investigations to understand the remaining gaps in MC research for a better quality of life for these young patients.

Mast cells (MCs) accumulate in the epithelium of patients with eosinophilic esophagitis (EoE), an inflammatory disorder characterized by extensive esophageal eosinophilic infiltration. Esophageal barrier dysfunction plays an important role in the pathophysiology of EoE. We hypothesized that MCs contribute to the observed impaired esophageal epithelial barrier. Herein, we demonstrate that coculture of differentiated esophageal epithelial cells with immunoglobulin E-activated MCs significanly decreased epithelial resistance by 30% and increased permeability by 22% compared with non-activated MCs. These changes were associated with decreased messenger RNA expression of barrier proteins filaggrin, desmoglein-1 and involucrin, and antiprotease serine peptidase inhibitor kazal type 7. Using targeted proteomics, we detected various cytokines in coculture supernatants, most notably granulocyte-macrophage colony-stimulating factor and oncostatin M (OSM). OSM expression was increased by 12-fold in active EoE and associated with MC marker genes. Furthermore, OSM receptor-expressing esophageal epithelial cells were found in the esophageal tissue of patients with EoE, suggesting that the epithelial cells may respond to OSM. Stimulation of esophageal epithelial cells with OSM resulted in a dose-dependent decrease in barrier function and expression of filaggrin and desmoglein-1 and an increase in protease calpain-14. Taken together, these data suggest a role for MCs in decreasing esophageal epithelial barrier function in EoE, which may in part be mediated by OSM.

The accumulation of senescent cells drives inflammaging and increases morbidity of chronic inflammatory lung diseases. Immune responses are built upon dynamic changes in cell metabolism that supply energy and substrates for cell proliferation, differentiation, and activation. Metabolic changes imposed by environmental stress and inflammation on immune cells and tissue microenvironment are thus chiefly involved in the pathophysiology of allergic and other immune-driven diseases. Altered cell metabolism is also a hallmark of cell senescence, a condition characterized by loss of proliferative activity in cells that remain metabolically active. Accelerated senescence can be triggered by acute or chronic stress and inflammatory responses. In contrast, replicative senescence occurs as part of the physiological aging process and has protective roles in cancer surveillance and wound healing. Importantly, cell senescence can also change or hamper response to diverse therapeutic treatments. Understanding the metabolic pathways of senescence in immune and structural cells is therefore critical to detect, prevent, or revert detrimental aspects of senescence-related immunopathology, by developing specific diagnostics and targeted therapies. In this paper, we review the main changes and metabolic alterations occurring in senescent immune cells (macrophages, B cells, T cells). Subsequently, we present the metabolic footprints described in translational studies in patients with chronic asthma and chronic obstructive pulmonary disease (COPD), and review the ongoing preclinical studies and clinical trials of therapeutic approaches aiming at targeting metabolic pathways to antagonize pathological senescence. Because this is a recently emerging field in allergy and clinical immunology, a better understanding of the metabolic profile of the complex landscape of cell senescence is needed. The progress achieved so far is already providing opportunities for new therapies, as well as for strategies aimed at disease prevention and supporting healthy aging.

AllergyEarly View LETTEROpen Access In vivo and ex vivo inflammatory responses of the esophageal mucosa to food challenge in adults with eosinophilic esophagitis Maria L. Haasnoot, Maria L. Haasnoot Department of Gastroenterology and Hepatology, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorMirelle T. A. Kleuskens, Mirelle T. A. Kleuskens orcid.org/0000-0002-1366-6034 Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this authorAlejandro Lopez-Rincon, Alejandro Lopez-Rincon Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Department of Data Science, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The NetherlandsSearch for more papers by this authorMara A. P. Diks, Mara A. P. Diks Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this authorIngrid Terreehorst, Ingrid Terreehorst Department of Ear Nose Throat, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorJaap H. Akkerdaas, Jaap H. Akkerdaas Department of Experimental Immunology, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorRonald van Ree, Ronald van Ree Department of Ear Nose Throat, Amsterdam UMC, Amsterdam, The Netherlands Department of Experimental Immunology, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorMarleen T. J. van Ampting, Marleen T. J. van Ampting Danone Nutricia Research, Utrecht, The NetherlandsSearch for more papers by this authorJohan Garssen, Johan Garssen Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Danone Nutricia Research, Utrecht, The NetherlandsSearch for more papers by this authorFrank A. Redegeld, Frank A. Redegeld Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this authorBetty C. A. M. van Esch, Betty C. A. M. van Esch Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Danone Nutricia Research, Utrecht, The NetherlandsSearch for more papers by this authorAlbert J. Bredenoord, Corresponding Author Albert J. Bredenoord [email protected] Department of Gastroenterology and Hepatology, Amsterdam UMC, Amsterdam, The Netherlands Correspondence Albert J. Bredenoord, Department of Gastroenterology and Hepatology, Amsterdam UMC, PO Box 22660, 1100 DD Amsterdam, The Netherlands. Email: [email protected] Maria L. Haasnoot and Mirelle T.A. Kleuskens are shared first author. Betty C.A.M. van Esch and Albert J. Bredenoord are shared last author.Search for more papers by this author Maria L. Haasnoot, Maria L. Haasnoot Department of Gastroenterology and Hepatology, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorMirelle T. A. Kleuskens, Mirelle T. A. Kleuskens orcid.org/0000-0002-1366-6034 Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this authorAlejandro Lopez-Rincon, Alejandro Lopez-Rincon Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Department of Data Science, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The NetherlandsSearch for more papers by this authorMara A. P. Diks, Mara A. P. Diks Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this authorIngrid Terreehorst, Ingrid Terreehorst Department of Ear Nose Throat, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorJaap H. Akkerdaas, Jaap H. Akkerdaas Department of Experimental Immunology, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorRonald van Ree, Ronald van Ree Department of Ear Nose Throat, Amsterdam UMC, Amsterdam, The Netherlands Department of Experimental Immunology, Amsterdam UMC, Amsterdam, The NetherlandsSearch for more papers by this authorMarleen T. J. van Ampting, Marleen T. J. van Ampting Danone Nutricia Research, Utrecht, The NetherlandsSearch for more papers by this authorJohan Garssen, Johan Garssen Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Danone Nutricia Research, Utrecht, The NetherlandsSearch for more papers by this authorFrank A. Redegeld, Frank A. Redegeld Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this authorBetty C. A. M. van Esch, Betty C. A. M. van Esch Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Danone Nutricia Research, Utrecht, The NetherlandsSearch for more papers by this authorAlbert J. Bredenoord, Corresponding Author Albert J. Bredenoord [email protected] Department of Gastroenterology and Hepatology, Amsterdam UMC, Amsterdam, The Netherlands Correspondence Albert J. Bredenoord, Department of Gastroenterology and Hepatology, Amsterdam UMC, PO Box 22660, 1100 DD Amsterdam, The Netherlands. Email: [email protected] Maria L. Haasnoot and Mirelle T.A. Kleuskens are shared first author. Betty C.A.M. van Esch and Albert J. Bredenoord are shared last author.Search for more papers by this author First published: 03 March 2023 https://doi.org/10.1111/all.15694AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL To the Editor, Elimination diets without the causative foods induce histological and clinical remission in patients with eosinophilic esophagitis (EoE), an allergen-driven type 2 inflammatory disease of the esophagus.1 However, current tests using skin or serum are poorly predictive of the causative foods,2 likely because the allergic inflammation may be restricted to the esophagus. We aimed to determine whether in vivo and ex vivo challenge of the esophageal mucosa with whole food extracts could yield clinically and immunologically relevant information about esophageal responses to specific foods. During endoscopy, the esophageal mucosa of 12 EoE patients was challenged by local injection of 3 common food triggers (cow's milk, wheat, and apple) and 3 foods based on patient's clinical history, and by local flush (i.e., spray) of a mixture of the 6 foods. Acute local responses were monitored for 20 min. Skin prick tests (SPT) and serum IgE measurements were also performed. Esophageal biopsies were exposed to foods in culture to analyze inflammatory mediator production, which was compared with 6 non-EoE controls. Methods are fully described in the Appendix S1. Patient characteristics are provided in Table 1. TABLE 1. EoE patient characteristics and sensitization patterns. Patient ID Sex Age (y) Atopic comorbidity PEC Patient's history Selected foods Skin prick test Serum sIgE test Esophageal mucosal injections Clinically suspected foods In addition to milk, wheat and apple Positive responses Positive responses (kU/L) Positive responses 1 M 26 Cata, doga, OASa, RhCa 45 Wheat, milk, apple, peanut, soy, tomato, Soy, peanut, tomato Wheat, soy, peanut, apple Wheat (0.44), apple (6.54), soy (0.66), peanut (1.16) Moderate narrowing/edema tomato/peanut 2 F 21 – 24 Milk, apple, hazelnut, cashew, galia melon Cashew, hazelnut, galia melon – – – 3 M 48 – 5 Wheat, apple, chicken Soy, peanut, chicken Wheat Milk (1.48), wheat (0.49) Moderate edema/rings apple/peanut, questionable milk 4 M 52 – 100 Wheat, apple, milk, orange, beer Soy, orange, beer Milk, beer Milk (2.93) Moderate edema wheat, questionable apple 5 M 44 RhCa 50 – Soy, peanut, tomatob – – – 6 M 48 OASa, RhCa 52 Wheat, apple, beer Soy, peanut, beer Peanut, soy, wheat, milk Peanut (0.72), malt (0.42) Strong response peanut/beer, moderate wheat 7 M 37 Hivesa, OASa, RhCa 14 – Soy, peanut, egg Inconclusive due to hives Wheat (0.55), apple (0.66), peanut (1.11) – 8 M 26 OASa, RhCa 30 Wheat, apple, grape, tomato, mango Mango, grape, tomato Grape, mango, wheat, apple, tomato – Moderate edema mango, questionable edema milk 9 F 28 OASa, RhCa NA Wheat, milk, apple, banana Soy, peanut, banana Wheat, banana – Not performedc 10 F 22 RhCa 50 Wheat, milk, egg Soy, peanut, egg Milk, egg Milk (2.85), wheat (0.40), egg (0.66) Moderate edema apple 11 M 41 Asthma, OASa, RhCa 20 Milk Soy, peanut, egg Apple, soy, milk, wheat Milk (3.08), apple (3.11) Contractile, muscular ring: apple, wheat and egg 12 M 30 Asthma, OASa, RhCa 12 Milk, peanut, wheat Soy, peanut egg Milk, soy, wheat Milk (2.38), wheat (1.27), apple (0.59), soy (0.84), peanut (0.85), egg (0.64) Questionable peanut Abbreviations: –, none; F, female; M, male; NA, not available; OAS, oral allergy syndrome; PEC, peak eosinophil count; RhC, rhinoconjunctivitis; sIgE, allergen-specific IgE. a SPT and/or serum sIgE proven. b Tomato instead of egg was used because egg extract was not available. c Not performed because the patient withdrew consent for the endoscopy. Foods presented in bold correspond with patient's history. All patients were previously diagnosed with EoE and presented with the typical symptoms and endoscopic signs of EoE at time of endoscopy. Of the 11 patients that underwent injections, acute responses characterized by edema, erythema, or smooth muscle contraction (determined by the formation of a muscular ring) at the injection site were observed in 8 patients after injection with apple (n = 4), peanut (n = 4), wheat (n = 3), milk (n = 2), tomato (n = 1), egg (n = 1), and mango (n = 1) (Table 1; Figure 1A). In addition, after the end of the endoscopy, 4 patients experienced dysphagia, cramping retrosternal pain or burning sensation that was similar to pain occurring after ingesting those foods. Of the in total 17 foods that induced acute responses following local injections, 9 foods (53%) corresponded with patient's clinical history, 6 (35%) with SPT results and 6 (35%) with serum IgE results. The local flush with a mixture of foods also induced acute responses but, unlike the injections, these responses were barely notable and were observed in only 4 patients (Table S1). Our results confirm the observations of our previous study that esophageal food challenge can trigger local responses in adult EoE patients.3 However, there was no clear relation between foods that induced a response by mucosal injection, and SPT or serum IgE. The fact that the foods that induce mucosal responses do not necessarily show positive SPT and/or serum IgE results, and the fact that SPT and serum IgE are poorly predictive of the causative foods,2 indicate that local esophageal challenge may indeed be needed for a better prediction of the causative foods. Nonetheless, given the moderate responsiveness to challenge by flush, the clinical challenges associated with injections, and the invasiveness for patients as endoscopic challenge can induce short lasting but severe symptoms, both challenge tests will not likely become a useful test in clinical practice. FIGURE 1Open in figure viewerPowerPoint In vivo and ex vivo responses to food challenge. (A) Acute responses to mucosal food injections. Patient 3 showed increased edema and more visible rings and furrows after injection of apple and peanut. Patient 6 showed increased edema and erythema after injection of wheat. Patient 11 showed a contractile muscular ring after injection of apple, wheat and egg. (B) Inflammatory protein levels in culture supernatant of non-challenged esophageal biopsies from EoE patients (EoE, n = 12) and controls (Ctrl, n = 6) cultured for 24 h. Mann–Whitney test: *p < .05, **p < .01, ***p < .001. (C) Inflammatory protein levels in culture supernatant of esophageal biopsies from EoE patients (EoE, n = 12) and controls (Ctrl, n = 6) exposed to saline (negative control) or the common EoE triggers apple, cow's milk or wheat extract for 24 h. Wilcoxon matched-pairs signed rank test: *p < .05, **p < .01. In contrast, a less invasive biopsy-based ex vivo food challenge test may be considered a promising tool for the identification of causative foods in EoE patients. Non-challenged EoE esophageal biopsies maintained in culture for 24 h showed increased production of total IgE (13.7 vs. 0.1 ng/mg, p = .0002), IL-5 (12.5 vs. 1.1 pg/mg, p = .0288), IL-6 (29.8 vs. 1.5 ng/mg, p = .0047), IL-8 (86.6 vs. 23.2 ng/mg, p = .0069), IL-13 (28.6 vs. 0.0 pg/mg, p = .0080), and MCP-1 (659 vs. 112 pg/mg, p = .0320) compared with non-challenged biopsies from controls (Figure 1B). Eotaxin, IL-9, and IFN-γ were below the detection limit. Analysis of protein levels based on peak eosinophil count did not provide additional insights (data not shown). Furthermore, when exposing biopsies to food in culture, an immunological response is triggered that may reflect the inflammatory cascade seen in EoE. Interestingly, IL-5 levels were increased after ex vivo exposure to milk (89.8 vs. 12.5 pg/mg, p = .0195), and IL-9 was increased after exposure to apple (132.3 vs. 0.0 pg/mg, p = .0039; Figure 1C). To our knowledge, we are the first to report food-specific induction of IL-5, an important factor in eosinophil trafficking,4 and IL-9, a promotor of mast cell expansion and function,5 in the inflamed esophagus of EoE patients, highlighting a potential role for both cytokines in the allergen-specific immune response in EoE. Lastly, we used a machine learning approach6 to study whether the ex vivo challenge test can better discriminate clinically suspected (as provided in Table 1) from non-suspected foods than the conventional SPT and serum IgE. Indeed, the ex vivo challenge test outperformed SPT/serum IgE with an AUC of 0.64 vs. 0.5 (Figure S1), evidencing sufficient discriminative scores.7 Performing food re-challenges based on the ex vivo results was beyond the scope of this study. This study has limitations. Our study was conducted in a small cohort, and the tested foods were not proven by elimination diets. Extending the current study in a larger cohort of EoE patients in which causative and safe foods have been identified is needed to shed more light on the usefulness of the ex vivo test to identify causative foods and guide elimination diets. Furthermore, EoE is patchy in biopsies. Normalization of cytokine levels for epithelial/immune cell composition of the biopsies is therefore needed for standardization of the ex vivo test. In conclusion, we demonstrated that results of food challenge using esophageal tissue provide distinct results from tests using skin and serum and may better reflect clinical response to food exposure. Esophageal biopsy tissue culture is a functional model of EoE and could potentially be used as an ex vivo model for esophageal food challenge to (i) study the food-induced immune response and (ii) identify causative foods to guide elimination diets, and therefore warrants further validation and development. FUNDING INFORMATION This research is funded within the Partnership between NWO domain Applied and Engineered Sciences and Danone Nutricia Research and with additional financial support from Topsector Agri and Food, project number 16495 with the acronym LOIRE. AJB is supported by Vidi grant 91718300 from the Netherlands Organization for Scientific Research NWO. CONFLICT OF INTEREST STATEMENT AJB received research funding from Norgine, Thelial, SST and received speaker and/or consulting fees from Laborie, Arena, EsoCap, Medtronic, Dr. Falk Pharma, Calypso Biotech, Alimentiv, Sanofi, Reckett, Regeneron and AstraZeneca (all unrelated to this work). RvR received consultancy fees from HAL Allergy, Citeq, Angany, Reacta Healthcare, Mission MightyMe and AB Enzymes, speaker fees from HAL Allergy, ThermoFisher Scientific and ALK, and has stock option of Angany (all unrelated to this work). MTJVA is employed by Danone Nutricia Research. JG and BCAMVE are partly employed by Danone Nutricia Research. All other authors report no conflict of interest. Supporting Information Filename Description all15694-sup-0001-supinfo.docxWord 2007 document , 854.8 KB Appendix S1: Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Gonsalves N, Yang GY, Doerfler B, Ritz S, Ditto AM, Hirano I. Elimination diet effectively treats eosinophilic esophagitis in adults; food reintroduction identifies causative factors. Gastroenterology. 2012; 142(7): 1451- 9.e1. quiz e14-5. 2Philpott H, Nandurkar S, Royce SG, Thien F, Gibson PR. Allergy tests do not predict food triggers in adult patients with eosinophilic oesophagitis. A comprehensive prospective study using five modalities. Aliment Pharmacol Ther. 2016; 44(3): 223- 233. 3Warners MJ, Terreehorst I, van den Wijngaard RM, et al. 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