Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility1. The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (TH2) cells within a distinct microanatomical niche. TH2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as TH2-interacting fascial fibroblasts (TIFFs), which expand in response to TH2 cytokines to form subcutaneous fibrous bands. Activation of the TH2-TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified TH2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life.
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.
Copyright © 2021 Üzülmez, Kalic, Mayr, Lengger, Tscheppe, Radauer, Hafner, Hemmer and Breiteneder.

Around 25% of food allergic persons in Central Europe suffer from carrot allergy caused by the major carrot allergen Dau c 1. Three different isoallergens, Dau c 1.01, Dau c 1.02 and Dau c 1.03 are identified. However, information about the qualitative and quantitative composition of natural (n)Dau c 1 is scarce.
The new carrot allergen Dau c 1.0401 is identified on the mRNA and protein level by RT-PCR and mass spectrometry. It displays only around 60% sequence identity to the other known Dau c 1 isoallergens. NMR and CD-spectra are typical for a well-folded protein containing both α-helices and β-strands. It showed a poor refolding capacity after incubation at 95 °C. IgE-binding is impaired in immunoblots, whereas in inhibition assays IgE binding to soluble Dau c 1.0401 is detected and it clearly provoked a response in mediator release assays.
Dau c 1.0401 is a new isoallergen which contributes to the allergenicity of carrots. The absence of immunoreactivity in immobilized assays indicates that IgE binding is impaired when the protein is blotted on a solid phase. Altogether, the results point out that its allergenicity can be reduced upon carrot processing.
© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.

The prevalence of fish allergy among fish-processing workers is higher than in the general population, possibly due to sensitization via inhalation and higher exposure. However, the response of the bronchial epithelium to fish allergens has never been explored. Parvalbumins (PVs) from bony fish are major sensitizers in fish allergy, while cartilaginous fish and their PVs are considered less allergenic. Increasing evidence demonstrates that components other than proteins from the allergen source, such as low molecular weight components smaller than 3 kDa (LMC) from pollen, may act as adjuvants during allergic sensitization. We investigated the response of bronchial epithelial cells to PVs and to LMC from Atlantic cod, a bony fish, and gummy shark, a cartilaginous fish. Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with fish PVs and/-or the corresponding fish LMC. Barrier integrity, transport of PVs across the monolayers and release of mediators were monitored. Intact PVs from both the bony and the cartilaginous fish were rapidly internalized by the cells and transported to the basolateral side of the monolayers. The PVs did not disrupt the epithelial barrier integrity nor did they modify the release of proinflammatory cytokines. In contrast, LMC from both fish species modified the physical and immunological properties of the epithelial barrier and the responses differed between bony and cartilaginous fish. While the barrier integrity was lowered by cod LMC 24 h after cell stimulation, it was increased by up to 2.3-fold by shark LMC. Furthermore, LMC from both fish species increased basolateral and apical release of IL-6 and IL-8, while CCL2 release was increased by cod but not by shark LMC. In summary, our study demonstrated the rapid transport of PVs across the epithelium which may result in their availability to antigen presenting cells required for allergic sensitization. Moreover, different cell responses to LMC derived from bony versus cartilaginous fish were observed, which may play a role in different allergenic potentials of these two fish classes.
Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

Allergen-specific immunotherapy is the only curative treatment of honeybee venom (HBV) allergy, which is able to protect against further anaphylactic sting reactions. Recent analyses on a molecular level have demonstrated that HBV represents a complex allergen source that contains more relevant major allergens than formerly anticipated. Moreover, allergic patients show very diverse sensitization profiles with the different allergens. HBV-specific immunotherapy is conducted with HBV extracts which are derived from pure venom. The allergen content of these therapeutic extracts might differ due to natural variations of the source material or different down-stream processing strategies of the manufacturers. Since variations of the allergen content of therapeutic HBV extracts might be associated with therapeutic failure, we adressed the component-resolved allergen composition of different therapeutic grade HBV extracts which are approved for immunotherapy in numerous countries. The extracts were analyzed for their content of the major allergens Api m 1, Api m 2, Api m 3, Api m 5 and Api m 10. Using allergen-specific antibodies we were able to demonstrate the underrepresentation of relevant major allergens such as Api m 3, Api m 5 and Api m 10 in particular therapeutic extracts. Taken together, standardization of therapeutic extracts by determination of the total allergenic potency might imply the intrinsic pitfall of losing information about particular major allergens. Moreover, the variable allergen composition of different therapeutic HBV extracts might have an impact on therapy outcome and the clinical management of HBV-allergic patients with specific IgE to particular allergens.