Nonatopic Donors Research Articles

Autologous Th2-polarized lymphocytes induce atopic dermatitis lesions in non-atopic human skin xenotransplants.

The key signals that suffice to induce atopic dermatitis (AD) in human skin remain incompletely understood. Also, current mouse models reflect human AD only unsatisfactorily. Therefore, we have asked whether a humanized AD mouse model can be developed that reflects human AD more faithfully and permit to identify key signals that suffice to induce AD lesions in previously healthy human skin in vivo. Healthy human skin from non-atopic donors was transplanted onto SCID/beige mice. After xenotransplant reinnervation by mouse sensory nerve fibers had occurred, mixed autologous human Th2 CD4+ and Tc2 CD8+ T cells that had been pretreated in vitro with IL-2, IL-4, and LPS were injected intradermally into the xenotransplants without skin barrier disruption. Injected non-atopic xenotransplants rapidly developed a morphological, functional, and immunological phenocopy of human AD lesions regarding skin barrier defects, immunopathology including intraepidermal eosinophils, mast cell activation, increase of thymic stromal lymphopoietin, eotaxin-1 and type 2 cytokine circuits, and even showed characteristic neuroimmunological abnormalities such as ß2-adrenergic receptor downregulation. The experimentally induced AD lesions in human skin responded to standard AD therapy (topical dexamethasone or tacrolimus; systemic anti-IL-4Rα antibody [dupilumab]), and relapsed when neurogenic skin inflammation was induced by exposing mice to perceived stress. This new animal model uniquely mimics the complexity of human AD and its clinical response to standard therapy and psychoemotional stressors in vivo, and shows that Th2-polarized lymphocytes associated with excessive IL-4Rα-mediated signaling suffice to induce human AD skin lesions, while atopy and epidermal barrier disruption are dispensable.

323 A Novel, Stress-Responsive and Clinically Predictive Humanized Mouse Model of Atopic Dermatitis: Autologous Th2-Polarized Lymphocytes Suffice to Induce Characteristic Lesions in Healthy Non-Atopic Human Skin Xenotransplants In Vivo

Clinically relevant in vivo models for atopic dermatitis (AD) are needed, in which the minimal signals required for AD induction in human skin can be defined and candidate therapeutics explored. Here, we report a novel humanized AD mouse model that uses split-thickness skin from healthy, non-atopic female human donors xenotransplanted onto SCID/beige mice and injected with autologous, Th2-polarized PBMCs. After intradermal injection of PBMCs preincubated only with IL-2, IL-4 and LPS, the xenotransplants develop a phenocopy of human AD lesions, including epidermal hyperplasia, hyperproliferation, erythema, and skin barrier impairment (reduced filaggrin and claudin-1 expression, altered corneocyte nanotexture [atomic force miocroscopy]).

Potent Anti-Inflammatory Effects of Tetracyclines on Human Eosinophils.

Eosinophils are potent pro-inflammatory cells. Not only in allergic diseases but also in other diseases there is a need for treatment strategies to induce resolution of eosinophil-mediated inflammation. During the last years beneficial non-antibiotic activities of tetracyclines (TCNs) have been shown in different diseases in which eosinophils play a role, for example, asthma and bullous pemphigoid. The working mechanism of these effects remains to be clarified. Aim of the present study was to investigate the effects of TCNs on eosinophils. Flow cytometry analysis of apoptosis, mitochondrial membrane potential, activation of caspases, intracellular H2O2 and calcium, surface expression of eosinophil activation markers was performed in highly purified peripheral blood eosinophils of non-atopic donors. Tetracycline hydrochloride, minocycline and doxycycline significantly induced eosinophil apoptosis. All TCNs were able to significantly overcome the strong survival enhancing effects of pro-eosinophilic cytokines and staphylococcus aureus enterotoxins. Tetracycline hydrochloride induced eosinophil apoptosis was accompanied by intracellular production of hydrogen peroxide, loss of mitochondrial membrane potential and activation of caspases. Moreover, tetracycline hydrochloride significantly down regulated eosinophil surface expression of CD9 and CD45, and of the activation markers CD11b and CD69, but not of CD54, CD63, or CD95. Our data, propably for the first time, point to a potent anti-inflammatory role of TCNs on eosinophils.

Distinction between peanut allergy and tolerance by characterization of B cell receptor repertoires.

BackgroundSpecific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin‐specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors.Methods2S albumin‐binding B‐cells were single‐cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non‐atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering.ResultsThe frequency of 2S albumin‐binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non‐atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non‐specific mAbs (9/10) were mainly derived from non‐atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non‐atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances.ConclusionsPeanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs).

Curability and transferability of atopy with allogeneic hematopoietic cell transplantation

Atopy is excessive production of IgE in response to allergens. We evaluated in patients undergoing allogeneic hematopoietic cell transplantation (HCT) the following hypotheses: (1) Atopy is "curable" in atopic patients receiving HCT from a nonatopic donor (D-R+), and (2) Atopy is transferable from atopic donors to nonatopic recipients (D+R-). Atopic patients with atopic donors (D+R+) and non-atopic patients with non-atopic donors (D-R-) served as controls. We measured levels of multiallergen-specific IgE (A-IgE, atopy defined as ≥0.35 kUA/L) in sera from 54 patients and their donors pre HCT and from the patients at ≥2 years post HCT. Only 7/12 (58%) D- R+ patients became nonatopic after HCT. Only 1/11 (9%) D+R- patients became atopic. Eleven of 13 (85%) D-R- patients remained nonatopic. Unexpectedly, 11/18 (61%) D+R+ patients became nonatopic. In conclusion, contrary to our hypothesis and previous reports, the "cure" of atopy may occur in only some D-R+ patients and the transfer of atopy may occur rarely. The "cure" may not be necessarily due to the exchange of atopic for nonatopic immune system, as the "cure" may also occur in D+R+ patients.

Physical and immunological barrier of human primary nasal epithelial cells from non-allergic and allergic donors

The epithelial cell-derived cytokine milieu has been discussed as a “master switch” in the development of allergic disease.To understand the role of innate immune response in nasal epithelial cells during allergic inflammation, we created and established a fast and minimally invasive method to isolate and culture human nasal epithelial cells from clinically and immunologically well characterized patients. Human nasal epithelial cells from non-atopic volunteers and from allergic rhinitis patients were compared in respect to their growth, barrier integrity, pattern recognition, receptor expression, and immune responses to allergens and an array of pathogen-associated molecular patterns and inflammasome activators.Cells from nasal scrapings were clearly identified as nasal epithelial cells by staining of pan-Cytokeratin, Cytokeratin-14 and Tubulin. Additionally, Mucin 5AC staining revealed the presence of goblet cells, while staining of tight-junction protein Claudin-1, Occludin and ZO-1 showed the ability of the cells to form a tight barrier. Cells of atopic donors grew slower than cells of non-atopic donors. All nasal epithelial cells expressed TLR1-6 and 9, yet the expression of TLR-9 was lower in cells from allergic rhinitis (AR) donors. Additionally, epithelial cells from AR donors responded with a different TLR expression pattern to stimulation with TLR ligands. TLR-3 was the most potent modulator of cytokine and chemokine secretion in all human nasal epithelial cells (HNECs). The secretion of IL-1β, CCL-5, IL-8, IL-18 and IL-33 was elevated in HNECs of AR donors as compared to cells of non-atopic donors. This was observed in the steady-state (IL-18, IL-33) as well as under stimulation with TLR ligands (IL-18, IL-33, CCL-5, IL-8), aqueous pollen extracts (IL-18, IL-33), or the inflammasome activator Nigericin (IL-1β).In conclusion, nasal epithelial cells of AR donors show altered physical barrier responses in steady-state and in response to allergen stimulation. Cells of AR donors show increased expression of pro-inflammatory and IL-1 family cytokines at baseline and under stimulation, which could contribute to a micromilieu which is favorable for Th2.

The pseudoallergen receptor MRGPRX2 on peripheral blood basophils and eosinophils: Expression and function.

Mas-related G protein-coupled receptor X2 (MRGPRX2) is regarded as a mast cell-specific receptor mediating non-IgE-dependent activation. We aimed to investigate whether human basophils and eosinophils express functional MRGPRX2. Flow cytometry, immunocytochemistry, immunofluorescence, Western blot, and RT-PCR were performed in highly purified peripheral blood basophils and eosinophils of atopic and nonatopic donors. To assess functional activity, fluorescent avidin-based degranulation assay, calcium mobilization, cytokine production in supernatants, assessment of viability/apoptosis, and tricolor granulocyte activation test were used. MRGPRX2 was significantly expressed by basophils and eosinophils but not neutrophils. Functional capacity was shown by anti-MRGPRX2 mAb-induced calcium influx and concentration-dependent induction of degranulation. Sequential stimulation in the calcium mobilization assay gave no evidence for desensitization or receptor internalization. Anti-MRGPRX2 mAb significantly promoted survival. Inhibition of apoptosis could be due to released IL-3, IL-5, and GM-CSF found in supernatants. Short-term incubation with IL-3 dose-dependently upregulated MRGPRX2 expression in both, stimulation for 24hours with anti-IgE, C5a, fMLP, and IL-3 in basophils and by IL-3, IL-5, and IL-33 in eosinophils. Among known mast cell MRGPRX2 agonists ciprofloxacin but not PMX-53 was functional on basophils and eosinophils. In basophils of allergic subjects, tricolor granulocyte activation test using grass pollen demonstrated MRGPRX2 upregulation associated with degranulation and CD63 expression. Unraveling the regulation and signaling mechanisms of MRGPRX2 on basophils and eosinophils might enable the development of new therapeutic strategies to prevent or inhibit allergic and nonallergic hypersensitivity. Moreover, addressing MRGPRX2 might have potential for diagnostic purposes in (drug) hypersensitivity.

Excretion of Dietary Cow's Milk Derived Peptides Into Breast Milk.

Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125–135) and β-casein f(81–92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.

Rapid and comprehensive discovery of unreported shellfish allergens using large-scale transcriptomic and proteomic resources

[Extract] Discovery of allergens in various food and inhalant sources is central to our understanding of the molecular mechanisms of allergic reactions. Allergen characterization is the most important underlying factor for better patient management and the design and development of novel immunotherapeutics.1 Of the "Big eight" allergen food groups, shellfish presents a unique challenge in terms of allergen discovery due to the large number and diversity of consumed species, leading to heterogeneity of allergen structure and cross-reactivity among various sources. At present, 31 crustacean allergens have been officially registered in the World Health Organization and International Union of Immunological Societies Allergen Nomenclature Database as compared with 4 mollusk allergens due to pitfalls in current allergy discovery approaches. Cosensitization of patients with crustacean and mollusk allergy is often described; however, the current diagnostic approaches to manage these patients are not based on sufficient molecular knowledge of these shellfish allergens.

The Innate Immune Cross Talk between NK Cells and Eosinophils Is Regulated by the Interaction of Natural Cytotoxicity Receptors with Eosinophil Surface Ligands.

Previous studies suggested that the cross talk between NK cells and other cell types is crucial for the regulation of both innate and adaptive immune responses. In the present study, we analyzed the phenotypic and functional outcome of the interaction between resting or cytokine-activated NK cells and eosinophils derived from non-atopic donors. Our results provide the first evidence that a natural cytotoxicity receptor (NCR)/NCR ligand-dependent cross talk between NK cells and eosinophils may be important to upregulate the activation state and the effector function of cytokine-primed NK cells. This interaction also promotes the NK-mediated editing process of dendritic cells that influence the process of Th1 polarization. In turn, this cross talk also resulted in eosinophil activation and acquisition of the characteristic features of antigen-presenting cells. At higher NK/eosinophil ratios, cytokine-primed NK cells were found to kill eosinophils via NKp46 and NKp30, thus suggesting a potential immunoregulatory role for NK cells in dampening inflammatory responses involving eosinophils.

The innate immune cross talk between NK cells and eosinophils is regulated by the interaction of Natural Cytotoxicity Receptors with eosinophil surface ligands

Previous studies suggested that the cross talk between NK cells and other cell types is crucial for the regulation of both innate and adaptive immune responses [1,2]. In the present study, we analyzed the phenotypic and functional outcome of the interaction between resting or cytokine-activated NK cells and eosinophils derived from non-atopic donors. Our results provide the first evidence that an NCR/NCR ligand-dependent cross talk between NK cells and eosinophils may be important to up regulate the activation state and the effector function of cytokine-primed NK cells. This interaction also promotes the NK-mediated editing process of DCs that influences the process of Th1 polarization. In turn, this cross talk also resulted in eosinophil activation and acquisition of the characteristic features of antigen presenting cells. At higher NK/eosinophil ratios, cytokine-primed NK cells were found to kill eosinophils via NKp46 and NKp30, thus suggesting a potential immunoregulatory role for NK cells in dampening inflammatory responses involving eosinophils.

α-NAC-Specific Autoreactive CD8+ T Cells in Atopic Dermatitis Are of an Effector Memory Type and Secrete IL-4 and IFN-γ.

Autoreactivity may play a critical role in the chronification of atopic dermatitis (AD). Several studies showed that AD patients produce IgE Abs specific for autoantigens, and we described Th as well as CD8(+) T cells specific for the autoallergen Hom s 2, the α-chain of the nascent polypeptide-associated complex (α-NAC). This study aimed to investigate the frequency and inflammatory phenotype of autoallergen-specific CD8(+) T cells. CD8(+) T cell immunodominant epitopes of α-NAC were mapped by applying prediction softwares, and binding affinity was confirmed by stabilization of empty MHC complexes. MHC class I tetramers were assembled and binding cells were analyzed directly ex vivo by flow cytometry and in terms of single-cell assessment by ChipCytometry. We report significantly elevated numbers of α-NAC-specific peripheral T cells in sensitized patients compared with nonatopic controls. These cells secrete IL-4 and IFN-γ, and surface markers revealed significantly elevated frequencies of circulating terminally differentiated α-NAC-specific CD8(+) T cells in patients with AD compared with nonatopic donors. The observed phenotype of α-NAC-specific CD8(+) T cells indicates a role in the pathogenesis of AD.

Basophil markers for identification and activation in the indirect basophil activation test by flow cytometry for diagnosis of autoimmune urticaria.

BackgroundThe indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c).MethodsDonor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20).ResultsFor basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity.ConclusionsFor basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

Tacrolimus use in renal and cardiac pediatric transplantation: no increased risk of food allergies

Food allergies have been shown to occur in increased prevalence in children after orthotopic liver transplantation from nonatopic donors. 1 Lebel M.J. Chapdelaine H. Paradis L. Des Roches A. Alvarez F. Increase in de novo food allergies after pediatric liver transplantation: tacrolimus vs. cyclosporine immunosuppression. Pediatr Transplant. 2014; 18: 733-739 Crossref PubMed Scopus (19) Google Scholar , 2 Nowak-Wegrzyn A.H. Sicherer S.H. Conover-Walker M.K. Wood R.A. Food allergy after pediatric organ transplantation with tacrolimus immunosuppression. J Allergy Clin Immunol. 2001; 108: 146-147 Abstract Full Text PDF PubMed Scopus (54) Google Scholar , 3 Levy Y. Davidovits M. Cleper R. Shapiro R. New-onset post-transplantation food allergy in children—is it attributable only to the immunosuppressive protocol?. Pediatr Transplant. 2009; 13: 63-69 Crossref PubMed Scopus (48) Google Scholar , 4 De Bruyne R. Dullaers M. Van Biervliet S. et al. Post-transplant food allergy in children is associated with liver and not with renal transplantation: a monocentric comparative study. Eur J Pediatr. 2013; 172: 1069-1075 Crossref PubMed Scopus (37) Google Scholar , 5 Ozdemir O. Arrey-Mensah A. Sorensen R.U. Development of multiple food allergies in children taking tacrolimus after heart and liver transplantation. Pediatr Transplant. 2006; 10: 380-383 Crossref PubMed Scopus (51) Google Scholar , 6 Brown C. Haringman N. Davies C. et al. High prevalence of food sensitisation in young children with liver disease: a clue to food allergy pathogenesis. Pediatr Allergy Immunol. 2012; 23: 771-778 Crossref PubMed Scopus (25) Google Scholar , 7 Wisniewski J. Lieberman J. Nowak-Wegrzyn A. et al. De novo food sensitization and eosinophilic gastrointestinal disease in children post liver transplantation. Clin Transplant. 2012; 26: E365-E371 Crossref PubMed Scopus (34) Google Scholar , 8 Frischmeyer-Guerrerio P.A. Wisniewski J. Wood R.A. Nowak-Wegrzyn A. Manifestations and long-term outcome of food allergy in children after solid organ transplantation. J Allergy Clin Immunol. 2008; 122: 1031-1033 Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Immunosuppression with tacrolimus (TAC) and young age at transplantation have been identified as important risk factors for the development of transplant-acquired food allergy (TAFA), although the pathophysiology remains unclear. 1 Lebel M.J. Chapdelaine H. Paradis L. Des Roches A. Alvarez F. Increase in de novo food allergies after pediatric liver transplantation: tacrolimus vs. cyclosporine immunosuppression. Pediatr Transplant. 2014; 18: 733-739 Crossref PubMed Scopus (19) Google Scholar , 2 Nowak-Wegrzyn A.H. Sicherer S.H. Conover-Walker M.K. Wood R.A. Food allergy after pediatric organ transplantation with tacrolimus immunosuppression. J Allergy Clin Immunol. 2001; 108: 146-147 Abstract Full Text PDF PubMed Scopus (54) Google Scholar , 3 Levy Y. Davidovits M. Cleper R. Shapiro R. New-onset post-transplantation food allergy in children—is it attributable only to the immunosuppressive protocol?. Pediatr Transplant. 2009; 13: 63-69 Crossref PubMed Scopus (48) Google Scholar , 4 De Bruyne R. Dullaers M. Van Biervliet S. et al. Post-transplant food allergy in children is associated with liver and not with renal transplantation: a monocentric comparative study. Eur J Pediatr. 2013; 172: 1069-1075 Crossref PubMed Scopus (37) Google Scholar , 5 Ozdemir O. Arrey-Mensah A. Sorensen R.U. Development of multiple food allergies in children taking tacrolimus after heart and liver transplantation. Pediatr Transplant. 2006; 10: 380-383 Crossref PubMed Scopus (51) Google Scholar , 6 Brown C. Haringman N. Davies C. et al. High prevalence of food sensitisation in young children with liver disease: a clue to food allergy pathogenesis. Pediatr Allergy Immunol. 2012; 23: 771-778 Crossref PubMed Scopus (25) Google Scholar , 7 Wisniewski J. Lieberman J. Nowak-Wegrzyn A. et al. De novo food sensitization and eosinophilic gastrointestinal disease in children post liver transplantation. Clin Transplant. 2012; 26: E365-E371 Crossref PubMed Scopus (34) Google Scholar , 8 Frischmeyer-Guerrerio P.A. Wisniewski J. Wood R.A. Nowak-Wegrzyn A. Manifestations and long-term outcome of food allergy in children after solid organ transplantation. J Allergy Clin Immunol. 2008; 122: 1031-1033 Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar The objective of this study was to establish whether TAFA was observed after renal and cardiac transplantations and to determine whether the type of solid organ transplanted and/or immunosuppression protocol played a role in the development of TAFA.

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor

Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

Transcriptional Analysis of Hair Follicle-Derived Keratinocytes from Donors with Atopic Dermatitis Reveals Enhanced Induction of IL32 Gene by IFN-γ

We cultured human hair follicle-derived keratinocytes (FDKs) from plucked hairs. To gain insight into gene expression signatures that can distinguish atopic dermatitis from non-atopic controls without skin biopsies, we undertook a comparative study of gene expression in FDKs from adult donors with atopic dermatitis and non-atopic donors. FDK primary cultures (atopic dermatitis, n = 11; non-atopic controls, n = 7) before and after interferon gamma (IFN-γ) treatment were used for microarray analysis and quantitative RT-PCR. Comparison of FDKs from atopic and non-atopic donors indicated that the former showed activated pathways with innate immunity and decreased pathways of cell growth, as indicated by increased NLRP2 expression and decreased DKK1 expression, respectively. Treatment with IFN-γ induced the enhanced expression of IL32, IL1B, IL8, and CXCL1 in the cells from atopic donors compared to that in cells from non-atopic donors at 24 h after treatment. IL1B expression in FDKs after IFN-γ treatment correlated with IL32 expression. We hypothesized that overexpression of IL32 in hair follicle keratinocytes of patients with atopic dermatitis would lead to the excessive production of pro-IL1β and that the activation of IL1β from pro-IL1β by inflammasome complex, in which NLRP2 protein might be involved, would be augmented. This is the first report to show enhanced induction of cytokine/chemokine genes by IFN-γ in atopic dermatitis using cultured FDKs.

Activin A and TGF-β promote TH9 cell–mediated pulmonary allergic pathology

IL-9-secreting (T(H)9) T cells are thought to represent a distinct T-cell subset. However, evidence for their functionality in disease is uncertain. To define a functional phenotype for T(H)9-driven pathology in vivo. We used fluorescence-activated cell sorting to identify circulating T(H)9 cells in atopic and nonatopic subjects. In mice we utilized a model of allergic airways disease induced by house dust mite to determine T(H)9 cell function in vivo and the role of activin A in T(H)9 generation. Allergic patients have elevated T(H)9 cell numbers in comparison to nonatopic donors, which correlates with elevated IgE levels. In a murine model, allergen challenge with house dust mite leads to rapid T(H)9 differentiation and proliferation, with much faster kinetics than for T(H)2 cell differentiation, resulting in the specific recruitment and activation of mast cells. The TGF-β superfamily member activin A replicates the function of TGF-β1 in driving the in vitro generation of T(H)9 cells. Importantly, the in vivo inhibition of T(H)9 differentiation induced by allergen was achieved only when activin A and TGF-β were blocked in conjunction but not alone, resulting in reduced airway hyperreactivity and collagen deposition. Conversely, adoptive transfer of T(H)9 cells results in enhanced pathology. Our data identify a distinct functional role for T(H)9 cells and outline a novel pathway for their generation in vitro and in vivo. Functionally, T(H)9 cells promote allergic responses resulting in enhanced pathology mediated by the specific recruitment and activation of mast cells in the lungs.

Phenotypic analysis of perennial airborne allergen‐specific CD4+ T cells in atopic and non‐atopic individuals

Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals.

Downregulation of basophil-derived IL-4 and in vivo T H2 IgE responses by serotonin and other organic cation transporter 3 ligands

Murine basophils can contribute to the T(H)2 polarization of the immune response by providing rapidly large amounts of IL-4, which suggests that pharmacologic downregulation of this cytokine might provide a strategy to attenuate pathologies associated with excessive production. We examined a number of physiological and pharmacologic ligands of the organic cation transporter 3 (OCT3), a membrane carrier of biogenic amines, for their inhibitory effect on IL-4 production by basophils, selecting the most efficient compounds for invivo evaluation in basophil-dependent experimental models. IL-4 production by basophils isolated exvivo or from bone marrow cultures was assessed in response to various stimuli with or without biogenic monoamines or pharmacologic analogs. Selected compounds were administered invivo to examine their effect on levels of circulating IgE generated during a basophil-dependent T(H)2 response and on basophil activation in mice receiving IL-33. We found a drastic decrease in IL-4 production by stimulated basophils on exposure to serotonin (5-hydroxytryptamine [5-HT]) that is taken up by basophils through the specific high-affinity transporters serotonin transporter and the polyspecific, high-capacity organic cation transporter 3 (OCT3; or Slc22a3) but inhibits their function exclusively through the latter. This downregulation is likewise observed invivo in response to 5-HT and other OCT3 ligands, as well as in human basophils sorted from PMBCs of nonatopic donors. We provide evidence for a new means of downregulating IL-4 production by basophils, both invitro and invivo, through OCT3 targeted by 5-HT and pharmacologic ligands.