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Background The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. Objective We aimed to evaluate the capacity of EPIT to provide protection against cashew‐induced anaphylaxis in a relevant mouse model. Methods The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew‐sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast‐cell proteases (mMCP)‐1/7, were quantified. Results Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast‐cell reactivity as attested by the reduction of mMCP‐1/7 in plasma, suggesting that EPIT specifically decrease IgE‐mediated anaphylaxis. Conclusion We demonstrate that EPIT markedly reduced IgE‐mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy., This study demonstrates a newly developed robust mouse model of IgE‐mediated cashew anaphylaxis. Cashew‐sensitized mice are treated with epicutaneous patches containing cashew protein extract (cashew patches) or excipient (placebo patches) for 16 weeks. Mice treated with cashew patches present a decrease in cashew‐specific Th2 response and a decrease in mast‐cell reactivity and anaphylaxis symptoms following oral challenge. Abbreviations: EPIT, epicutaneous immunotherapy.

Epicutaneous immunotherapy (EPIT) protocols have recently been developed to restore tolerance in patients with food allergy. The mechanisms by which EPIT protocols promote desensitization rely on a profound immune deviation of pathogenic T- and B-cell responses.To date, little is known about the contribution of skin dendritic cells (skDCs) to T-cell remodeling and EPIT efficacy.We capitalized on a preclinical model of food allergy to ovalbumin (OVA) to characterize the phenotype and functions of OVAsup+/supskDCs throughout the course of EPIT.Our results showed that both Langerhans cells and dermal conventional cDC1 and cDC2 subsets retained their ability to capture OVA in the skin and to migrate toward the skin-draining lymph nodes during EPIT. However, their activation/maturation status was significantly impaired, as evidenced by the gradual and selective reduction of CD86, CD40, and OVA protein expression in respective subsets. Phenotypic changes during EPIT were also characterized by a progressive diversification of single-cell gene signatures within each DC subset. Interestingly, we observed that OVAsup+/supLangerhans cells progressively lost their capacity to prime CD4sup+/supTsubEFF/subcells, but gained regulatory T-cell stimulatory properties. In contrast, cDC1 were inefficient in priming CD4sup+/supTsubEFF/subcells or in reactivating TsubMEM/subcells in vitro, whereas cDC2 retained moderate stimulatory properties, and progressively biased type 2 immunity toward type 1 and type 17 responses.Our results therefore emphasize that the acquisition of distinct phenotypic and functional specializations by skDCs during EPIT is at the cornerstone of the desensitization process.

Background: Epicutaneous immunotherapy (EPIT®), currently investigated in the treatment of food allergy, needs the integrity of the skin to warrant safety and efficacy. Mutations in the gene encoding the key epidermal protein filaggrin (FLG) are risk factors for peanut allergy and disrupt the skin intergrity. We investigated the association between FLG deficiency and peanut EPIT® efficacy in a murine model. Methods: FLG mutant mice deficient in filaggrin (FLG-/-) or wild-type (WT) mice were sensitized with peanut protein extract (peanut protein) and cholera toxin. Sensitized mice received a patch per week during 8 weeks for EPIT®, using Viaskin®, and were then submitted to sustained peanut oral exposure. We assessed blood humoral and cellular responses and evaluated eosinophil infiltration in the gut mucosa. The different steps of allergen capture and transportation following deposition on the skin was also analyzed in sensitized mice. Results: Sensitization of mice was confirmed by a significant increase of specific Th2 biaised immunological responses. In sensitized mice, EPIT® significantly reduced IgE levels, splenocytes secretion of Th2 cytokines and recruitment of eosinophils in esophagus, compared to sensitized mice without epicutaneous immunotherapy. The allergen applied onto the skin of FLG-/- mice did not undergo passive skin passage or systemic delivery. Instead, the allergen was captured by skin CD205highDCs, which migrated to afferent lymph nodes, as already described in WT mice. Conclusions: EPIT® was efficient and safe in FLG-/- mice, suggesting that in Humans EPIT® keeps efficacy and safety in the presence of loss of function of FLG.

Allergen-specific immunotherapy has been proposed as an attractive strategy to actively treat food allergy using the following three different immunotherapy routes: oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy. Regulatory T cells (Tregs) have been shown to have a pivotal role in the mechanisms of immunotherapy. The aim of this study was to compare the phenotype and function of Tregs induced in peanut-sensitized BALB/c mice using these three routes of treatment. We show that although EPIT, OIT and SLIT were all able to effectively desensitize peanut-sensitized mice, they induced different subsets of Tregs. Foxp3+ Tregs were induced by the three treatment routes but with greater numbers induced by EPIT. EPIT and OIT also increased the level of LAP+ Tregs, whereas SLIT induced IL-10+ cells. The suppressive activity of EPIT-induced Tregs did not depend on IL-10 but required CTLA-4, whereas OIT acted through both mechanisms and SLIT was strictly dependent on IL-10. Moreover, the three routes influenced the homing properties of induced Tregs differently, with a larger repertoire of chemokine receptors expressed by EPIT-induced Tregs compared with OIT- and SLIT- induced cells, resulting in different protective consequences against allergen exposure. Furthermore, whereas OIT- or SLIT-induced Tregs lost their suppressive activities after treatment was discontinued, the suppressive activities of EPIT-induced Tregs were still effective 8 weeks after the end of treatment, suggesting the induction of a more long-lasting tolerance. In summary, EPIT, OIT and SLIT mediated desensitization through the induction of different subsets of Tregs, leading to important differences in the subsequent protection against allergen exposure and the possible induction of tolerance.

The skin is a major immunologic organ that may induce protection, sensitization or tolerance. Epicutaneous immunotherapy (EPIT) has been proposed as an attractive strategy to actively treat food allergy and has been shown to induce tolerance in sensitized mice through the induction of Foxp3+ regulatory T cells (Tregs), especially CD62L+ Tregs. Among immune cells in the skin, dendritic cells are key players in antigen-specific immune activation or regulation. The role of different populations of skin DCs in tolerance induction remains to be elucidated. Using OVA-sensitized BALB/c mice, we demonstrated that the application of a patch containing OVA-A647 to the skin resulted in allergen uptake by Langerhans cells (LCs) and CD11b+ dermal cDC2 and subsequent migration into skin draining lymph nodes. These 2 populations induced Foxp3 expression in CD4+ cells in vitro. Only LCs induced LAP+ cells and CD62L+ Tregs. Using Langerin-eGFP-DTR mice, we analyzed the role of LCs in the mechanisms of tolerance induction by EPIT in vivo. Following complete depletion of LCs, a dramatic decrease in the number of OVA+ DCs and OVA+ CD11b+ dermal cDC2 was observed in skin draining lymph nodes 48 h after epicutaneous application. Likewise, 2 weeks of EPIT in non-depleted mice induced Foxp3+ Tregs, especially CD62L+, and LAP+ Tregs in skin draining lymph nodes and spleen, whereas no induction of Tregs was observed in LC-depleted mice. Following 8 weeks of treatment, EPIT-treated mice showed significant protection against anaphylaxis accompanied by a significant increase of Foxp3+ Tregs, especially CD62L+ Tregs, which was not seen in the absence of LCs. In summary, although both LCs and CD11b+ dermal cDC2s could induce regulatory T cells, the absence of LCs during EPIT impaired treatment efficacy, indicating their crucial role in skin-induced tolerance.

Background Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). Methods BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE‐patch or by PPE‐OIT. Another set of peanut‐sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analyzed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. Results Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4th week and a significant hypomethylation of the Foxp3 promoter in CD62L+ Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. Conclusions Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T‐cell compartments with downregulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect.

Epicutaneous immunotherapy is a developing technique, aiming at desensitizing patients with food allergy with less risks that oral ingestion or injection could generate. Several clinical trials have been performed and are currently running, in milk and peanut allergy, assessing the safety of the technique and its efficacy. Preclinical models indicate a major role in the mechanisms of desensitization, for example, Tregs and epigenetic modifications.

Background Allergen-specific immunotherapy favours immune deviation from a Th2 to a Th1 response and increases the number of regulatory T cells (Tregs). Epicutaneous immunotherapy (EPIT) of sensitized mice decreases the clinical and the allergen-specific Th2 responses and increases local and peripheral Foxp3(+) Tregs. Objective To investigate the role of Tregs in EPIT and characterize their phenotype and maintenance following EPIT. Methods Tregs were investigated using in vivo depletion or adoptive transfer into BALB/c mice. Tregs were depleted using anti-CD25 antibody injection during EPIT, and allergen-specific responses were compared with Sham, EPIT alone and naive mice. To demonstrate that Tregs can mediate protection by their own, and to study their maintenance following the end of EPIT, CD25(+) CD4(+) Tregs isolated from mice just after or 8 weeks after EPIT were transferred into peanut-sensitized mice. Foxp3-IRES-mRFP mice were transferred with EPIT-induced Tregs to analyse the induction of host Tregs. Results The anti-CD25 antibody injection to EPIT mice abrogated the induction of Tregs in spleen and the expression of Foxp3 in oesophagus. This resulted in levels of peanut-induced eosinophilic infiltration in oesophagus similar to Sham and significantly higher than EPIT. Whereas the transfer of Tregs from Sham-treated mice demonstrated no effect, the transfer of Tregs isolated just after EPIT prevented peanut-induced eosinophil infiltration and eotaxin expression and induced Foxp3 in oesophagus. The transfer of Tregs isolated 8 weeks after EPIT suppressed allergen-specific responses as efficiently as did Tregs isolated just after EPIT and increased spleen Foxp3(+) CD25(+) CD4(+) cells similarly. The use of reporter mice demonstrated an increase in host Tregs. Conclusions These results confirm the Tregs-mediated mechanism of EPIT and demonstrate the persistence of efficient Tregs during a long period of time after treatment cessation. This suggests that EPIT induces long-term tolerance in peanut-sensitized mice.

Background. The aim of this study was to compare the efficacy of epicutaneous immunotherapy (EPIT) to sublingual immunotherapy (SLIT) in a model of mice sensitized to Phleum pratense pollen. Methods. BALB/c mice were sensitized by sub-cutaneous route to pollen protein extract mixed treated for 8 weeks, using sham, EPIT, or SLIT. Measurements involved the serological response and cytokine profile from reactivated splenocytes, plethysmography after aerosol challenge to pollen, cell, and cytokine contents in the bronchoalveolar lavages (BALs). Results. After immunotherapy, sIgE was significantly decreased in the treated groups compared to sham (), whereas sIgG2a increased with EPIT and SLIT ( and versus sham). Reactivated splenocytes secreted higher levels of Th2 cytokines with sham (). Penh values were higher in sham than EPIT and SLIT. Eosinophil recruitment in BAL was significantly reduced only by EPIT (). Conclusion. In this model of mice sensitized to pollen, EPIT was at least as efficient as SLIT.

Introduction L’immunotherapie epicutanee (EPIT) constitue une voie therapeutique nouvelle pour l’allergie alimentaire. La peau contient une grande quantite de cellules presentatrice d’antigenes dont les cellules de Langerhans dans l’epiderme et les cellules dendritiques du derme. Le but de cette etude etait de caracteriser les cellules impliquees dans la prise en charge de l’allergene et la dynamique de leur migration vers les ganglions drainant la peau lors de l’application epicutanee chez des souris sensibilisees a l’ovalbumine. Methodes Des souris sensibilisees a l’ovalbumine (OVA) par voie sous-cutanee ont recu un patch (Viaskin®) contenant de l’OVA couple a un fluorochrome (OVA-A488) sur peau intacte ou abrasee, et les cellules ayant capte l’allergene ont ete analysees par cytometrie en flux apres 2, 6, 24, 48 et 72 h dans la peau et les ganglions drainants et comparee a des souris naives (controle). Resultats Apres deux heures d’application, un passage passif important est detecte dans les ganglions drainants apres application sur peau abrasee contrairement a la peau intacte. Apres application sur peau intacte, l’OVA est prise en charge par les cellules de Langerhans dans l’epiderme plus rapidement chez les souris sensibilisees que chez les souris naives. Chez les souris sensibilisees, le nombre de cellules OVA+ dans l’epiderme diminue significativement apres 24 h et augmente transitoirement dans le derme entre 24 et 48 h, suggerant une migration des cellules de Langerhans vers les ganglions drainants. Apres 48 h, une augmentation significative des cellules dendritiques OVA+ est observee dans les ganglions drainants. Les cellules de Langerhans ayant migre dans les ganglions presentent une forte expression de CMH-II et une augmentation de CD86, PD-L1 et de PD-L2. L’abrasion de la peau induit une expression plus importante de CD86 et PD-L1 mais plus faible de PD-L2. Discussion Ces resultats montrent que l’application epicutanee d’allergene sur peau intacte chez des souris sensibilisees permet sa prise en charge par les cellules de Langerhans, leur maturation et leur migration rapide vers les ganglions drainants avec un profil d’activation tolerogene par rapport a l’application sur peau abrasee.

Introduction L’immunotherapie epicutanee sur peau intacte (EPIT) est une voie therapeutique des allergies alimentaires en cours de developpement. Dans les modeles animaux, la desensibilisation obtenue par l’EPIT est maintenue apres arret du traitement (tolerance) et previent de sensibilisations a d’autres allergenes. Le but de ce travail etait d’evaluer, dans un modele de souris sensibilisees, la cinetique des modifications epigenetiques etayant l’effet therapeutique de l’EPIT et l’induction de tolerance. Methodes Des souris BALB/c femelles sensibilisees a l’arachide ont ete traitees par EPIT ou immunotherapie orale (OIT) ou non traitees pendant 8 semaines. Les prelevements de rate et sang ont ete realises aux semaines 1, 2, 4 et 8 du traitement et 8 semaines apres arret du traitement. Un autre panel de groupes de souris a ete analyse apres une phase de sensibilisation a un autre allergene, l’ovalbumine. La methylation de l’ADN a ete analysee par pyrosequencage dans les cellules CD4+ purifiees de la rate et du sang. Resultats Dans les cellules CD4+ (rate et sang), une hypermethylation des ilots CpG associes au promoteur de Gata3 apparait des la 4e semaine d’EPIT, persiste jusqu’a la fin du traitement (p Conclusion L’EPIT comparee a l’OIT conduit a une signature epigenetique unique et stable permettant une regulation negative de l’expression des genes de la voie du Th2 et une activation des cellules T regulatrices. Ces mecanismes seraient impliques dans l’induction de tolerance par EPIT.

Background. Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC). Methods. Primary HPMC were isolated from omental biopsies and TLR expression detected by real-time polymerase chain reaction (PCR), reverse transcription (RT)–PCR and flow cytometry. The responsiveness of HPMC to specific bacterial TLR agonists was determined using chemokine production as a biological readout. The requirement for CD14 in HPMC responses to a clinically relevant Staphylococcus epidermidis cell-free supernatant (SES) was investigated using soluble CD14 or anti-CD14-blocking antibodies. Results. Real-time PCR detected TLR1-6 messenger RNA expression in HPMC and responses to TLR2/1 and TLR2/6 ligands and SES. No cell surface TLR4 expression or responses to lipopolysaccharide were detectable in HPMC, but they did respond to flagellin, a TLR5 ligand. SES-mediated responses were dependent on TLR2 but did not require CD14 in HPMC for optimal efficiency, unlike peripheral blood mononuclear cells. HPMC expression of TLR2 was also modulated by TLR2 ligands and inflammatory cytokines. Conclusions. These data suggest that mesothelial cell activation by TLR2/1, TLR2/6 and TLR5 contributes to bacterial recognition influencing the course of the infective process and has implications for improving treatment of infection in PD patients.

Summary Background Allergen-specific immunotherapy, subcutaneous immunotherapy (SCIT) or oral, has been used for almost a century to redirect inappropriate immune responses in atopic patients. A new mode of administration through the intact skin [epicutaneous immunotherapy (EPIT)], using an original epicutaneous delivery system, may represent an alternative to these classical methods. Objective Proof of concept of efficacy of EPIT on intact skin in mice sensitized to aeroallergens or food allergens. Methods Mice were sensitized to pollen (n=18), house dust mite (HDM, n=24), ovalbumin (OVA, n=18) or peanut (n=18), and allocated to four groups: EPIT, SCIT, not treated (NT) and control. Specific Ig (sIg)E, sIgG1 and sIgG2a were monitored. After 8 weeks of treatment, plethysmography was performed after aerosol provocation with appropriate allergens. Results At the highest doses of methacholine, pause enhancement (Penh) values were significantly decreased in the EPIT group vs. the sensitized NT groups (7.5 vs. 12.3 – pollen, 7.6 vs. 8.9 – HDM, 11.5 vs. 14.5 – OVA, 7.6 vs. 12.8 – peanut, respectively) (P

The peritoneal macrophage (Mφ) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11bhigh cells, with the remaining 5% being 12/15-LOX−. 12/15-LOX+ cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX− cells are a dendritic cell population. Resident peritoneal Mφ numbers were significantly increased in 12/15-LOX−/− mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mφ from 12/15-LOX−/− mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX−/− mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX−/− adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX+ cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX−/− mice showed elevated TGF-β1, along with increased immigration of monocytes/Mφ, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-α. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MΦ population and regulates both the recruitment of monocytes/Mφ and cytokine response to bacterial products in vivo.

Whether leukocytes exert an influence on vascular function in vivo is not known. Here, genetic and pharmacologic approaches show that the absence of neutrophils leads to acute blood pressure dysregulation. Following neutrophil depletion, systolic blood pressure falls significantly over 3 days (88.0 ± 3.5 vs 104.0 ± 2.8 mm Hg, day 3 vs day 0, mean ± SEM, P < .001), and aortic rings from neutropenic mice do not constrict properly. The constriction defect is corrected using l-nitroarginine-methyl ester (L-NAME) or the specific inducible nitric oxide synthase (iNOS) inhibitor 1400W, while acetylcholine relaxation is normal. iNOS- or IFNγ-deficient mice are protected from neutropenia-induced hypotension, indicating that iNOS-derived nitric oxide (NO) is responsible and that its induction involves IFNγ. Oral enrofloxacin partially inhibited hypotension, implicating bacterial products. Roles for cyclooxygenase, complement C5, or endotoxin were excluded, although urinary prostacyclin metabolites were elevated. Neutrophil depletion required complement opsinization, with no evidence for intravascular degranulation. In summary, circulating neutrophils contribute to maintaining physiological tone in the vasculature, at least in part through suppressing early proinflammatory effects of infection. The speed with which hypotension developed provides insight into early changes that occur in the absence of neutrophils and illustrates the importance of constant surveillance of mucosal sites by granulocytes in healthy mice.

Background Allergy to cow's milk increases the risk of sensitization to other foods in young children. Objectives We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk. Methods BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT–treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays. Results In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced T H 2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region. Conclusions Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell–dependent mechanism.

Introduction L’immunotherapie epicutanee sur peau saine (EPIT®) constitue une voie therapeutique nouvelle des allergies alimentaires. Ses mecanismes d’actions ont ete mis en evidence dans plusieurs modeles precliniques et sont largement lies aux cellules T regulatrices (Tregs). Par son profil d’efficacite et sa securite d’emploi, elle est adaptee au traitement precoce des allergies. Le but de ce travail est d’evaluer, dans un modele de souris sensibilisees, l’impact du traitement precoce par EPIT sur l’evolution naturelle des allergies. Methodes Des souris BALB/c femelles sensibilisees au lait ont ete traitees par EPIT ou non traitees (NT) pendant 8 semaines avant d’etre sensibilisees a l’arachide. Les animaux ont ensuite ete testes par injection intraveineuse (IV) d’arachide afin de declencher une anaphylaxie evaluee par la baisse de temperature et la liberation de mMCP-1dans le plasma. Des souris uniquement sensibilisees a l’arachide et des souris naives constituent respectivement les groupes controles positifs et negatifs. Dans un deuxieme temps, des Tregs provenant de souris sensibilisees au lait EPIT ou NT ont ete transferees a des souris naives avant sensibilisation a l’arachide. La reponse a l’injection IV d’arachide a ete etudiee dans les memes conditions. Resultats Les souris NT sensibilisees successivement au lait puis a l’arachide presentaient, comme les souris sensibilisees a l’arachide, une forte chute de la temperature corporelle apres le challenge IV (4,1 °C ; p Conclusion Le traitement precoce par EPIT pourrait prevenir les sensibilisations ulterieures par un mecanisme dependant des Tregs. Des etudes cliniques devront confirmer cet effet protecteur chez l’Homme.

BackgroundFood allergy may affect the gastrointestinal tract and eosinophilia is often associated with allergic gastrointestinal disorders. Allergy to peanuts is a life-threatening condition and effective and safe treatments still need to be developed. The present study aimed to evaluate the effects of sustained oral exposure to peanuts on the esophageal and jejunal mucosa in sensitized mice. We also evaluated the effects of desensitization with epicutaneous immunotherapy (EPIT) on these processes.MethodsMice were sensitized by gavages with whole peanut protein extract (PPE) given with cholera toxin. Sensitized mice were subsequently exposed to peanuts via a specific regimen and were then analysed for eosinophilia in the esophagus and gut. We also assessed mRNA expression in the esophagus, antibody levels, and peripheral T-cell response. The effects of EPIT were tested when intercalated with sensitization and sustained oral peanut exposure.ResultsSustained oral exposure to peanuts in sensitized mice led to severe esophageal eosinophilia and intestinal villus sub-atrophia, i.e. significantly increased influx of eosinophils into the esophageal mucosa (136 eosinophils/mm(2)) and reduced villus/crypt ratios (1.6±0.15). In the sera, specific IgE levels significantly increased as did secretion of Th2 cytokines by peanut-reactivated splenocytes. EPIT of sensitized mice significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (50 eosinophils/mm(2), pConclusionsGastro-intestinal lesions induced by sustained oral exposure in sensitized mice are efficaciously treated by allergen specific EPIT.

Background Eosinophilia is often linked to allergic gastrointestinal disorders linked to food allergy. EPIT using Viaskin® device has been described as a therapeutic method in food allergy. We developed a model of mice sensitized to peanut, exhibiting EoE and AE after exclusive feeding with peanut protein extracts (PPE). This study was conducted in order to evaluate the efficacy of EPIT. Methods After oral sensitization with PPE and cholera toxin, 30 BALB/c mice were treated weekly during 8 weeks by PPE skin applications (EPIT), 20 mice were not treated (Sham) and 10 mice constituted the control group (C). Mice were then exclusively fed with PPE. Specific IgE, IgG1 and IgG2a were monitored during immunotherapy. Esophageal and jejunal samples were taken for histological analyses. Results sIgE increased after oral sensitization, respectively 0.207 ±0.03 and 0.214±0.04 μg/ml, in EPIT and Sham, with undetectable values in C. Following EPIT, sIgE decreased and sIgG2a increased, respectively 0.139±0.01 vs 0.166±0.01 μg/ml (EPIT vs Sham, p

Epicutaneous immunotherapy onto intact skin has proved to be an efficient and safe alternative treatment of allergy in an animal model with various allergens and in children for cow’s milk allergy. The aim of this study was to analyze the different steps of the immunological handling of the allergen when deposited on intact skin using an epicutaneous delivery system and its immune consequences in sensitized BALB/c mice. As expected, when applied on intact skin, OVA exhibits neither a passive passage through the skin nor any detectable systemic delivery. The current study demonstrates that, after a prolonged application on intact skin, OVA is taken up by dendritic cells in the superficial layers of the stratum corneum and transported, after internalization, to the draining lymph nodes, with variations according to the previous level of sensitization of the mice. When OVA is applied with the epicutaneous delivery system repeatedly, specific local and systemic responses are down-modulated in association with the induction of regulatory T cells. Besides providing new insights into skin function in the presence of allergens, this study indicates that the skin might have a tolerogenic role, at least when kept intact.

Background: Peanut allergy is a life-threatening condition for which new efficient and safe treatment is expected. We evaluated epicutaneous immunotherapy (EPIT) as a new alternative treatment for peanut allergy in sensitized mice. Methods: Sixty BALB/c mice were sensitized by gavages with peanut protein extract (PPE) mixed with cholera toxin. An epicutaneous delivery system, coated with 100 µg PPE (Viaskin®, DBV Technologies, Paris, France), was applied to intact skin every week during 48 h (EPIT; n = 20). This group was compared with sensitized mice treated with subcutaneous immunotherapy (SCIT; n = 20), untreated sensitized mice (sham, n = 20), and naive mice (naive; n = 20). After the 8-week treatment, a histamine release test, airway hyperreactivity measurement by plethysmography, and a resistance-compliance measurement after the challenge were performed. Blood and bronchoalveolar lavage were sampled for serology, cytokines, and cytology. Results: Specific IgE (sIgE) increased after sensitization in the EPIT (0.26 µg/ml) and SCIT (0.21 µg/ml) groups and decreased after treatment (0.09 µg/ml, p < 0.001 and 0.06 µg/ml, p < 0.001, respectively). The IgG1/IgG2a ratio decreased in the EPIT and SCIT groups versus the sham group (3.7; p < 0.001 and 2.7; p < 0.01 and 15.1, respectively). At the higher metacholine concentration, enhanced pause values were lower in the EPIT and SCIT groups than in the sham group (7.29, 6.74, and 10.99, p < 0.01, respectively), and did not differ from that of the naive group (5.06). Resistance-compliance was reversed in the treated groups versus the sham group (p < 0.001). IL-4, IL-5, IL-13, eotaxin, and eosinophils were reduced in the BAL of the EPIT and SCIT groups versus the sham group (p < 0.001). Conclusion: In peanut-sensitized mice, based on biological and physiological responses, EPIT is as efficient as subcutaneous treatment which is the reference method in immunotherapy.

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Although the IL-6-related cytokine oncostatin M (OSM) affects processes associated with disease progression, the specific function of OSM in the face of an inflammatory challenge remains unclear. In this report, a peritoneal model of acute inflammation was used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared with wild-type and IL-6-deficient mice, peritoneal inflammation in oncostatin M receptor-β-deficient (OSMR-KO) mice resulted in enhanced monocytic cell trafficking. In contrast to IL-6-deficient mice, OSMR-KO mice displayed no difference in neutrophil and lymphocyte migration. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression after peritoneal inflammation identified OSM as a prominent regulator of CCL5 expression. Specifically, OSM inhibited IL-1β-mediated NF-κB activity and CCL5 expression in human mesothelial cells. This was substantiated in vivo where peritoneal inflammation in OSMR-KO mice resulted in a temporal increase in both CCL5 secretion and NF-κB activation. These findings suggest that IL-6 and OSM individually affect the profile of leukocyte trafficking, and they point to a hitherto unidentified interplay between OSM signaling and the inflammatory activation of NF-κB.

Introduction Les cellules T regulatrices (Tregs) jouent un role primordial dans les mecanismes d’action de l’immunotherapie specifique. La capacite des Tregs a migrer vers les organes cibles est liee a l’expression de recepteurs specifiques. Le but de cette etude est la caracterisation de l’expression des recepteurs de migration vers la peau (CLA) et le tube digestif (CCR9) par les Tregs induit par l’immunotherapie epicutanee (EPIT) qui constitue une voie therapeutique nouvelle pour le traitement des allergies alimentaires. Methodes Des souris BALB/c femelles sensibilisees a l’arachide par voie orale ont ete traitees par EPIT ou non traitees (NT). Apres 8 semaines de traitement, les souris ont ete sacrifiees afin de prelever la rate, les ganglions mesenteriques (mLNs) et les ganglions inguinaux (iLNs) drainant la peau. Apres extractions des cellules, la proportion de Tregs et l’expression des recepteurs de migration specifique CLA + CCR9– (peau), CLA-CCR9+ (tube digestif) et CLA + CCR9+ (double tropisme) ont ete analyses par cytometrie en flux. Resultats Apres 8 semaines de traitement, la proportion de cellules Tregs CD4 + CD25 + Foxp3+ dans la rate et les iLNs est augmentee par EPIT par rapport au NT ( p p p p Discussion Les Tregs Foxp3+ jouent un role essentiel au cours du traitement de l’allergie alimentaire. Le tropisme a la fois digestif et cutane des Tregs induits par l’EPIT aussi bien dans les ganglions drainant la peau que le tube digestif, fait de cette methode un traitement de choix de l’allergie alimentaire.

Results: In spleen, expression of CCR4 increased on Tregs induced by all 3 therapies whereas the expression of CXCR3, CCR6 and CCR8 increased on EPIT-induced Tregs only. Skin homing receptor (CLA) increased on EPITbut not SLITor OIT-induced Tregs. Gut homing receptor CCR9 increased on EPITand OITbut not SLIT-induced Tregs. Interestingly, 81% of the CCR9+ Tregs induced by EPIT expressed CLA whereas only 43% did after OIT. In iLN, Tregs level increased only after EPIT with induction of CLA+CCR9-(22%) and CLA+CCR9+ (12%) Tregs. In mLN, EPIT and OIT significantly induced CCR9+ Tregs, 25% and 18% of them expressing also CLA in EPIT and OIT respectively.

Methods: After sensitization to milk, mice were treated by EPIT or Sham, and 2 weeks later, submitted to a peanut-sensitization procedure. Mice were then intravenously challenged with peanut. In a second experiment, CD4+CD25+ T cells were isolated from spleen of milk-sensitized mice after EPIT or Sham, and transferred into naive mice. Recipient mice were submitted to peanut sensitization and intravenously challenged with peanut. In a third experiment, Tregs were obtained from Foxp3+-gfp mice sensitized to peanut and EPIT-treated, then adoptively transferred. Recipient mice were submitted or not to peanut sensitization before transfer, then all sensitized and IV challenged to ovalbumin. Outcome tools were rectal temperature, hypersensitivity reactions and blood mouse mast cell protease-1 (mMCP1).

Cross-sectional studies have shown that the capacity of CD8+cells from human immunodeficiency virus (HIV)-infected patients and simian immunodeficiency virus (SIV) SIVmac-infected macaques to suppress the replication of human and simian immunodeficiency viruses in vitro depends on the clinical stage of disease, but little is known about changes in this antiviral activity over time in individual HIV-infected patients or SIV-infected macaques. We assessed changes in the soluble factor-mediated noncytolytic antiviral activity of CD8+cells over time in eight cynomolgus macaques infected with SIVmac251 to determine the pathophysiological role of this activity. CD8+cell-associated antiviral activity increased rapidly in the first week after viral inoculation and remained detectable during the early phase of infection. The net increase in antiviral activity of CD8+cells was correlated with plasma viral load throughout the 15 months of follow-up. CD8+cells gradually lost their antiviral activity over time and acquired virus replication-enhancing capacity. Levels of antiviral activity correlated with CD4+T-cell counts after viral set point. Concentrations of β-chemokines and interleukin-16 in CD8+cell supernatants were not correlated with this antiviral activity, and α-defensins were not detected. The soluble factor-mediated antiviral activity of CD8+cells was neither cytolytic nor restricted to major histocompatibility complex. This longitudinal study strongly suggests that the increase in noncytolytic antiviral activity from baseline and the maintenance of this increase over time in cynomolgus macaques depend on both viral replication and CD4+T cells.

The aim of this study was to evaluate the kinetics of lymphocyte proliferation during primary infection of macaques with pathogenic simian immunodeficiency virus (SIV) and to study the impact of short-term postexposure highly active antiretroviral therapy (HAART) prophylaxis. Twelve macaques were infected by intravenous route with SIVmac251 and given treatment for 28 days starting 4 h postexposure. Group 1 received a placebo, and groups 2 and 3 received combinations of zidovudine (AZT), lamivudine (3TC), and indinavir. Macaques in group 2 received AZT (4.5 mg/kg of body weight), 3TC (2.5 mg/kg), and indinavir (20 mg/kg) twice per day by the oral route whereas macaques in group 3 were given AZT (4.5 mg/kg) and 3TC (2.5 mg/kg) subcutaneously twice per day, to improve the pharmacokinetic action of these drugs, and a higher dose of indinavir (60 mg/kg). The kinetics of lymphocyte proliferation were analyzed by monitoring 5-bromo-2′-deoxyuridine (BrdU) uptake ex vivo and by fluorescence-activated cell sorting analysis. HAART did not protect against SIV infection but did strongly impact on virus loads: viremia was delayed and lowered during antiviral therapy in group 2, with better control after treatment was stopped, and in group 3, viremia was maintained at lower levels during treatment, with virus even undetectable in the blood of some macaques, but there was no evidence of improved control of the virus after treatment. We provide direct evidence that dividing NK cells are detected earlier than dividing T cells in the blood (mostly in CD45RA−T cells), mirroring plasma viremia. Dividing CD8+T cells were detected earlier than dividing CD4+T cells, and the highest percentages of proliferating T cells coincided with the first evidence of partial control of peak viremia and with an increase in the percentage of circulating gamma interferon-positive CD8+T cells. The level of cell proliferation in the blood during SIV primary infection was clearly associated with viral replication levels because the inhibition of viral replication by postexposure HAART strongly reduced lymphocyte proliferation. The results and conclusions in this study are based on experiments in a small numbers of animals and are thus preliminary.