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Postoperative pain affects most patients after major surgery and can transition to chronic pain. Here, we discovered that postoperative pain hypersensitivity correlated with markedly increased local levels of the metabolite BH4. Gene transcription and reporter mouse analyses after skin injury identified neutrophils, macrophages and mast cells as primary postoperative sources of GTP cyclohydrolase-1 (Gch1) expression, the rate-limiting enzyme in BH4 production. While specificGch1deficiency in neutrophils or macrophages had no effect, mice deficient in mast cells or mast cell-specificGch1showed drastically decreased postoperative pain after surgery. Skin injury induced the nociceptive neuropeptide substance P, which directly triggers the release of BH4-dependent serotonin in mouse and human mast cells. Substance P receptor blockade substantially ameliorated postoperative pain. Our findings underline the unique position of mast cells at the neuro-immune interface and highlight substance P-driven mast cell BH4 production as promising therapeutic targets for the treatment of postoperative pain.

Routine clinical assays, such as conventional immunohistochemistry, often fail to resolve the regional heterogeneity of complex inflammatory skin conditions. Here we introduce MANTIS (Multiplexed Annotated Tissue Imaging System), a flexible analytic pipeline compatible with routine practice, specifically-designed for spatially-resolved immune phenotyping of the skin in experimental or clinical samples. Based on phenotype attribution matrices coupled to α-shape algorithms, MANTIS projects a representative digital immune landscape, while enabling automated detection of major inflammatory clusters and concomitant single-cell data quantification of biomarkers. We observed that severe pathological lesions from systemic lupus erythematosus, Kawasaki syndrome or COVID-19-associated skin manifestations share common quantitative immune features, while displaying a non-random distribution of cells with the formation of disease-specific dermal immune structures. Given its accuracy and flexibility, MANTIS is designed to solve the spatial organization of complex immune environments to better apprehend the pathophysiology of skin manifestations.

Mast cells (MCs) are heterogenous tissue-resident immune cells associated with blood vessels and neurons. However, the extent to which specialized MCs can exhibit micro-environment-specific functions is unclear. Using single cell RNA sequencing, we characterize connective tissue-type and mucosal MCs across organs and found that they can be discriminated based on MrgprB2 expression. While MrgprB2+ connective MCs are enriched in neuroreceptors, develop during the embryogenesis and renew independently of the bone marrow (BM); MrgprB2neg mucosal MCs arise postnatally, are sensitive to signals from the microbiome and renewed by BM progenitors. Importantly, MrgprB2+, but not MrgprB2neg MCs are required for food-induced anaphylactic shock. In the gut, MrgprB2+ MCs occur in contact with substance P-secreting enteric neurons and the specific depletion of either MrgprB2+ MCs or substance P results in dysregulation of gut contractions. Thus, we demonstrate that two independent MC populations exist in multiple organs, with different developmental dynamics and biological programming.

Interfering with age-related neuroimmune interactions promotes nerve regeneration

International audience; Mast cells have existed long before the development of adaptive immunity, although they have been given different names. Thus, in the marine urochordate Styela plicata, they have been designated as test cells. However, based on their morphological characteristics (including prominent cytoplasmic granules) and mediator content (including heparin, histamine, and neutral proteases), test cells are thought to represent members of the lineage known in vertebrates as mast cells. So this lineage presumably had important functions that preceded the development of antibodies, including IgE. Yet mast cells are best known, in humans, as key sources of mediators responsible for acute allergic reactions, notably including anaphylaxis, a severe and potentially fatal IgE-dependent immediate hypersensitivity reaction to apparently harmless antigens, including many found in foods and medicines. In this review, we briefly describe the origins of tissue mast cells and outline evidence that these cells can have beneficial as well as detrimental functions, both innately and as participants in adaptive immune responses. We also discuss aspects of mast cell heterogeneity and comment on how the plasticity of this lineage may provide insight into its roles in health and disease. Finally, we consider some currently open questions that are yet unresolved.

BACKGROUND: Mastocytosis is associated with mast cell (MC) mediator-related symptoms for which limited therapies are available. OBJECTIVE: Our aim was to assess the efficacy and safety of omalizumab in the treatment of MC mediator-related symptoms in adult patients with mastocytosis. RESULTS: We identified one multi-centre retrospective cohort study (39 patients), one retrospective cohort study (13 patients), 4 case series and 10 case reports. No published controlled randomized study was identified. We included 69 patients (13 patients with cutaneous mastocytosis and 56 with systemic mastocytosis). The mean age was 48 years. Omalizumab maintenance dose was 300 mg for the majority of patients. The mean duration of treatment was 17 months. Treatment led to a tolerability of venom immunotherapy and to a complete resolution of severe reactions in all patients with post-honeybee sting anaphylaxis. Complete resolution of idiopathic anaphylaxis episodes was noted in 84% of the patients. Complete resolution of palpitations, gastrointestinal, cutaneous, neuropsychiatric, respiratory and musculoskeletal symptoms was observed at a rate of 43%, 29%, 27%, 11%, 9% and 0%, respectively. Efficacy was maintained for the entire duration of the treatment in all but four responders. Adverse events were reported for 13 patients. CONCLUSIONS AND CLINICAL RELEVANCE: Omalizumab appears to prevent some life-threatening reactions associated with mastocytosis and may be a good option to treat the associated symptoms. However, the evidence relied upon is observational, uncontrolled and from a small number of patients. A randomized controlled trial is needed to better understand the place of omalizumab in mastocytosis treatment.

Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR-Cas 9 gene editing. By using high resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the suppression of functional responses, i.e. degranulation, in single human mast cells. The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures and the short hands-on time of the functional analysis step enables the high-throughput screening of shRNA or CRISPR-Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyse single cells significantly reduces the total number of cells required, and allows for the parallel visualization of the degranulation profile of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference and CRISPR-Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data, and image analysis can be completed within 5 days, requiring only standard laboratory equipment and expertise., EDITORIAL SUMMARY This protocol presents an an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR-Cas 9 gene editing using high resolution confocal microscopy with a fluorochrome-labeled avidin probe.

The peripheral nervous system and the immune system are virtually distributed in all organs in mouse and human. They act in concert to constitute an exceptional sensory system capable of sensing threatening stimuli, including allergens, and triggering appropriate biological responses. Recent advances in the field have revealed that tissue-resident mast cells and nearby sensory neurons could form functional neuroimmune clusters driven by bidirectional interactions and predisposed to significantly influence major allergic conditions. In this review, we highlight recent discoveries that paved the way toward a better understanding of how mast cells and sensory neurons can communicate to regulate basic features of allergic disorders in different organs.

International audience; Peripheral neurons (including sensory neurons) are ubiquitously distributed in all tissues, particularly at the interface with the environment. The primary function of sensory neurons is the transmission of sensations of temperature, pain and itch to elicit appropriate behavioral responses. More recently, sensory neurons have emerged as potent regulators of type 2 immune responses and allergic inflammation. There is increasing evidence showing that neurons can express receptors previously thought to be restricted to the immune compartment. In addition, certain subtypes of immune cells (e.g. mast cells, ILC2s or macrophages) also express specific neuroreceptors that provide them with the capacity to integrate neuron-derived signals and modulate their activation status during the development of allergic inflammation.

International audience; Allergic skin diseases, such as atopic dermatitis, are clinically characterized by severe itching and type 2 immunity-associated hypersensitivity to widely distributed allergens, including those derived from house dust mites (HDMs). Here we found that HDMs with cysteine protease activity directly activated peptidergic nociceptors, which are neuropeptide-producing nociceptive sensory neurons that express the ion channel TRPV1 and Tac1, the gene encoding the precursor for the neuropeptide substance P. Intravital imaging and genetic approaches indicated that HDM-activated nociceptors drive the development of allergic skin inflammation by inducing the degranulation of mast cells contiguous to such nociceptors, through the release of substance P and the activation of the cationic molecule receptor MRGPRB2 on mast cells. These data indicate that, after exposure to HDM allergens, activation of TRPV1+Tac1+ nociceptor–MRGPRB2+ mast cell sensory clusters represents a key early event in the development of allergic skin reactions.

SummaryCD4+ T helper cells (Th) infiltrate sites of inflammation and orchestrate the immune response by instructing local leukocytes. Mast cells (MCs) are tissue sentinel cells particularly abundant in skin and mucosa. Here, we analyzed the interplay between human MCs and Th cells and, through the application of RNAseq and functional assays, showed that Th cells induced a specific transcriptomic program in helped MCs (named here MCTH) driving them toward an inflammatory phenotype. The gene signature of MCTH indicated that MCs helped by Th cell acquired in turn the capacity to regulate effector T cell response through wide-range of soluble and membrane ligands. Accordingly, we showed that MCTH promoted Th17 cells and notably an inflammatory subset of Th17, producing both IFN-γ and GM-CSF, through a PGE2 and IL-1β axis. Our findings demonstrate that activated effector/memory CD4+ T cells activate and instruct resting MCs toward a specific differentiated pro-inflammatory phenotype endowed with the capacity to speak back to effector T cells and to mold their functions.

Taniguchi et al . (Research Articles, 17 July 2020, p. 269) claim that the cytokine interleukin-33 induces accumulation of tumor-associated macrophages expressing the immunoglobulin E receptor FcεRI. Although these findings hold great therapeutic promise, we provide evidence that the anti-FcεRI antibody used in this study is not specific for FcεRI on macrophages, which raises concerns about the validity of some of the conclusions.

International audience; Background: In contrast to their clearly defined roles in allergic diseases, the physiologic functions of Immunoglobulin E antibodies (IgEs) and mast cells (MCs) remain enigmatic. Recent research supports the toxin hypothesis, showing that MCs and IgE-related type 2 immune responses can enhance host defense against certain noxious substances, including honeybee venom (BV). However, the mechanisms by which MCs can interfere with BV toxicity are unknown. In this study, we assessed the role of IgE and certain MC products in MC-mediated BV detoxification.Methods: We applied in vitro and in vivo fluorescence microscopyimaging, and flow cytometry, fibroblast-based toxicity assays and mass spectrometry to investigate IgE-mediated detoxification of BV cytotoxicity by mouse and human MCs in vitro. Pharmacologic strategies to interfere with MC-derived heparin and proteases helped to define the importance of specific detoxification mechanisms.Results: Venom-specific IgE increased the degranulation and cytokine responses of MCs to BV in vitro. Passive serum sensitization enhanced MC degranulation in vivo. IgE-activated mouse or human MCs exhibited enhanced potential for detoxifying BV by both proteolytic degradation and heparin-related interference with toxicity. Mediators released by IgE-activated human MCs efficiently degraded multiple BV toxins.Conclusions: Our results both reveal that IgE sensitization enhances the MC's ability to detoxify BV and also assign efficient toxin-neutralizing activity to MC-derived heparin and proteases. Our study thus highlights the potential importance of IgE, MCs, and particular MC products in defense against BV.

International audience; Allergies are considered to represent mal-directed type 2 immune responses against mostly innocuous exogenous compounds. Immunoglobulin E (IgE) antibodies are a characteristic feature of allergies and mediate hypersensitivity against allergens through activation of effector cells, particularly mast cells (MCs). Although the physiological functions of this dangerous branch of immunity have remained enigmatic, recent evidence shows that allergic immune reactions can help to protect against the toxicity of venoms. Because bacteria are a potent alternative source of toxins, we assessed the possible role of allergy-like type 2 immunity in antibacterial host defense. We discovered that the adaptive immune response against Staphylococcus aureus (SA) skin infection substantially improved systemic host defense against secondary SA infections in mice. Moreover, this acquired protection depended on IgE effector mechanisms and MCs. Importantly, our results reveal a previously unknown physiological function of allergic immune responses, IgE antibodies, and MCs in host defense against a pathogenic bacterium.

International audience; Gain-of-function mutations in NLRP3 are responsible for a spectrum of autoinflammatory diseases collectively referred to as “cryopyrin-associated periodic syndromes” (CAPS). Treatment of CAPS patients with IL-1–targeted therapies is effective, confirming a central pathogenic role for IL-1β. However, the specific myeloid cell population(s) exhibiting inflammasome activity and sustained IL-1β production in CAPS remains elusive. Previous reports suggested an important role for mast cells (MCs) in this process. Here, we report that, in mice, gain-of-function mutations in Nlrp3 restricted to neutrophils, and to a lesser extent macrophages/dendritic cells, but not MCs, are sufficient to trigger severe CAPS. Furthermore, in patients with clinically established CAPS, we show that skin-infiltrating neutrophils represent a substantial biological source of IL-1β. Together, our data indicate that neutrophils, rather than MCs, can represent the main cellular drivers of CAPS pathology.

Introduction L’histologie standard et l’immunohistochimie sont les outils classiquement utilises dans le diagnostic des dermatoses inflammatoires mais ne permettent pas une etude exhaustive du tissu. D’autres methodes, telles que le sequencage d’ARN en cellule unique, fournissent des donnees quantitatives, mais necessitent une dissociation tissulaire complete a l’origine d’une perte de l’information cruciale concernant l’organisation tissulaire. Materiel et methodes Nous avons mis au point une technique d’imagerie analytique capable d’exploiter de multiples donnees de fluorescence, appelee « Unbiased cLustering of specTRal emissiON » (ULTRON). Elle permet de capturer de nombreux parametres cellulaires, tout en preservant leur localisation spatiale a partir de coupes de peau. ULTRON permet d’acquerir jusqu’a 12 marqueurs simultanement au moyen d’un microscope confocal conventionnel. Dans un second temps, les donnees acquises sont traitees par des techniques poussees de modelisation informatique associees a du « machine-learning » et analysees de maniere quantitative. Cette approche permet une identification automatique des sous-populations immunitaires et la conception d’une carte numerique du tissu, chaque sous-population etant associee a ses coordonnees spatiales precises sur l’image d’origine. Elle permet ainsi l’analyse exhaustive de la distribution, du phenotype, de la localisation des cellules immunitaires et de leur rapport entre elles et avec les elements structuraux (resolution : 200 nm). Trois panels d’anticorps specifiques ont ete valides. Resultats Le contexte sanitaire nous a conduit a observer avec interet l’apparition de cas de pseudo-engelures (et autres dermatoses) secondaires a l’infection par le SARS-CoV-2, chez des patients le plus souvent jeunes et tres peu symptomatiques sur le plan respiratoire. Nous avons mene une etude multicentrique des biopsies cutanees realisees en routine chez des patients ayant presente des pseudo-engelures (n = 15). Des patients controles, atteints de lupus engelures (n = 5) ont egalement ete etudies. La technologie ULTRON a permis d’etablir une signature immunitaire dans cette pathologie dermatologique emergente. Discussion Au-dela du developpement d’une technique d’imagerie de pointe, ces travaux ont permis la caracterisation de populations immunitaires specifiquement enrichies au cours des pseudo-engelures contemporaines de la COVID-19 et cela dans leur environnement tissulaire preserve.

International audience; Mast cells and basophils are best known for their key role during allergic responses [1]. Yet, they also have pivotal functions in innate and acquired (via IgE) defense mechanisms against microbes and parasites as well as in the neutralization of certain toxins such as snake venoms, through the release of proteases and other mediators contained in their cytoplasmic granules [2, 3] (Figure 1, example of a mast cell releasing cytoplasmic granules stained using fluorochrome‐labeled avidin, red). Recent work indicates that these cells have also important functions in a much broader range of inflammatory and pathological conditions including autoimmune diseases, cardiovascular diseases, skin diseases, neuroinflammatory disease and even cancer. However, the mechanisms by which mast cells and basophils mediate their functions in these settings remain largely unknown. Not surprisingly, these new aspects of mast cell and basophil biology have raised interest in the immunology community, with the emergence of new groups focused on studying their involvement in the maintenance of homeostasis and the promotion of disease.

International audience; Recent studies revealed the existence of unique functional links between mast cells and nociceptors in the skin. Here, we propose that mast cells and nociceptors form a single regulatory unit in both physiology and disease. In this model, MrgprB2/X2 signaling is a primary mechanism by which mast cells functionally interact with nociceptors to form specialized neuroimmune clusters that regulate pain, inflammation, and itch.

International audience; Interplay between physiological systems in the body plays a prominent role in health and disease. At the cellular level, such interplay is orchestrated through the binding of specific ligands to their receptors expressed on cell surface. G protein-coupled receptors (GPCR) are seven-transmembrane domain receptors that initiate various cellular responses and regulate homeostasis. In this review, we focus on particular GPCRs named Mas-related G protein-coupled receptors (Mrgprs) mainly expressed by sensory neurons and specialized immune cells. We describe the different subfamilies of Mrgprs and their specific ligands, as well as recent advances in the field that illustrate the role played by these receptors in neuro-immune biological processes, including itch, pain and inflammation in diverse organs.

International audience; Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target.

International audience

International audience

International audience; Contact hypersensitivity (CHS) is a common skin disease induced by epicutaneous sensitization to haptens. Conflicting results have been obtained regarding pathogenic versus protective roles of mast cells (MCs) in CHS, and this has been attributed in part to the limitations of certain models for studying MC functions in vivo. Here we describe a fluorescent imaging approach that enables in vivo selective labeling and tracking of MC secretory granules by real-time intravital 2-photon microscopy in living mice, and permits the identification of such MCs as a potential source of cytokines in different disease models. We show using this method that dermal MCs release their granules progressively into the surrounding microenvironment, but also represent an initial source of the antiinflammatory cytokine IL-10, during the early phase of severe CHS reactions. Finally, using 3 different types of MC-deficient mice, as well as mice in which IL-10 is ablated specifically in MCs, we show that IL-10 production by MCs can significantly limit the inflammation and tissue pathology observed in severe CHS reactions.

International audience; Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Background Conflicting results have been obtained regarding the roles of Fc receptors and effector cells in models of active systemic anaphylaxis (ASA). In part, this might reflect the choice of adjuvant used during sensitization because various adjuvants might differentially influence the production of particular antibody isotypes. Objective We developed an "adjuvant-free" mouse model of ASA and assessed the contributions of components of the "classical" and "alternative" pathways in this model. Methods Mice were sensitized intraperitoneally with ovalbumin at weekly intervals for 6 weeks and challenged intraperitoneally with ovalbumin 2 weeks later. Results Wild-type animals had immediate hypothermia and late-phase intraperitoneal inflammation in this model. These features were reduced in mice lacking the IgE receptor FceRI, the IgG receptor FcγRIII or the common γ-chain FcRγ. FcγRIV blockade resulted in a partial reduction of inflammation without any effect on hypothermia. Depletion of monocytes/macrophages with clodronate liposomes significantly reduced the hypothermia response. By contrast, depletion of neutrophils or basophils had no significant effects in this ASA model. Both the hypothermia and inflammation were dependent on platelet-activating factor and histamine and were reduced in 2 types of mast cell (MC)–deficient mice. Finally, engraftment of MC-deficient mice with bone marrow–derived cultured MCs significantly exacerbated the hypothermia response and restored inflammation to levels similar to those observed in wild-type mice. Conclusion Components of the classical and alternative pathways contribute to anaphylaxis in this adjuvant-free model, with key roles for MCs and monocytes/macrophages.

Objective Gouty arthritis is caused by precipitation in joints of crystals of monosodium urate (MSU). While it has been reported that mast cells (MCs) infiltrate gouty tophi, little is known about the actual roles of MCs during acute attacks of gout. This study was undertaken to assess the role of MCs in a mouse model of MSU crystal-induced acute arthritis.

International audience; Quorum sensing molecules (QSMs) are secreted by bacteria to signal population density. Upon reaching a critical concentration, QSMs induce transcriptional alterations in bacteria that enable virulence factor expression and biofilm formation. It is unclear whether mammalian hosts can recognize QSMs to trigger responsive antibacterial immunity. We report that mouse mast cell-specific G protein-coupled-receptor Mrgprb2, and its human homologue MRGPRX2, are receptors for Gram-positive QSMs, including competence-stimulating peptide (CSP)-1. CSP-1 activates Mrgprb2/MRGPRX2, triggering mast cell degranulation which inhibits bacterial growth and prevents biofilm formation. Such antibacterial functions are reduced in Mrgprb2-deficient mast cells, while wildtype mast cells fail to inhibit the growth of bacterial strains lacking CSP-1. Mrgprb2-knockout mice exhibit reduced bacterial clearance, while pharmacologically activating Mrgprb2 in vivo eliminates bacteria and improves disease score. These findings identify a host defense mechanism that uses QSMs as an “Achilles heel” and suggest MRGPRX2 as a potential therapeutic target for controlling bacterial infections.

International audience; Gouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib) can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

Asthma has multiple features, including airway hyperreactivity, inflammation and remodelling. The TNF superfamily member TNFSF14 (LIGHT), via interactions with the receptor TNFRSF14 (HVEM), can support TH2 cell generation and longevity and promote airway remodelling in mouse models of asthma, but the mechanisms by which TNFSF14 functions in this setting are incompletely understood. Here we find that mouse and human mast cells (MCs) express TNFRSF14 and that TNFSF14:TNFRSF14 interactions can enhance IgE-mediated MC signalling and mediator production. In mouse models of asthma, TNFRSF14 blockade with a neutralizing antibody administered after antigen sensitization, or genetic deletion of Tnfrsf14, diminishes plasma levels of antigen-specific IgG1 and IgE antibodies, airway hyperreactivity, airway inflammation and airway remodelling. Finally, by analysing two types of genetically MC-deficient mice after engrafting MCs that either do or do not express TNFRSF14, we show that TNFRSF14 expression on MCs significantly contributes to the development of multiple features of asthma pathology., TNFSF14 (LIGHT) contributes to airway inflammation and remodelling. Here the authors show that TNFSF14 acting on its receptor TNFRSF14 on mast cells enhances their IgE-dependent activation and that interference with this pathway attenuates features of asthma pathology in mice.

International audience; Comment on Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Schauer C, Janko C, Munoz LE, Zhao Y, Kienhöfer D, Frey B, Lell M, Manger B, Rech J, Naschberger E, Holmdahl R, Krenn V, Harrer T, Jeremic I, Bilyy R, Schett G, Hoffmann M, Herrmann M. Nat Med. 2014 May;20(5):511-7. doi: 10.1038/nm.3547. Epub 2014 Apr 28. PMID: 24784231

International audience; Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P–dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients., Cytotoxic T lymphocytes recognise and eliminate tumour cells. Here, the authors show that on contact with these immune cells melanoma cells can resist T cell cytotoxicity by modulating the trafficking of their lysosomal compartment, this results in the degradation of the T cell protein perforin by the protease cathepsin B.

Background Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen-specific immunotherapy. Nevertheless, its cell-activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side-by-side comparison of the immune-modulating properties of MPLA and LPS on different immune cells. Methods Immune-activating properties of MPLA and LPS were compared in human monocytes and mast cells (MCs), a mouse endotoxin shock model (ESM), and mouse bone marrow (BM)-derived myeloid dendritic cells (mDCs), T cells (TCs), B cells, and MCs. Results In a mouse in vivo ESM and a human ex vivo monocyte activation test (MAT), MPLA induced the same cytokine secretion pattern as LPS (ESM: IL-6, IL-12, TNF-α; MAT: IL-1β, IL-6, TNF-α), albeit at lower levels. Mouse mDCs and ex vivo isolated B cells stimulated with MPLA required a higher threshold to induce TRIF-dependent cytokine secretion (IL-1β, IL-6, IL-10, and TNF-α) than did LPS-stimulated cells. In mDC:DO11.10 CD4 TC cocultures, stimulation with MPLA, but not with LPS, resulted in enhanced OVA-specific IL-4 and IL-5 secretion from DO11.10 CD4 TCs. Unexpectedly, in both human and mouse MCs, MPLA, unlike LPS, did not elicit secretion of pro-inflammatory cytokines. Conclusions Compared to LPS, MPLA induced a qualitatively similar, but less potent pro-inflammatory immune response, but was unable to activate human or mouse MCs.

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the 'mast cell knock-in' approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

International audience; Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes, such as tryptase and chymase, at the cell-cell contact site. This previously unidentified mast cell effector mechanism, which we name the antibody-dependent degranulatory synapse (ADDS), is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably, IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence.

Background Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies. Objective We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample. Methods Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs. Results Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63 hi population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63 hi basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C. Conclusion BATs to measure upregulation of basophil CD203c and induction of a CD63 hi basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.

Background Mast cells are key components of the skin microenvironment in psoriasis, yet their functional role in this T-cell–mediated inflammatory disorder remains to be elucidated. Objective To define the impact of T-cell/mast-cell cognate interactions on the cytokines produced by T H cells. Methods We used human primary mast cells and effector/memory CD4 + T cells for in vitro coculture experiments, and we analyzed T H cells responses by using cytometry. CD4 + T-cell/mast-cell conjugates in skin lesions from patients with psoriasis were analyzed by using 3-color immunohistochemistry and confocal microscopy. Results We show that IFN-γ–primed human mast cells formed productive immunologic synapses with antigen-experienced CD4 + T cells. These interactions promoted the generation of T H 22 and IL-22/IFN-γ–producing T H cells from the circulating memory CD4 + T-cell pool via a TNF-α/IL-6–dependent mechanism. An analysis of human psoriatic skin biopsies showed a rich infiltrate of IL-22 + CD4 + T cells frequently found in contact with mast cells. Moreover, most of these mast-cell–conjugated lymphocytes coexpressed IFN-γ, suggesting that IL-22 + IFN-γ + CD4 + T cells are generated in vivo on interaction with mast cells. Conclusions Our findings identify human mast cells as functional partners of T H cells, shaping their responses toward IL-22 production.

It has been suggested that mast cells might serve, under certain circumstances, as antigen-presenting cells (APCs) for T cells. However, whether cognate interactions between mast cells and class II–restricted CD4+ T cells actually occur is still an open question. We addressed this question by using peritoneal cell–derived mast cells (PCMCs) and freshly isolated peritoneal mast cells as APC models. Our results show that in vitro treatment of PCMCs with interferon-γ and interleukin-4 induced surface expression of mature major histocompatibility complex class II molecules and CD86. When interferon-γ/interleukin-4–primed PCMCs were used as APCs for CD4+ T cells, they induced activation of effector T cells but not of their naive counterparts as evidenced by CD69 up-regulation, proliferation, and cytokine production. Confocal laser scanning microscopy showed that CD4+ T cells formed immunological synapses and polarized their secretory machinery toward both antigen-loaded PCMCs and freshly isolated peritoneal mast cells. Finally, on cognate interaction with CD4+ T cells, mast cells lowered their threshold of activation via FcϵRI. Our results show that mast cells can establish cognate interactions with class II–restricted helper T cells, implying that they can actually serve as resident APCs in inflamed tissues.