House dust mites activate nociceptor-mast cell clusters to drive type 2 skin inflammation.

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.

Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice.

Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short life span, suggesting that they have beneficial roles in maintaining health. However, these roles are not fully defined. Here we demonstrate that basophil-deficient mice exhibit reduced bacterial clearance and increased morbidity and mortality in the cecal ligation and puncture (CLP) model of sepsis. Among the several proinflammatory mediators that we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. In accordance with that observation, we found that mice with genetic ablation of Tnf in basophils exhibited reduced systemic concentrations of TNF during endotoxemia. Moreover, after CLP, mice whose basophils could not produce TNF, exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality. Taken together, our results show that basophils can enhance the innate immune response to bacterial infection and help prevent sepsis.

IgE Effector Mechanisms, in Concert with Mast Cells, Contribute to Acquired Host Defense against Staphylococcusaureus.

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.

Widespread monoclonal IgE antibody convergence to an immunodominant, proanaphylactic Ara h 2 epitope in peanut allergy.

BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.

IgG Subclass-Dependent Pulmonary Antigen Retention during Acute IgG-Dependent Systemic Anaphylaxis in Mice.

Mouse models of active systemic anaphylaxis rely predominantly on IgG Abs forming IgG-allergen immune complexes that induce IgG receptor-expressing neutrophils and monocytes/macrophages to release potent mediators, leading to systemic effects. Whether anaphylaxis initiates locally or systemically remains unknown. In this study, we aimed at identifying the anatomical location of IgG-allergen immune complexes during anaphylaxis. Active systemic anaphylaxis was induced following immunization with BSA and i.v. challenge with fluorescently labeled BSA. Ag retention across different organs was examined using whole-body fluorescence imaging, comparing immunized and naive animals. Various mouse models and in vivo deletion strategies were employed to determine the contribution of IgG receptors, complement component C1q, myeloid cell types, and anaphylaxis mediators. We found that following challenge, Ag diffused systemically, but specifically accumulated in the lungs of mice sensitized to that Ag, where it formed large Ab-dependent aggregates in the vasculature. Ag retention in the lungs did not rely on IgG receptors, C1q, neutrophils, or macrophages. IgG2a-mediated, but neither IgG1- nor IgG2b-mediated, passive systemic anaphylaxis led to Ag retention in the lung. Neutrophils and monocytes significantly accumulated in the lungs after challenge and captured high amounts of Ag, which led to downmodulation of surface IgG receptors and triggered their activation. Thus, within minutes of systemic injection in sensitized mice, Ag formed aggregates in the lung and liver vasculature, but accumulated specifically and dose-dependently in the lung. Neutrophils and monocytes recruited to the lung captured Ag and became activated. However, Ag aggregation in the lung vasculature was not necessary for anaphylaxis induction.

Autoreactive IgE: Pathogenic role and therapeutic target in autoimmune diseases.

Autoimmunity is the break of tolerance to self-antigens that leads to organ-specific or systemic diseases often characterized by the presence of pathogenic autoreactive antibodies (AAb) produced by plasmablast and/or plasma cells. AAb are prevalent in the general population and not systematically associated with clinical symptoms. In contrast, in some individuals, these AAb are pathogenic and drive the development of signs and symptoms of antibody-mediated autoimmune diseases (AbAID). AAb production, isotype profiles, and glycosylations are promoted by pro-inflammatory triggers linked to genetic, environmental, and hormonal parameters. Recent evidence supports a role for pathogenic AAb of the IgE isotype in a number of AbAID. Autoreactive IgE can drive the activation of mast cells, basophils, and other types of FcεRI-bearing cells and may play a role in promoting autoantibody production and other pro-inflammatory pathways. In this review, we discuss the current knowledge on the pathogenicity of autoreactive IgE in AbAID and their status as therapeutic targets. We also highlight unresolved issues including the need for assays that reproducibly quantify IgE AAbs, to validate their diagnostic and prognostic value, and to further study their pathophysiological contributions to AbAID.

IgE in the pathophysiology and therapy of food allergy.

Food allergy is becoming a major public health issue, with no regulatory approved therapy to date. Food allergy symptoms range from skin rash and gastrointestinal symptoms to anaphylaxis, a potentially fatal systemic allergic shock reaction. IgE antibodies are thought to contribute importantly to key features of food allergy and anaphylaxis, and measurement of allergen-specific IgE is fundamental in diagnosing food allergy. This review will discuss recent advances in the regulation of IgE production and IgE repertoires in food allergy. We will describe the current understanding of the role of IgE and its high-affinity receptor FcεRI in food allergy and anaphylaxis, by reviewing insights gained from analyses of mouse models. Finally, we will review data derived from clinical studies of the effect of anti-IgE therapeutic monoclonal antibodies (mAbs) in food allergy, and recent insight on the efficiency and mechanisms through which these mAbs block IgE effector functions.

IgE antibodies increase honeybee venom responsiveness and detoxification efficiency of mast cells.

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.

A beneficial role for immunoglobulin E in host defense against honeybee venom.

Allergies are widely considered to be misdirected type 2 immune responses, in which immunoglobulin E (IgE) antibodies are produced against any of a broad range of seemingly harmless antigens. However, components of insect venoms also can sensitize individuals to develop severe IgE-associated allergic reactions, including fatal anaphylaxis, upon subsequent venom exposure. We found that mice injected with amounts of honeybee venom similar to that which could be delivered in one or two stings developed a specific type 2 immune response that increased their resistance to subsequent challenge with potentially lethal amounts of the venom. Our data indicate that IgE antibodies and the high affinity IgE receptor, FcεRI, were essential for such acquired resistance to honeybee venom. The evidence that IgE-dependent immune responses against venom can enhance survival in mice supports the hypothesis that IgE, which also contributes to allergic disorders, has an important function in protection of the host against noxious substances.

The pathophysiology of anaphylaxis.

Anaphylaxis is a severe systemic hypersensitivity reaction that is rapid in onset; characterized by life-threatening airway, breathing, and/or circulatory problems; and usually associated with skin and mucosal changes. Because it can be triggered in some persons by minute amounts of antigen (eg, certain foods or single insect stings), anaphylaxis can be considered the most aberrant example of an imbalance between the cost and benefit of an immune response. This review will describe current understanding of the immunopathogenesis and pathophysiology of anaphylaxis, focusing on the roles of IgE and IgG antibodies, immune effector cells, and mediators thought to contribute to examples of the disorder. Evidence from studies of anaphylaxis in human subjects will be discussed, as well as insights gained from analyses of animal models, including mice genetically deficient in the antibodies, antibody receptors, effector cells, or mediators implicated in anaphylaxis and mice that have been "humanized" for some of these elements. We also review possible host factors that might influence the occurrence or severity of anaphylaxis. Finally, we will speculate about anaphylaxis from an evolutionary perspective and argue that, in the context of severe envenomation by arthropods or reptiles, anaphylaxis might even provide a survival advantage.

Pathways of immediate hypothermia and leukocyte infiltration in an adjuvant-free mouse model of anaphylaxis.

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 FcεRI, 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.

IgG subclasses determine pathways of anaphylaxis in mice.

BACKGROUND: Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (FcγRs). Mouse IgG2a and IgG2b bind activating FcγRI, FcγRIII, and FcγRIV and inhibitory FcγRIIB; mouse IgG1 binds only FcγRIII and FcγRIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. OBJECTIVE: We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependent passive systemic anaphylaxis. METHODS: Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for FcγRs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. FcγR expression was evaluated on these cells before and after anaphylaxis. RESULTS: Activating FcγRIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory FcγRIIB to negatively regulate these models of anaphylaxis. CONCLUSION: We propose that the differential expression of inhibitory FcγRIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass-dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions in vivo.

Mast cells contribute to bleomycin-induced lung inflammation and injury in mice through a chymase/mast cell protease 4-dependent mechanism.

Mast cells (MCs) are found in large numbers in lungs of patients with pulmonary fibrosis. However, the functions of MCs in lung fibrosis remain largely unknown. We assessed the role of MCs and MC protease 4 (MCPT4), the mouse counterpart of human MC chymase, in a mouse model of bleomycin (BLM)-induced lung injury. We found that levels of inflammation in the bronchoalveolar lavage and the lung, as well as levels of lung fibrosis, were reduced 7 d after intranasal delivery of BLM MC-deficient Kit(W-sh/W-sh) mice compared with wild-type (WT) mice. Confirming the implication of MCs in these processes, we report that the levels of inflammation and fibrosis observed in Kit(W-sh/W-sh) mice can be restored to those observed in WT mice after the adoptive transfer of bone marrow-derived cultured MCs into Kit(W-sh/W-sh) mice. Additionally, we show that levels of inflammation and fibrosis are also reduced in MC chymase MCPT4-deficient mice as compared with WT mice at day 7, suggesting a role for MC-derived MCPT4 in these processes. Our results support the conclusion that MCs can contribute to the initial lung injury induced by BLM through release of the MCPT4 chymase.