Mast cells contribute to the resolution of allergic inflammation by releasing resolvin D1.

BACKGROUND: Mast cells are initiators and main effectors of allergic inflammation, together with eosinophils, with whom they can interact in a physical and soluble cross-talk with marked pro-inflammatory features, the Allergic Effector Unit. The pro-resolution role of mast cells, alone or in co-culture with eosinophils, has not been characterized yet. OBJECTIVES: We aimed to investigate select pro-resolution pathways in mast cells in vitro and in vivo in allergic inflammation. METHODS: In vitro, we employed human and murine mast cells and analyzed release of resolvin D1 and expression of 15-lipoxygenase after IgE-mediated activation. We performed co-culture of IgE-activated mast cells with peripheral blood eosinophils and investigated 15-lipoxygenase expression and Resolvin D1 release. In vivo, we performed Ovalbumin/Alum and Ovalbumin/S. aureus enterotoxin B allergic peritonitis model in Wild Type mice following a MC "overshoot" protocol. RESULTS: We found that IgE-activated mast cells release significant amounts of resolvin D1 30 min after activation, while 15-lipoxygenase expression remained unchanged. Resolvin D1 release was found to be decreased in IgE-activated mast cells co-cultured with peripheral blood eosinophils for 30 min In vivo, mast cell-overshoot mice exhibited a trend of reduced inflammation, together with increased peritoneal resolvin D1 release. CONCLUSIONS: Mast cells can actively contribute to resolution of allergic inflammation by releasing resolvin D1.

Patients with coronavirus disease 2019 characterized by dysregulated levels of membrane and soluble cluster of differentiation 48.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can progress into a severe form of acute lung injury. The cosignaling receptor cluster of differentiation 48 (CD48) exists in membrane-bound (mCD48) and soluble (sCD48) forms and has been reported to be implicated in antiviral immunity and dysregulated in several inflammatory conditions. Therefore, CD48 dysregulation may be a putative feature in COVID-19-associated inflammation that deserves consideration. OBJECTIVE: To analyze CD48 expression in lung autopsies and peripheral blood leukocytes and sera of patients with COVID-19. The expression of the CD48 ligand 2B4 on the membrane of peripheral blood leukocytes was also assessed. METHODS: Twenty-eight lung tissue samples obtained from COVID-19 autopsies were assessed for CD48 expression using gene expression profiling immunohistochemistry (HTG autoimmune panel). Peripheral whole blood was collected from 111 patients with COVID-19, and the expression of mCD48 and of membrane-bound 2B4 was analyzed by flow cytometry. Serum levels of sCD48 were assessed by enzyme-linked immunosorbent assay. RESULTS: Lung tissue of patients with COVID-19 showed increased CD48 messenger RNA expression and infiltration of CD48+ lymphocytes. In the peripheral blood, mCD48 was considerably increased on all evaluated cell types. In addition, sCD48 levels were significantly higher in patients with COVID-19, independently of disease severity. CONCLUSION: Considering the changes of mCD48 and sCD48, a role for CD48 in COVID-19 can be assumed and needs to be further investigated.

An mTORC1 to HRI signaling axis promotes cytotoxicity of proteasome inhibitors in multiple myeloma.

Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs.

Cromolyn Sodium differentially regulates human mast cell and mouse leukocyte responses to control allergic inflammation.

BACKGROUND: Cromolyn Sodium (CS) has been used in the past as an anti-allergy drug owing to its mast cell (MC) stabilizing properties that impair histamine release. However, additional mechanisms for its clinical actions are likely and might help to identify new roles for MCs and leukocytes in regulating inflammation. Here, using human cord blood-derived MCs (CBMCs), mouse bone marrow-derived MCs (BMMCs) and eosinophils (BMEos), and in vivo mouse models of allergic inflammation (AI), additional actions of CS on MCs were determined. METHODS: The in vitro effects of CS on IgE-activated human and mouse MCs were assessed by measuring the levels of pro-inflammatory (tryptase, LTC4, IL-8, CD48) and pro-resolution effectors (IL-10, CD300a, Annexin A1) before and after CS treatment. The in vivo effects of daily CS injections on parameters of inflammation were assessed using mouse models of allergic peritonitis (AP) (Ovalbumin/Alum- or Ovalbumin/S. aureus enterotoxin B) and allergic airways inflammation (AAI) (house dust mite (HDM)). RESULTS: In vitro, CS did not affect pro-inflammatory effectors but significantly increased the anti-inflammatory/pro-resolution CD300a levels and IL-10 release from IgE-activated CBMCs. BMMCs were not affected by CS. In vivo, CS injections decreased total cell and Eos numbers in the peritoneal cavity in the AP models and bronchoalveolar lavage and lungs in the AAI model. CS reduced EPX release from PAF-activated BMEos in vitro, possibly explaining the in vivo findings. CONCLUSION: Together, these results demonstrate immunomodulatory actions for CS in AI that are broader than only MC stabilization.

A novel mast cell-dependent allergic peritonitis model.

Typical murine models of allergic inflammation are induced by the combination of ovalbumin and aluminum hydroxide. However, accumulating evidence indicates that, in models of asthma and atopic dermatitis, allergic inflammation can be generated in the absence of aluminum hydroxide. Moreover, co-administration of Staphylococcus aureus enterotoxin B with ovalbumin can enhance inflammation. The objective of this study was to establish a rapid and mast cell-dependent murine model of allergic inflammation by inducing allergic peritonitis using ovalbumin and S. aureus enterotoxin B. Allergic peritonitis was induced in C57BL/6 mice by subcutaneous sensitization and intraperitoneal challenge with ovalbumin and S. aureus enterotoxin B. Disease characteristics were assessed by flow cytometry, enzyme-linked immunosorbent assay (ELISA), trypan blue exclusion and colorimetric assays. The time-course of the allergic peritonitis revealed a peak of peritoneal inflammation 48 h after challenge, as assessed by total cells and eosinophil counts. The decrease of cell numbers started 96 h post-challenge, with complete clearance within 168 h. Moreover, significantly higher levels of tryptase and increased vascular permeability were found 30 min following challenge. Allergic inflammation induction by ovalbumin and S. aureus enterotoxin B was impaired in mast cell-deficient mice and partially restored by mice reconstitution with bone marrow-derived mast cells, indicating the mast cell role in this model. We present a novel model of allergic peritonitis that is mast cell-dependent, simple and robust. Moreover, the use of S. aureus enterotoxin B better resembles human allergic inflammation, which is known to be characterized by the colonization of S. aureus.

Update on Eosinophil Interaction with Mast Cells: The Allergic Effector Unit.

Mast cells and eosinophils are the key effector cells of allergy [1]. In general, allergic reactions are composed of two phases, namely an early phase and a late phase, and after that resolution occurs. If the allergic reactions fail to resolve after the late phase, allergic inflammation (AI) can evolve into a chronic phase mainly involving mast cells and eosinophils that abundantly coexist in the inflamed tissue in the late and chronic phases and cross-talk in a bidirectional manner. We defined these bidirectional interactions between MCs and Eos, as the "allergic effector unit." This cross talk is mediated by both physical cell-cell contacts through cell surface receptors such as CD48, 2B4, and respective ligands and through released mediators such as various specific granular mediators, arachidonic acid metabolites, cytokines, and chemokines [2, 3]. The allergic effector unit can be studied in vitro in a customized co-culture system using mast cells and eosinophils derived from either mouse or human sources.

Granulocyte-targeted therapies for airway diseases.

The average respiration rate for an adult is 12-20 breaths per minute, which constantly exposes the lungs to allergens and harmful particles. As a result, respiratory diseases, which includes asthma, chronic obstructive pulmonary disease (COPD) and acute lower respiratory tract infections (LTRI), are a major cause of death worldwide. Although asthma, COPD and LTRI are distinctly different diseases with separate mechanisms of disease progression, they do share a common feature - airway inflammation with intense recruitment and activation of granulocytes and mast cells. Neutrophils, eosinophils, basophils, and mast cells are crucial players in host defense against pathogens and maintenance of lung homeostasis. Upon contact with harmful particles, part of the pulmonary defense mechanism is to recruit these cells into the airways. Despite their protective nature, overactivation or accumulation of granulocytes and mast cells in the lungs results in unwanted chronic airway inflammation and damage. As such, understanding the bright and the dark side of these leukocytes in lung physiology paves the way for the development of therapies targeting this important mechanism of disease. Here we discuss the role of granulocytes in respiratory diseases and summarize therapeutic strategies focused on granulocyte recruitment and activation in the lungs.

Tezepelumab: a novel biological therapy for the treatment of severe uncontrolled asthma.

Introduction: Thymic stromal lymphopoietin (TSLP) is overexpressed in the airways of severe asthmatics and is an upstream cytokine that orchestrates inflammatory responses in asthma. TSLP exerts its effects by binding to a high affinity heteromeric receptor complex composed of TSLPR and IL-7Rα. An association of polymorphisms in TSLP with airway hyperresponsiveness, IgE, eosinophilia and asthma has been documented. TSLP has been implicated in asthma pathophysiology. Tezepelumab is a first-in-class human monoclonal antibody that binds to TSLP, thus inhibiting its interaction with TSLP receptor complex. Tezepelumab given as an add-on-therapy to patients with severe uncontrolled asthma has shown safety, tolerability and efficacy. Several trials are evaluating the long-term safety and the efficacy of tezepelumab in adults and adolescents with severe uncontrolled asthma.Areas covered: We provide an overview of the monoclonal antibody therapeutics market for severe uncontrolled asthma, examine the underlying pathophysiology that drives TSLP and discuss the use of tezepelumab for the treatment of severe uncontrolled asthma,Expert opinion: TSLP is a promising target for T2-high and perhaps some patients with T2-low asthma. The results of preliminary clinical trials are encouraging. Several unanswered questions concerning basic pathophysiological aspects of TSLP variants, the long-term safety and efficacy of tezepelumab with different phenotypes/endotypes of asthma should be addressed.

2B4 and CD48: A powerful couple of the immune system.

The signaling lymphocytic activation molecule (SLAM) family of receptors (SLAMF) is a group of receptors belonging to the CD2 family. It is composed of several members expressed on many hematopoietic cells. Most of the receptors interact in a homophilic fashion with neighboring cells. Their distribution and binding properties, together with their ability to function as both activating and inhibitory receptors, put them as key players in the immune system regulation. Several SLAM family receptors have been extensively investigated. This review mainly focuses on CD244 (2B4 or SLAMF4,) and CD48, particularly as expressed by the key cells of allergy, mast cells and eosinophils.

A Hexokinase 2 Modulator for Field-Directed Treatment of Experimental Actinic Keratoses.

Overexpression of hexokinase 2, and its binding to VDAC1 on the outer mitochondrial membrane of cancer cells, is key to their metabolic reprogramming to aerobic glycolysis, which enables them to proliferate. We describe Comp-1, an allosteric small molecule that selectively detaches hexokinase 2 from the mitochondria. Detachment of hexokinase 2 reduces glycolysis and triggers apoptosis in cancer cells, without affecting hexokinase 1-expressing normal cells. The anti-cancer activity of Comp-1 was demonstrated in the UVB-damaged skin model in SKH-1 mice. Topical treatment with Comp-1 led to 70% reduction in lesion number and area. This in vivo efficacy was obtained without local skin reactions or other safety findings. Mechanism-related pharmacodynamic markers, including hexokinase 2 and cleaved caspase 3 levels, are affected by Comp-1 treatment in vivo. Good Laboratory Practice toxicology studies in minipigs for 28 days and 13 weeks established no systemic toxicities and minimal dermal reaction for once-daily application of up to 20% and 15% ointment strengths, respectively. Thus, Comp-1 may address a significant unmet medical need for a non-irritating efficacious topical actinic keratosis treatment.

Hypoxic-induced truncation of voltage-dependent anion channel 1 is mediated by both asparagine endopeptidase and calpain 1 activities.

The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane (OMM) protein, serves as a mitochondrial gatekeeper, mediating the transport of nucleotides, Ca2+ and other metabolites across the OMM. VDAC1 also plays a central role in mitochondria-mediated apoptosis by facilitating the release of apoptotic proteins and by association with both pro- and anti-apoptotic proteins. Tumor cells, which are constantly exposed to hypoxic conditions, affect the cell via the transcription factor hypoxia-inducible factor (HIF) that induces transcriptional activity. In cultured cells and in lung cancer patients, hypoxia induces VDAC1 truncation at the C-terminus (VDAC1-ΔC). However, the molecular mechanisms involved in VDAC1-ΔC formation are unknown. Here, we show that hypoxia-induced VDAC1-ΔC formation is inhibited by the Ca2+ chelator BAPTA-AM, by calpain inhibitor-1, by inhibitor of the asparagine endopeptidase (AEP) and by si-RNA targeting HIF1-α or Ca2+-activated protease calpain-1 expression but not that of calpain-2. Finally, VDAC1-ΔC expressed in bacteria and reconstituted into a planar lipid bilayer exhibited decreased channel conductance relative to the full-length protein, yet retained voltage-dependent conductance. These findings suggest that hypoxia, acting via HIF-1α expression, leads to VDAC1 cleavage involving the activation of calpain 1 and AEP.

Human eosinophils and mast cells: Birds of a feather flock together.

While the origin of the phrase "birds of a feather flock together" is unclear, it has been in use for centuries and is typically employed to describe the phenomenon that people with similar tastes or interests tend to seek each other out and congregate together. In this review, we have co-opted this phrase to compare innate immune cells of related origin, the eosinophil and mast cell, because they very often accumulate together in tissue sites under both homeostatic and inflammatory conditions. To highlight overlapping yet distinct features, their hematopoietic development, cell surface phenotype, mediator release profiles and roles in diseases have been compared and contrasted. What emerges is a sense that these two cell types often interact with each other and their tissue environment to provide synergistic contributions to a variety of normal and pathologic immune responses.

The role of calcium in VDAC1 oligomerization and mitochondria-mediated apoptosis.

The voltage-dependent anion channel (VDAC), located at the outer mitochondria membrane (OMM), mediates interactions between mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca(2+), and metabolites. Substantial evidence points to VDAC1 as being a key player in apoptosis, regulating the release of apoptogenic proteins from mitochondria, such as cytochrome c, and interacting with anti-apoptotic proteins. Recently, we demonstrated that VDAC1 oligomerization is a general mechanism common to numerous apoptogens acting via different initiating cascades and proposed that a protein-conducting channel formed within a VDAC1 homo/hetero oligomer mediates cytochrome c release. However, the molecular mechanism responsible for VDAC1 oligomerization remains unclear. Several studies have shown that mitochondrial Ca(2+) is involved in apoptosis induction and that VDAC1 possesses Ca(2+)-binding sites and mediates Ca(2+) transport across the OMM. Here, the relationship between the cellular Ca(2+) level, [Ca(2+)]i, VDAC1 oligomerization and apoptosis was studied. Decreasing [Ca(2+)]i using the cell-permeable Ca(2+) chelating reagent BAPTA-AM was found to inhibit VDAC1 oligomerization and apoptosis, while increasing [Ca(2+)]i using Ca(2+) ionophore resulted in VDAC1 oligomerization and apoptosis induction in the absence of apoptotic stimuli. Moreover, induction of apoptosis elevated [Ca(2+)]i, concomitantly with VDAC1 oligomerization. AzRu-mediated inhibition of mitochondrial Ca(2+) transport decreased VDAC1 oligomerization, suggesting that mitochondrial Ca(2+) is required for VDAC1 oligomerization. In addition, increased [Ca(2+)]i levels up-regulate VDAC1 expression. These results suggest that Ca(2+) promotes VDAC1 oligomerization via activation of a yet unknown signaling pathway or by increasing VDAC1 expression, leading to apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.