CD300a Regulates Mouse Macrophage Functionality in Allergic Inflammation.

BACKGROUND: CD300a is an inhibitory receptor (IR) expressed on several leukocytes, including mast cells (MCs) and macrophages (MΦ), important cells in allergic inflammation (AI). We have previously characterized CD300a role on MCs and in vivo in mouse models of allergy, in which the absence of CD300a resulted in increased inflammatory features and delayed resolution. However, the exact mechanism of this delayed resolution is unclear. Our hypothesis is that MΦ, important players in resolution, might be impaired when CD300a is absent. OBJECTIVES: The aim of the study was to investigate CD300a-dependent functionality of mouse MΦ. METHOD: MΦ were purified from the peritoneum of wild-type (WT) and CD300a-/- mice naïve and 48 h and 96 h after challenge with ovalbumin/alum. Phenotype switching was analyzed via specific M1-M2 inducers and markers. MΦ phagocytotic ability was assessed via Staphylococcus aureus pHrodo-conjugated bioparticles. The influence of MCs on MΦ was investigated by incubating WT MΦ with supernatants from non-activated and IgE-activated bone marrow-derived MCs (BMMCs) and analyzing functional responses. RESULTS: Naïve CD300a-/- MΦ presented with increased sensitivity to activation when treated with LPS. Absence of CD300a results in increased Arg1 expression and increased IL-6 release when MΦ are purified from allergic peritonitis-induced mice. Similar results were obtained when CD300a-/- MΦ were purified 96 h after challenge. On the other hand, CD300a absence did not affect phagocytosis. WT MΦ incubated with supernatants of non-activated and IgE-activated BMMCs presented with increased iNOS expression and decreased Arg1 levels. CONCLUSIONS: The IR CD300a controls the activation state of MΦ, and its absence could augment the inflammatory state seen in CD300a-/- mice. Moreover, MCs can also influence MΦ phenotype switching. This may partially explain the delayed AI resolution seen in these mice.

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.

Glucocorticoids and COVID-19.

Coronavirus Disease 19 (COVID-19) is associated with high morbidity and mortality rates globally, representing the greatest health and economic challenge today. Several drugs are currently approved for the treatment of COVID-19. Among these, glucocorticoids (GCs) have received particular attention due to their anti-inflammatory and immunosuppressive effects. In fact, GC are widely used in current clinical practice to treat inflammatory, allergic and autoimmune diseases. Major mechanisms of GC action include inhibition of innate and adaptive immune activity. In particular, an important role is played by the inhibition of pro-inflammatory cytokines and chemokines, and the induction of proteins with anti-inflammatory activity. Overall, as indicated by various national and international regulatory agencies, GCs are recommended for the treatment of COVID-19 in patients requiring oxygen therapy, with or without mechanical ventilation. Regarding the use of GCs for the COVID-19 treatment of non-hospitalized patients at an early stage of the disease, many controversial studies have been reported and regulatory agencies have not recommended their use. The decision to start GC therapy should be based not only on the severity of COVID-19 disease, but also on careful considerations of the benefit/risk profile in individual patients, including monitoring of adverse events. In this review we summarize the effects of GCs on the major cellular and molecular components of the inflammatory/immune system, the benefits and the adverse common reactions in the treatment of inflammatory/autoimmune diseases, as well as in the management of COVID-19.

Selected recent advances in understanding the role of human mast cells in health and disease.

Mast cells are highly granular tissue-resident cells and key drivers of inflammation, particularly in allergies as well as in other inflammatory diseases. Most mast cell research was initially conducted in rodents but has increasingly shifted to the human system, with the advancement of research technologies and methodologies. Today we can analyze primary human cells including rare subpopulations, we can produce and maintain mast cells isolated from human tissues, and there are several human mast cell lines. These tools have substantially facilitated our understanding of their role and function in different organs in both health and disease. We can now define more clearly where human mast cells originate from, how they develop, which mediators they store, produce de novo, and release, how they are activated and by which receptors, and which neighboring cells they interact with and by which mechanisms. Considerable progress has also been made regarding the potential contribution of mast cells to disease, which, in turn, has led to the development of novel approaches for preventing key pathogenic effects of mast cells, heralding the era of mast cell-targeted therapeutics. In this review, we present and discuss a selection of some of the most significant advancements and remaining gaps in our understanding of human mast cells during the last 25 years, with a focus on clinical relevance.

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.

Mimicking human riboflavin responsive neuromuscular disorders by silencing flad-1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission.

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

Latest Progresses in Allergic Diseases Biomarkers: Asthma and Atopic Dermatitis.

In the last years, the understanding of the pathologic mechanisms of asthma and atopic dermatitis, both characterized by allergic inflammation, has greatly improved. However, it is evident that both diseases present with high heterogeneity, which complicates the diagnosis and the therapeutic approach of the patients. Moreover, some of the currently available strategies to treat asthma and atopic dermatitis are still mostly controlling the symptoms, but not to lead towards full healing, thus having these two diseases labelled as unmet clinical needs by WHO. Therefore, the "one-size-fits-all" strategy is outdated for asthma and atopic dermatitis, and there is the need of better methods to clearly diagnose the disease and tailor the therapy according to the specific symptomatology. In this regard, the use of biomarkers has been advanced in order to characterize both diseases according to their clinical signs and to facilitate the subsequent treatment. Despite the advancements made in this regard, there is still need for better and more sensitive biomarkers and for less invasive sampling methodologies, with the aim to diagnose specifically each manifestation of asthma and atopic dermatitis and to provide the best treatment with the least suffering for the patients.

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.

Comparing biologicals and small molecule drug therapies for chronic respiratory diseases: An EAACI Taskforce on Immunopharmacology position paper.

Chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), together with their comorbidities, bear a significant burden on public health. Increased appreciation of molecular networks underlying inflammatory airway disease needs to be translated into new therapies for distinct phenotypes not controlled by current treatment regimens. On the other hand, development of new safe and effective therapies for such respiratory diseases is an arduous and expensive process. Antibody-based (biological) therapies are successful in treating certain respiratory conditions not controlled by standard therapies such as severe allergic and refractory eosinophilic severe asthma, while in other inflammatory respiratory diseases, such as COPD, biologicals are having a more limited impact. Small molecule drug (SMD)-based therapies represent an active field in pharmaceutical research and development. SMDs expand biologicals' therapeutic targets by reaching the intracellular compartment by delivery as either an oral or topically based formulation, offering both convenience and lower costs. Aim of this review was to compare and contrast the distinct pharmacological properties and clinical applications of SMDs- and antibody-based treatment strategies, their limitations and challenges, in order to highlight how they should be integrated for their optimal utilization and to fill the critical gaps in current treatment for these chronic inflammatory respiratory diseases.

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.

Leukocyte CD300a Contributes to the Resolution of Murine Allergic Inflammation.

CD300a is an inhibitory receptor for mast cells and eosinophils in allergic inflammation (AI); however, the spatiotemporal expression of CD300a and its potential roles in the resolution of AI are still to be determined. In this study, employing a mouse model of allergic peritonitis, we demonstrate that CD300a expression on peritoneal cells is regulated from inflammation to resolution. Allergic peritonitis-induced CD300a-/- mice had a rapid increase in their inflammatory cell infiltrates and tryptase content in the peritoneal cavity compared with wild type, and their resolution process was significantly delayed. CD300a-/- mice expressed lower levels of ALX/FPR2 receptor on peritoneal cells and had higher levels of LXA4 in the peritoneal lavage. CD300a activation on mouse bone marrow-derived mast cells regulated ALX/FPR2 expression levels following IgE-mediated activation. Together, these findings indicate a role for CD300a in AI and its resolution, in part via the specialized proresolving mediator LXA4 and ALX/FPR2 receptor pathway activation.

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.