Metabolic pathways in immune senescence and inflammaging: Novel therapeutic strategy for chronic inflammatory lung diseases. An EAACI position paper from the Task Force for Immunopharmacology.

The accumulation of senescent cells drives inflammaging and increases morbidity of chronic inflammatory lung diseases. Immune responses are built upon dynamic changes in cell metabolism that supply energy and substrates for cell proliferation, differentiation, and activation. Metabolic changes imposed by environmental stress and inflammation on immune cells and tissue microenvironment are thus chiefly involved in the pathophysiology of allergic and other immune-driven diseases. Altered cell metabolism is also a hallmark of cell senescence, a condition characterized by loss of proliferative activity in cells that remain metabolically active. Accelerated senescence can be triggered by acute or chronic stress and inflammatory responses. In contrast, replicative senescence occurs as part of the physiological aging process and has protective roles in cancer surveillance and wound healing. Importantly, cell senescence can also change or hamper response to diverse therapeutic treatments. Understanding the metabolic pathways of senescence in immune and structural cells is therefore critical to detect, prevent, or revert detrimental aspects of senescence-related immunopathology, by developing specific diagnostics and targeted therapies. In this paper, we review the main changes and metabolic alterations occurring in senescent immune cells (macrophages, B cells, T cells). Subsequently, we present the metabolic footprints described in translational studies in patients with chronic asthma and chronic obstructive pulmonary disease (COPD), and review the ongoing preclinical studies and clinical trials of therapeutic approaches aiming at targeting metabolic pathways to antagonize pathological senescence. Because this is a recently emerging field in allergy and clinical immunology, a better understanding of the metabolic profile of the complex landscape of cell senescence is needed. The progress achieved so far is already providing opportunities for new therapies, as well as for strategies aimed at disease prevention and supporting healthy aging.

Modulation of allergic responses by mitochondrial STAT3 inhibitors.

BACKGROUND: Recently, we have shown that mast cell mitochondrial STAT3 could serve as a new target for the regulation of the allergic response as it plays an essential role in immunologically mediated degranulation of mast cells. In the present work, we explored how two recently developed mitochondrial STAT3 inhibitors (Mitocur-1 and Mitocur-3) modulate the allergic response. METHODS: Experiments were performed both in vitro in cultured human/mouse mast cells and with rat basophilic leukemia (RBL) cells and also in vivo in mice. The effect of mitochondrial STAT3 inhibition on mast cell function was determined via checking degranulation and several cytokines secretion levels. RESULTS: Here, we show that treatment of rodent and human cultured mast cells with low concentrations of mitochondrial STAT3 inhibitors had no effect on STAT3 target gene expression. However, these inhibitors caused a significant reduction in mast cell exocytosis and cytokine release, due to a decrease in OXPHOS activity and STAT3 serine 727 phosphorylation. It was also observed in an OVA mouse model of allergic asthma that one of the inhibitors used significantly reduced eosinophilia and neutrophilia compared to the control mice group. Furthermore, it was observed that treatment with this inhibitor resulted in a significant reduction in blood histamine levels in mice after IgE-Ag challenge. CONCLUSION: The present data strongly suggest that the development of mitochondrial STAT3 inhibitors could serve as a potential treatment for allergy-associated diseases.

AllergoOncology: Opposite outcomes of immune tolerance in allergy and cancer.

While desired for the cure of allergy, regulatory immune cell subsets and nonclassical Th2-biased inflammatory mediators in the tumour microenvironment can contribute to immune suppression and escape of tumours from immunological detection and clearance. A key aim in the cancer field is therefore to design interventions that can break immunological tolerance and halt cancer progression, whereas on the contrary allergen immunotherapy exactly aims to induce tolerance. In this position paper, we review insights on immune tolerance derived from allergy and from cancer inflammation, focusing on what is known about the roles of key immune cells and mediators. We propose that research in the field of AllergoOncology that aims to delineate these immunological mechanisms with juxtaposed clinical consequences in allergy and cancer may point to novel avenues for therapeutic interventions that stand to benefit both disciplines.

CD48 on blood leukocytes and in serum of asthma patients varies with severity.

BACKGROUND: CD48 is a membrane receptor (mCD48) on eosinophils and mast cells and exists in a soluble form (sCD48). CD48 has a pivotal role in murine asthma and in the proinflammatory interactions of mast cells with eosinophils via its ligand CD244. Thus, CD48 might be important in human asthma. METHODS: Therefore, two separate cohorts (IL and UK) comprising mild, moderate, and severe asthma and healthy volunteers were evaluated for blood leukocyte mCD48 expression and sCD48 in serum. Asthmatic bronchial biopsies were immunostained for CD48. sCD48 effect on CD244-dependent eosinophil activation was evaluated. RESULTS: Eosinophil mCD48 expression was significantly elevated in moderate while downregulated in severe asthma. mCD48 expression on B, T, and NK cells and monocytes in severe asthma was significantly increased. sCD48 levels were significantly higher in mild while reduced in severe asthma. sCD48 optimal cutoff values for differentiating asthma from health were identified as >1482 pg/ml (IL) and >1619 pg/ml (UK). In asthmatic bronchial biopsies, mCD48 was expressed predominantly by eosinophils. sCD48 inhibited anti-CD244-induced eosinophil activation. CONCLUSIONS: mCD48 and sCD48 are differentially expressed in the peripheral blood of asthma patients of varying severity. sCD48 inhibits CD244-mediated eosinophil activation. These findings suggest that CD48 may play an important role in human asthma.

AllergoOncology - the impact of allergy in oncology: EAACI position paper.

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

sCD48 is anti-inflammatory in Staphylococcus aureus Enterotoxin B-induced eosinophilic inflammation.

BACKGROUND: Staphylococcus aureus, one of the most important pathogens, is heavily associated with allergy. S. aureus and its toxins interact with eosinophils through CD48, a GPI-anchored receptor important in allergy mainly as expressed by the eosinophils (mCD48). CD48 can exist in a soluble form (sCD48). Our aim was to investigate SEB-induced regulation of eosinophil CD48 and the possible formation and role of sCD48 in SEB-mediated eosinophil activation in vitro and in vivo. METHODS: Human peripheral blood eosinophils were activated by SEB with or without inhibitors for phospholipases (PL) (-C or -D), or cycloheximide, or brefeldin A. We evaluated eosinophil activation (CD11b expression or EPO/IL-8 release), mCD48 (flow cytometry), sCD48 (ELISA), SEB binding to sCD48 (ELISA), and chemotaxis toward SEB. C57BL/6 mice were pre-injected (ip.) with sCD48, and then, peritonitis was induced by SEB injection; peritoneal lavages were collected after 48 h and analyzed by flow cytometry and ELISA. RESULTS: SEB-activated human eosinophils formed sCD48, directly correlating with CD11b expression, through cell-associated PL-C and -D. mCD48 remained stable due to up-regulation in CD48 transcription and cellular trafficking. sCD48 bound to SEB and down-regulated SEB stimulatory effects on eosinophils as assessed by EPO and IL-8 release and eosinophil chemotaxis toward SEB. sCD48 showed anti-inflammatory activity in a SEB-induced mouse peritonitis model. CONCLUSIONS: SEB regulates CD48 dynamics on eosinophils. Our data indicate sCD48 as a SEB-induced 'decoy' receptor derived from eosinophil and therefore as a potential anti-inflammatory tool in S. aureus-induced eosinophil inflammation often associated with allergy.

The CD48 receptor mediates Staphylococcus aureus human and murine eosinophil activation.

BACKGROUND: Allergy is characterized by eosinophilia and an increased susceptibility to microbial infection. Atopic dermatitis (AD) is typically associated with Staphylococcus aureus (SA) colonization. Some of the mechanisms by which SA and its exotoxins interact with eosinophils remain elusive. CD48, a glycosylphosphatidylinositol-anchored receptor belonging to the CD2 family, participates in mast cells-SA stimulating cross-talk, facilitates the formation of the mast cell/eosinophils effector unit and as expressed by eosinophils, mediates experimental asthma. OBJECTIVE: To investigate the role of CD48 expressed on human peripheral blood and mouse bone marrow-derived eosinophils (BMEos) in their interaction with heat-killed SA and its three exotoxins, Staphylococcal enterotoxin B (SEB), protein A (PtA) and peptidoglycan (PGN). METHODS: Eosinophils were obtained from human peripheral blood and BM of WT and CD48-/- mice. SA was heat killed and eosinophils-SA/exotoxins interactions were analyzed by confocal microscopy, adhesion and degranulation, cell viability, cytokine release and cell signalling. In addition, peritonitis was induced by SEB injection into CD48-/- and WT mice. CD48 expression was studied in AD patients' skin and as expressed on their leucocytes in the peripheral blood. RESULTS: We provide evidence for the recognition and direct physical interaction between eosinophils and SA/exotoxins. Skin of AD patients showed a striking increase of eosinophil-associated CD48 expression while on peripheral blood leucocytes it was down-regulated. SA/exotoxins enhanced CD48 eosinophil expression, bound to CD48 and caused eosinophil activation and signal transduction. These effects were significantly decreased by blocking CD48 on human eosinophils or in BMEos from CD48-/- mice. We have also explored the role of CD48 in a SEB-induced peritonitis model in CD48-/- mice by evaluating inflammatory peritoneal cells, eosinophil numbers and activation. CONCLUSIONS: These data demonstrate the important role of CD48 in SA/exotoxins-eosinophil activating interactions that can take place during allergic responses and indicate CD48 as a novel therapeutic target for allergy and especially of AD.

Interacting mast cells and eosinophils acquire an enhanced activation state in vitro.

BACKGROUND: Mast cells (MCs) and eosinophils (Eos), the key effector cells in allergy, are abundantly co-localized particularly in the late and chronic stages of allergic inflammation. Recent evidence has outlined a specialized 'allergic effector unit' in which MCs and Eos communicate via both soluble mediators and physical contact. However, the functional impact of this bi-directional crosstalk on the cells' effector activities has not yet been revealed. We aimed to investigate whether MC/eosinophil interactions can influence the immediate and late activation phenotypes of these cells. METHODS: Human and murine MCs and Eos were co-cultured under various conditions for 1-2 h or 1-3 days, and in selected experiments cell-cell contact was blocked. Cell migration and mediator release were examined, and flow cytometry was applied to stain intracellular signaling molecules and surface receptors. RESULTS: Eosinophils enhanced basal MCs mediator release and co-stimulated IgE-activated MCs through physical contact involving CD48-2B4 interactions. Reciprocally, resting and IgE-stimulated MCs led to eosinophil migration and activation through a paracrine-dependent mechanism. Increased phosphorylation of activation-associated signaling molecules, and enhanced release of tumor necrosis factor α, was observed in long-term co-cultures. Eosinophils also showed enhanced expression of intercellular adhesion molecule 1, which depended on direct contact with MCs. CONCLUSIONS: Our findings reveal a new role for MC/eosinophil interplay in augmenting short- and long-term activation in both cells, in a combined physical/paracrine manner. This enhanced functional activity may thus critically contribute to the perpetuation of the inflammatory response in allergic conditions.

The inhibitory receptor CD300a is up-regulated by hypoxia and GM-CSF in human peripheral blood eosinophils.

BACKGROUND: Eosinophils are involved in several inflammatory processes including allergic inflammation. It has been shown that eosinophil functions may be regulated by activating or inhibitory receptors. Hypoxia is a feature of inflamed tissues and has recently been shown to regulate eosinophil viability and pro-angiogenic potential. In this study, the effect of hypoxia and GM-CSF on the inhibitory receptor CD300a in human peripheral blood eosinophils was investigated. METHODS: CD300a expression on eosinophils was analyzed by flow cytometry and evaluated by immuno-fluorescence; mRNA levels were evaluated by RT-PCR. RESULTS: An increase in the expression of CD300a was observed in hypoxic eosinophils compared to the normoxic ones. GM-CSF strongly induced CD300a increase also after 3 h in culture. In addition, hypoxia augmented mRNA levels of CD300a. Inhibition of hypoxia-inducible factor (HIF)-1 abolished the hypoxia-/GM-CSF-induced CD300a increase. CONCLUSION: CD300a expression is up-regulated by hypoxia, and GM-CSF where HIF-1 might play an important role. These results are important for the understanding of eosinophils behavior in inflamed tissue and suggest a new effect on their function in allergic inflammation. Taken together our data point out CD300a as a novel target for the treatment of allergy.

Eosinophil major basic protein activates human cord blood mast cells primed with fibroblast membranes by integrin-ß1.

BACKGROUND: Mast cell (MC) - eosinophil (Eos) activating cross-talk might be critical for the severity and chronicity of allergy. Among soluble mediators, eosinophil major basic protein (MBP), a hallmark of allergy, is particularly important because it was shown to activate specific MC subtypes. We previously demonstrated that MBP activates IgE-desensitized rat MC and human lung and cord blood-derived MC (CBMC) after priming with fibroblast membranal stem cell factor. However, a distinct mechanism for this activation was missing. Therefore, we aimed to investigate it. METHODS: Major basic protein-1 activation of CBMC primed with fibroblast-derived membranes (FBM) was measured by ß-hexosaminidase and tryptase release. Chemical cross-linking followed by micrometric flow cytometry probed direct interactions. Antibodies neutralized integrin-ß1 and recognized its active form. Pertussis toxin (Ptx) was used to decrease integrin-ß1 active form expression. Hematopoietic cell kinase (Hck) was identified by immunoprecipitation (IP) and silenced by siRNA. RESULTS: Major basic protein-1-induced CBMC activation is mediated partly by MBP1-integrin-ß1 interaction on the MC surface. FBM prime CBMC via a G protein, as confirmed by Ptx, to shift integrin-ß1 to its active form. Following MBP1 binding, integrin-ß1 binds Hck that further transduces the activation signal. MC priming with FBM leads to up-regulation in Hck protein level. MC integrin-ß1 neutralization inhibits MBP1-induced activation and uptake. Hck silencing results with reduced MBP1-induced activation. CONCLUSIONS: Fibroblast-derived membranes, integrin-ß1, and Hck are involved in MBP1-induced activation of CBMC and therefore represent a distinct mechanism for this activation. This finding might implicate integrin-ß1 and Hck as targets for decreasing MC - Eos activating cross-talk in allergy.

Physical interactions between mast cells and eosinophils: a novel mechanism enhancing eosinophil survival in vitro.

BACKGROUND: Mast cells (MCs) and eosinophils (Eos) are the key effector cells of the allergic reaction. Although classically associated with different stages of the response, the cells co-exist in the inflamed tissue in the late and chronic phases in high numbers and are likely to cross-talk. While some mediators of MCs are known to affect Eos biology and vice versa, paracrine and physical interplay between the two cells has not been described yet. We aimed to investigate whether intercellular MC-Eos communication could take place in the allergic response and exert functional bidirectional changes on the cells. METHODS: Tissue sections from various allergic disorders were specifically stained for both cells. Human cord blood-derived MCs and peripheral blood Eos, co-cultured under different conditions, were studied by advanced microscopy and flow cytometry. RESULTS: Several co-localized MC-Eos pairs were detected in human nasal polyps and asthmatic bronchi, as well in mouse atopic dermatitis. In vitro, MCs and Eos formed stable conjugates at high rates, with clear membrane contact. In the presence of MCs, Eos were significantly more viable under several co-culture conditions and at both IgE-activated and steroid-inhibited settings. MC regulation of Eos survival required communication through soluble mediators but was even more dependent on physical cell-cell contact. CONCLUSIONS: Our findings provide the first evidence for a complex network of paracrine and membrane interactions between MCs and Eos. The prosurvival phenotype induced by this MC-Eos interplay may be critical for sustaining chronic allergic inflammation.

Osteopontin is expressed and functional in human eosinophils.

BACKGROUND: Eosinophils are critically involved in allergic inflammation and tissue remodeling. Osteopontin (OPN) is a glycoprotein molecule which exhibits pro-fibrogenic and pro-angiogenic properties and has recently also been implicated in allergic diseases. In this study, we investigated the expression and function of OPN in human eosinophils. METHODS: Osteopontin mRNA (RT-PCR) and protein (immunofluorescence) expression in peripheral blood eosinophils from atopic human subjects were evaluated. Soluble OPN release was determined in resting and activated eosinophils. The contribution of OPN to eosinophil-induced angiogenesis was determined using the chick embryo chorio- allantoic membrane (CAM) assay and OPN-induced eosinophil chemotaxis was determined (ChemoTx System microplate wells). Finally, OPN expression in bronchoalveolar lavage (BAL) fluids from mild asthmatic and normal control subjects was determined. RESULTS: Osteopontin is expressed in human eosinophils and is increased following GM-CSF and IL-5 activation. Eosinophil-derived OPN contributes to eosinophil-induced angiogenesis. Recombinant OPN promotes eosinophil chemotaxis in vitro and this effect is mediated by alpha(4)beta(1) integrin binding. Soluble OPN is increased in the bronchoalveolar lavage fluid from mild asthmatic subjects and correlates with eosinophil counts. CONCLUSIONS: We therefore conclude that OPN is likely to contribute to the process of angiogenesis observed in the airways in asthma.

AllergoOncology: ultra-low IgE, a potential novel biomarker in cancer-a Position Paper of the European Academy of Allergy and Clinical Immunology (EAACI).

Elevated serum IgE levels are associated with allergic disorders, parasitosis and specific immunologic abnormalities. In addition, epidemiological and mechanistic evidence indicates an association between IgE-mediated immune surveillance and protection from tumour growth. Intriguingly, recent studies reveal a correlation between IgE deficiency and increased malignancy risk. This is the first review discussing IgE levels and links to pathological conditions, with special focus on the potential clinical significance of ultra-low serum IgE levels and risk of malignancy. In this Position Paper we discuss: (a) the utility of measuring total IgE levels in the management of allergies, parasitosis, and immunodeficiencies, (b) factors that may influence serum IgE levels, (c) IgE as a marker of different disorders, and d) the relationship between ultra-low IgE levels and malignancy susceptibility. While elevated serum IgE is generally associated with allergic/atopic conditions, very low or absent IgE may hamper anti-tumour surveillance, indicating the importance of a balanced IgE-mediated immune function. Ultra-low IgE may prove to be an unexpected biomarker for cancer risk. Nevertheless, given the early stage of investigations conducted mostly in patients with diseases that influence IgE levels, in-depth mechanistic studies and stratification of malignancy risk based on associated demographic, immunological and clinical co-factors are warranted.

Angiogenesis in asthma.

Asthma is a chronic inflammatory disease of the airways characterized by infiltration and activation of inflammatory cells and by structural changes, including subepithelial fibrosis, smooth muscle cells hypertrophy/hyperplasia, epithelial cell metaplasia and angiogenesis. These structural changes are thought to correlate with asthma severity and to account for the development of progressive lung function deterioration. The mechanism underlying airway angiogenesis in asthma and its precise clinical relevance have not yet been completely elucidated. This review provides recent data showing the contribution of allergic inflammation in increased airway vascularity and potential therapeutical approaches in asthma treatment by acting on bronchial microvascular changes.

The role of eosinophil major basic protein in angiogenesis.

BACKGROUND: Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. METHODS: Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. RESULTS: Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly-L-arginine does not induce any angiogenic effects. CONCLUSIONS: These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.

TRAIL mediated signaling in human mast cells: the influence of IgE-dependent activation.

BACKGROUND: Mast cells activation through FcepsilonRI cross-linking has a pivotal role in the initiation of allergic reactions. The influence of this activation on programmed cell death of human mast cells has not yet been clarified. This study evaluates the influence of IgE-dependent activation alone and in synergy with TRAIL on the expression of molecules involved in the apoptotic signal transduction. METHODS: Human cord blood derived mast cells (CBMC) were cultured with myeloma IgE followed by activation with anti-human IgE. The expression of proteins involved in apoptotic signal transduction was assessed by immunoblot analysis. To test the effect of activation on a pro-apoptotic stimulus, activated, IgE-treated and resting CBMC were incubated with TRAIL, or in a medium with suboptimal concentrations of stem cell factor (SCF). RESULTS: In accordance with a previous study of ours, it was found that IgE-dependent activation increased TRAIL-induced caspase-8 and caspase-3 cleavage. However, it did not have a significant influence on CBMC death induced by SCF withdrawal. IgE-dependent activation increased the expression of FLIP and myeloid cell leukemia 1 (MCL-1) anti-apoptotic molecules as well as the pro-apoptotic one, BIM. In addition, a decrease in BID expression was observed. TRAIL could reverse the increase in FLIP but did not influence the upregulation of MCL-1 and of BIM. CONCLUSIONS: These findings suggest that IgE-dependent activation of human mast cells induces an increase in both pro-survival and pro-apoptotic molecules. We therefore hypothesized that IgE-dependent activation may regulate human mast cell apoptosis by fine-tuning anti-apoptotic and pro-apoptotic factors.

Monoclonal antibodies: the new magic bullets for allergy: IUPHAR Review 17.

Allergic diseases and conditions are widespread and their incidence is on the increase. They are characterized by the activation of mast cells resident in tissues and the consequent infiltration and stimulation of several inflammatory cells, predominantly eosinophils. Cell-cell cross-talk and the release of mediators are responsible for the symptoms and for the modulation of the response. The gold standard of therapeutic intervention is still glucocorticosteroids, although they are not effective in all patients and may cause numerous side effects. Symptomatic medications are also widespread. As research has led to deeper insights into the mechanisms governing the diseases, new avenues have been opened resulting in recent years in the development of monoclonal antibodies (mAbs) such as anti-IgE mAbs (omalizumab) and others still undergoing clinical trials aimed to specifically target molecules involved in the migration and stimulation of inflammatory cells. In this review, we summarize new developments in the field of anti-allergic mAbs with special emphasis on the treatment of asthma, particularly severe forms of this condition, and atopic dermatitis, which are two unmet clinical needs.