AllergoOncology: Biomarkers and refined classification for research in the allergy and glioma nexus-A joint EAACI-EANO position paper.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

Understanding Systemic and Local Inflammation Induced by Nasal Polyposis: Role of the Allergic Phenotype.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by persistent symptoms associated to the development of nasal polyps. To this day, the molecular mechanisms involved are still not well defined. However, it has been suggested that a sustained inflammation as allergy is involved in its onset. In this exploratory study, the aim was to investigate the effect of the allergic status in the development of CRSwNP. To achieve this, we recruited 22 patients with CRSwNP and classified them in non-allergic and allergic using ImmunoCAP ISAC molecular diagnosis. Plasma samples were analyzed using liquid chromatography coupled to mass spectrometry (LC-MS). Subsequently, significant metabolites from plasma that were commercially available were then analyzed by targeted analysis in some nasal polyps. Additionally, nasal polyp and nasal mucosa samples were examined for eosinophils, neutrophils, CD3+ and CD11c+ cells, as well as collagen deposition and goblet cell hyperplasia. We found that 9 out of the 22 patients were sensitized to some aeroallergens (named as allergic CRSwNP). The other 13 patients had no sensitizations (non-allergic CRSwNP). Regarding metabolomics, bilirubin, cortisol, lysophosphatidylcholines (LPCs) 16:0, 18:0 and 20:4 and lysophosphatidylinositol (LPI) 20:4, which are usually related to a sustained allergic inflammation, were unexpectedly increased in plasma of non-allergic CRSwNP compared to allergic CRSwNP. LPC 16:0, LPC 18:0 and LPI 20:4 followed the same trend in nasal polyp as they did in plasma. Comparison of nasal polyps with nasal mucosa showed a significant increase in eosinophils (p < 0.001) and neutrophils (p < 0.01) in allergic CRSwNP. There were more eosinophils in polyps of non-allergic CRSwNP than in their nasal mucosa (p < 0.01). Polyps from non-allergic CRSwNP had less eosinophils than the polyps of allergic CRSwNP (p < 0.05) and reduced amounts of collagen compared to their nasal mucosa (p < 0.001). Our data suggests that there is a systemic inflammatory response associated to CRSwNP in the absence of allergy, which could be accountable for the nasal polyp development. Allergic CRSwNP presented a higher number of eosinophils in nasal polyps, suggesting that eosinophilia might be connected to the development of nasal polyps in this phenotype.

The Role of Sphingolipids in Allergic Disorders.

Allergy is defined as a complex chronic inflammatory condition in which genetic and environmental factors are implicated. Sphingolipids are involved in multiple biological functions, from cell membrane components to critical signaling molecules. To date, sphingolipids have been studied in different human pathologies such as neurological disorders, cancer, autoimmunity, and infections. Sphingolipid metabolites, in particular, ceramide and sphingosine-1-phosphate (S1P), regulate a diverse range of cellular processes that are important in immunity and inflammation. Moreover, variations in the sphingolipid concentrations have been strongly associated with allergic diseases. This review will focus on the role of sphingolipids in the development of allergic sensitization and allergic inflammation through the activation of immune cells resident in tissues, as well as their role in barrier remodeling and anaphylaxis. The knowledge gained in this emerging field will help to develop new therapeutic options for allergic disorders.

Targeting Macrophages: Friends or Foes in Disease?

Macrophages occupy a prominent position during immune responses. They are considered the final effectors of any given immune response since they can be activated by a wide range of surface ligands and cytokines to acquire a continuum of functional states. Macrophages are involved in tissue homeostasis and in the promotion or resolution of inflammatory responses, causing tissue damage or helping in tissue repair. Knowledge in macrophage polarization has significantly increased in the last decade. Biomarkers, functions, and metabolic states associated with macrophage polarization status have been defined both in murine and human models. Moreover, a large body of evidence demonstrated that macrophage status is a dynamic process that can be modified. Macrophages orchestrate virtually all major diseases-sepsis, infection, chronic inflammatory diseases (rheumatoid arthritis), neurodegenerative disease, and cancer-and thus they represent attractive therapeutic targets. In fact, the possibility to "reprogram" macrophage status is considered as a promising strategy for designing novel therapies. Here, we will review the role of different tissue macrophage populations in the instauration and progression of inflammatory and non-inflammatory pathologies, as exemplified by rheumatoid arthritis, osteoporosis, glioblastoma, and tumor metastasis. We will analyze: 1) the potential as therapeutic targets of recently described macrophage populations, such as osteomacs, reported to play an important role in bone formation and homeostasis or metastasis-associated macrophages (MAMs), key players in the generation of premetastatic niche; 2) the current and potential future approaches to target monocytes/macrophages and their inflammation-causing products in rheumatoid arthritis; and 3) the development of novel intervention strategies using oncolytic viruses, immunomodulatory agents, and checkpoint inhibitors aiming to boost M1-associated anti-tumor immunity. In this review, we will focus on the potential of macrophages as therapeutic targets and discuss their involvement in state-of-the-art strategies to modulate prevalent pathologies of aging societies.

Human glutathione-S-transferase pi potentiates the cysteine-protease activity of the Der p 1 allergen from house dust mite through a cysteine redox mechanism.

Environmental proteases have been widely associated to the pathogenesis of allergic disorders. Der p 1, a cysteine-protease from house dust mite (HDM) Dermatophagoides pteronyssinus, constitutes one of the most clinically relevant indoor aeroallergens worldwide. Der p 1 protease activity depends on the redox status of its catalytic cysteine residue, which has to be in the reduced state to be active. So far, it is unknown whether Der p 1-protease activity could be regulated by host redox microenvironment once it reaches the lung epithelial lining fluid in addition to endogenous mite components. In this sense, Glutathione-S-transferase pi (GSTpi), an enzyme traditionally linked to phase II detoxification, is highly expressed in human lung epithelial cells, which represent the first line of defence against aeroallergens. Moreover, GSTpi is a generalist catalyst of protein S-glutathionylation reactions, and some polymorphic variants of this enzyme has been associated to the development of allergic asthma. Here, we showed that human GSTpi increased the cysteine-protease activity of Der p 1, while GSTmu (the isoenzyme produced by the mite) did not alter it. GSTpi induces the reduction of Cys residues in Der p 1, probably by rearranging its disulphide bridges. Furthermore, GSTpi was detected in the apical medium collected from human bronchial epithelial cell cultures, and more interesting, it increased cysteine-protease activity of Der p 1. Our findings support the role of human GSTpi from airways in modulating of Der p 1 cysteine-protease activity, which may have important clinical implications for immune response to this aeroallergen in genetically susceptible individuals.

The Activin A-Peroxisome Proliferator-Activated Receptor Gamma Axis Contributes to the Transcriptome of GM-CSF-Conditioned Human Macrophages.

GM-CSF promotes the functional maturation of lung alveolar macrophages (A-MØ), whose differentiation is dependent on the peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor. In fact, blockade of GM-CSF-initiated signaling or deletion of the PPARγ-encoding gene PPARG leads to functionally defective A-MØ and the onset of pulmonary alveolar proteinosis. In vitro, macrophages generated in the presence of GM-CSF display potent proinflammatory, immunogenic and tumor growth-limiting activities. Since GM-CSF upregulates PPARγ expression, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To verify this hypothesis, PPARγ expression and activity was assessed in human monocyte-derived macrophages generated in the presence of GM-CSF [proinflammatory GM-CSF-conditioned human monocyte-derived macrophages (GM-MØ)] or M-CSF (anti-inflammatory M-MØ), as well as in ex vivo isolated human A-MØ. GM-MØ showed higher PPARγ expression than M-MØ, and the expression of PPARγ in GM-MØ was found to largely depend on activin A. Ligand-induced activation of PPARγ also resulted in distinct transcriptional and functional outcomes in GM-MØ and M-MØ. Moreover, and in the absence of exogenous activating ligands, PPARγ knockdown significantly altered the GM-MØ transcriptome, causing a global upregulation of proinflammatory genes and significantly modulating the expression of genes involved in cell proliferation and migration. Similar effects were observed in ex vivo isolated human A-MØ, where PPARγ silencing led to enhanced expression of genes coding for growth factors and chemokines and downregulation of cell surface pathogen receptors. Therefore, PPARγ shapes the transcriptome of GM-CSF-dependent human macrophages (in vitro derived GM-MØ and ex vivo isolated A-MØ) in the absence of exogenous activating ligands, and its expression is primarily regulated by activin A. These results suggest that activin A, through enhancement of PPARγ expression, help macrophages to switch from a proinflammatory to an anti-inflammatory polarization state, thus contributing to limit tissue damage and restore homeostasis.

MAFB Determines Human Macrophage Anti-Inflammatory Polarization: Relevance for the Pathogenic Mechanisms Operating in Multicentric Carpotarsal Osteolysis.

Macrophage phenotypic and functional heterogeneity derives from tissue-specific transcriptional signatures shaped by the local microenvironment. Most studies addressing the molecular basis for macrophage heterogeneity have focused on murine cells, whereas the factors controlling the functional specialization of human macrophages are less known. M-CSF drives the generation of human monocyte-derived macrophages with a potent anti-inflammatory activity upon stimulation. We now report that knockdown of MAFB impairs the acquisition of the anti-inflammatory profile of human macrophages, identify the MAFB-dependent gene signature in human macrophages and illustrate the coexpression of MAFB and MAFB-target genes in CD163+ tissue-resident and tumor-associated macrophages. The contribution of MAFB to the homeostatic/anti-inflammatory macrophage profile is further supported by the skewed polarization of monocyte-derived macrophages from multicentric carpotarsal osteolysis (Online Mendelian Inheritance in Man #166300), a pathology caused by mutations in the MAFB gene. Our results demonstrate that MAFB critically determines the acquisition of the anti-inflammatory transcriptional and functional profiles of human macrophages.

Reshaping of Human Macrophage Polarization through Modulation of Glucose Catabolic Pathways.

Macrophages integrate information from the tissue microenvironment and adjust their effector functions according to the prevalent extracellular stimuli. Therefore, macrophages can acquire a variety of activation (polarization) states, and this functional plasticity allows the adequate initiation, regulation, and resolution of inflammatory responses. Modulation of the glucose metabolism contributes to the macrophage adaptation to the surrounding cytokine milieu, as exemplified by the distinct glucose catabolism of macrophages exposed to LPS/IFN-γ or IL-4. To dissect the acquisition of macrophage effector functions in the absence of activating cytokines, we assessed the bioenergetic profile of macrophages generated in the presence of GM-CSF (GM-MØ) or M-CSF (M-MØ), which do not release pro- or anti-inflammatory cytokines unless subjected to additional activating stimuli. Compared to M-MØ, GM-MØ displayed higher oxygen consumption rate and aerobic glycolysis (extracellular acidification rate [ECAR]), as well as higher expression of genes encoding glycolytic enzymes. However, M-MØ exhibited a significantly higher oxygen consumption rate/ECAR ratio. Surprisingly, whereas aerobic glycolysis positively regulated IL1B, TNF, and INHBA mRNA expression in both macrophage subtypes, mitochondrial respiration negatively affected IL6, IL1B, TNF, and CXCL10 mRNA expression in M-MØ. The physiological significance of these results became evident under low oxygen tensions, as hypoxia enhanced ECAR in M-MØ via HIF-1α and HIF-2α, increased expression of glycolytic enzymes and GM-MØ-specific genes, and diminished expression of M-MØ-associated genes. Therefore, our data indicate that GM-MØ and M-MØ display distinct bioenergetic profiles, and that hypoxia triggers a transcriptomic switch in macrophages by promoting a HIF-1α/HIF-2α-dependent increase in ECAR.

Influence of low oxygen tensions on macrophage polarization.

Microenvironmental conditions in infected, inflamed or damaged tissues are characterized by low levels of oxygen (hypoxia) and nutrients. Myeloid cells (mostly macrophages and neutrophils) account for 95% of the cells newly recruited into inflammatory sites, and exert their effector functions under these restrictive conditions. In the case of macrophages, adaptation to the surrounding tissue environment is underlined by their huge metabolic and functional plasticity, which allows them to critically participate in the maintenance of tissue homeostasis and the initiation and resolution of inflammatory processes under hypoxic conditions. Therefore, alterations in oxygen availability directly affect the macrophage functional state (polarization), a phenomenon that has been already illustrated in pathologies like cancer, atherosclerosis and obesity. This review summarizes recent advances on the molecular basis of macrophage sensing and response to changes in oxygen pressure, emphasizing the link among the hypoxia-induced signalling pathways, macrophage polarization and inflammatory pathologies.

The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro.

Alpha-defensins 1-3 release by dendritic cells is reduced by estrogen.

BACKGROUND: During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC) are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response. METHODS: We compare culture-generated monocyte derived DCs (MDDCs) with directly isolated myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) and measure their alpha-defensins 1-3 secretion by ELISA both, in basal situations and after hormone (E2 or PG) treatments. Moreover, using a cohort of pregnant women we isolated mDCs from blood and also measure the levels of these anti-microbial peptides along pregnancy. RESULTS: We show that mDCs and pDCs constitutively produce alpha-defensins 1-3 and at much higher levels than MDDCs. Alpha-defensins 1-3 production from mDCs and MDDCs but not pDCs is inhibited by E2. PG does not affect alpha-defensins 1-3 in any of the populations. Moreover, alpha-defensins 1-3 production by mDCs was reduced in the later stages of pregnancy in 40% of the patients. CONCLUSIONS: Here, we demonstrate that mDCs and pDCs secrete alpha-defensins 1-3 and present a novel effect of E2 on the secretion of alpha-defensins 1-3 by dendritic cells.

Differential resolution of inflammation and recovery after renal ischemia-reperfusion injury in Brown Norway compared with Sprague Dawley rats.

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury.

ERK1/2 mediates cytoskeleton and focal adhesion impairment in proximal epithelial cells after renal ischemia.

ERK1/2 has been reported to be activated in the postischemic kidney but its precise role in ischemia/reperfusion (I/R) injury remains unclear. Therefore, we have studied the expression of ERK1/2 and its contribution to cytoskeleton organization and cell adhesion structures in proximal tubular cells, all affected during I/R. We observe ERK1/2 activation at 24 hours of reperfusion in an in vivo model of I/R, when acute tubular necrosis (ATN) is most prominent. In addition, by means of an in vitro model of hypoxia/reoxygenation (H/R) in rat proximal NRK-52E cells we show that p-ERK1/2 is strongly induced early during reoxygenation. Moreover, we also demonstrate that ROS generation contributed to this induction. ERK1/2 activation is contemporary with cell-cell adhesion disruption during reoxygenation but the use of U0126 did not have effect on adherens junctions (AJ) and tight junctions (TJ) disassembly, neither on epithelial monolayer permeability. On the contrary, ERK1/2 affects cytoskeleton organization and focal complexes assembly during H/R, since U0126 improved actin and tubulin cytoskeleton structure, reduced cell contraction and prevented paxillin redistribution. In summary, ERK1/2 signalling plays an essential role in I/R induced injury, mediating proximal cell adhesive alterations which lead to tubular damage and ultimately might compromise renal function.

Estrogen inhibits dendritic cell maturation to RNA viruses.

Dendritic cells (DCs) play a central role in initiating and polarizing the immune response. Therefore, DC maturation represents a key control point in the shift from innate to adaptive immunity. It is suspected that during pregnancy, hormones are critical factors that modulate changes reported to occur in maternal immunity. Here we examined the effect of 17-beta-estradiol (E2) on the maturational response triggered by virus in human DCs and its influence on their ability to activate naive T cells. We developed an in vitro system to measure the response of DCs to virus infection with Newcastle disease virus (NDV) after a 24-hour E2 treatment. Using this system, we demonstrated that E2 pretreatment down-regulated the antiviral response to RNA viruses in DCs by profoundly suppressing type I interferon (IFN) synthesis and other important inflammatory products. In addition, the DCs capacity to stimulate naive CD4 T cells was also reduced. These results suggest an important role for E2 in suppressing the antiviral response and provide a mechanism for the reduced immunity to virus infection observed during pregnancy.

Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress.

Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, beta-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H(2)O(2), as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell-matrix but not cell-cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.

Renal ischemia/reperfusion injury: functional tissue preservation by anti-activated {beta}1 integrin therapy.

Renal ischemia/reperfusion injury (IRI) is an important cause of acute renal failure. Cellular and molecular responses of the kidney to IRI are complex and not fully understood. beta1 integrins localize to the basal surface of tubular epithelium interacting with extracellular matrix components of the basal membrane, including collagen IV. Whether preservation of tubular epithelium integrity could be a therapeutic approach for IRI was assessed. The effects of HUTS-21 mAb administration, which recognizes an activation-dependent epitope of beta1 integrins, in a rat model of IRI were investigated. Preischemic HUTS-21 administration resulted in the preservation of renal functional and histopathologic parameters. Analyses of activated beta1 integrins expression and focal adhesion kinase phosphorylation suggest that its deactivation after IRI was prevented by HUTS-21 treatment. Moreover, HUTS-21 impaired the inflammatory response in vivo, as indicated by inhibition of proinflammatory mediators and the absence of infiltrating cells. Ex vivo adhesion assays using reperfused kidneys revealed that HUTS-21 induced a significant increase of epithelial cell attachment to collagen IV. In conclusion, the data provide evidence that HUTS-21 has a protective effect in renal IRI, preventing tubular epithelial cell detachment by preserving activated beta1 integrins functions.