Histoplasma capsulatum-induced extracellular DNA trap release in human neutrophils.

Neutrophils are leukocytes that are capable of eliminating both intra- and extracellular pathogens by mechanisms such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). Histoplasma capsulatum var. capsulatum (H. capsulatum) is a dimorphic fungus with a global distribution that causes histoplasmosis, a disease that is endemic in different geographic areas and is spreading worldwide. The release of NETs has been described as an important host defense mechanism against different fungi; however, there are no reports demonstrating that this process is implicated in neutrophil response to H. capsulatum infection. Therefore, the aim of this work is to investigate whether isolated human neutrophils release NETs in response to H. capsulatum and the potential mechanisms involved, as well as delineate the NETs antifungal activity. Using both confocal fluorescence and scanning electron microscopy techniques, we determined that NETs are released in vitro in response to H. capsulatum via an oxidative mechanism that is downstream of activation of the Syk and Src kinase pathways and is also dependent on CD18. NETs released in response to H. capsulatum yeasts involve the loss of neutrophil viability and are associated with elastase and citrullinated histones, however also can occur in a PAD4 histone citrullination independent pathway. This NETs also presented fungicidal activity against H. capsulatum yeasts. Our findings may contribute to the understanding of how neutrophils recognize and respond as immune effector cells to H. capsulatum, which may lead to better knowledge of histoplasmosis pathophysiology and treatment.

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice.

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor ß (TGFß), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1ß (IL-1ß) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

Eosinophils release extracellular DNA traps in response to Aspergillus fumigatus.

BACKGROUND: Eosinophils mediate the immune response in different infectious conditions. The release of extracellular DNA traps (ETs) by leukocytes has been described as an innate immune response mechanism that is relevant in many disorders including fungal diseases. Different stimuli induce the release of human eosinophil ETs (EETs). Aspergillus fumigatus is an opportunistic fungus that may cause eosinophilic allergic bronchopulmonary aspergillosis (ABPA). It has been reported that eosinophils are important to the clearance of A fumigatus in infected mice lungs. However, the immunological mechanisms that underlie the molecular interactions between A fumigatus and eosinophils are poorly understood. OBJECTIVE: Here, we investigated the presence of EETs in the bronchial mucus plugs of patients with ABPA. We also determined whether A fumigatus induced the release of human eosinophil EETs in vitro. METHODS: Mucus samples of patients with ABPA were analyzed by light and confocal fluorescence microscopy. The release of EETs by human blood eosinophils was evaluated using different pharmacological tools and neutralizing antibodies by fluorescence microscopy and a fluorimetric method. RESULTS: We identified abundant nuclear histone-bearing EETs in the bronchial secretions obtained from patients with ABPA. In vitro, we demonstrated that A fumigatus induces the release of EETs through a mechanism independent of reactive oxygen species but associated with eosinophil death, histone citrullination, CD11b, and the Syk tyrosine kinase pathway. EETs lack the killing or fungistatic activities against A fumigatus. CONCLUSIONS: Our findings may contribute to the understanding of how eosinophils recognize and act as immune cells in response to A fumigatus, which may lead to novel insights regarding the treatment of patients with ABPA.

JM25-1, a Lidocaine Analog Combining Airway Relaxant and Antiinflammatory Properties: Implications for New Bronchospasm Therapy.

BACKGROUND: Inhaled lidocaine antagonized bronchospasm in animal models and patients, but adverse effects limited its efficacy. This study evaluated the antibronchospasm potential of the analog JM25-1, exploring in vitro mechanisms and translation to an animal model. METHODS: The effectiveness of JM25-1 was assessed in GH3 cells, rat tracheal rings, mouse lymphocytes, and human eosinophil systems in vitro, assessing changes in Na current, contraction, proliferation, and survival, respectively. Lung function and inflammatory changes were studied in ovalbumin-sensitized mice. RESULTS: The efficacy of JM25-1 was higher than lidocaine in inhibiting carbachol-induced and calcium-induced tracheal contractions (maximum effect inhibition at 1 mM [%]: 67 ± 10 [JM25-1] vs. 41 ± 11 [lidocaine] [P < 0.001] for carbachol; 100 ± 3 [JM25-1] vs. 36 ± 26 [lidocaine] [P < 0.001] for Ca; mean ± SD; n = 9 each) but lower in Na current (50% inhibitory concentration = 151.5, n = 8 vs. 0.2 mM; n = 5; P < 0.001). JM25-1 also inhibited eosinophil survival (dead cells [%]: 65 ± 6; n = 4; P < 0.001 at 1 mM) and lymphocyte proliferation (cells in phase S + G2 [%]: 94 ± 10; n = 6; P < 0.001) at 0.6 mM. Aerosolized JM25-1 (1%) decreased lung eosinophil numbers from 13.2 ± 2.4 to 1.7 ± 0.7 × 10/µm (n = 6; P < 0.001) and neutrophils from 1.9 ± 0.4 to 0.2 ± 0.1 × 10/µm (n = 7; P < 0.001). Other parameters, including airway hyperreactivity, cytokines, mucus, and extracellular matrix deposition, were also sensitive to aerosolized JM25-1. CONCLUSION: These findings highlight the potential of JM25-1, emphasizing its putative value in drug development for clinical conditions where there is bronchospasm.

Extracellular sombrero vesicles are hallmarks of eosinophilic cytolytic degranulation in tissue sites of human diseases.

Eosinophil sombrero vesicles are large tubular carriers resident in the cytoplasm of human eosinophils, identifiable by transmission electron microscopy, and important for immune mediator transport. Increased formation of sombrero vesicles occurs in activated eosinophils in vitro and in vivo. In tissue sites of eosinophilic cytolytic inflammation, extracellular eosinophil sombrero vesicles are noted, but their frequency and significance in eosinophil-associated diseases remain unclear. Here, we performed comprehensive quantitative transmission electron microscopy analyses and electron tomography to investigate the numbers, density, integrity, and 3-dimensional structure of eosinophil sombrero vesicles in different biopsy tissues from 5 prototypic eosinophil-associated diseases (eosinophilic chronic rhinosinusitis/nasal sinuses, ulcerative colitis/intestines, hypereosinophilic syndrome/skin, dermatitis/skin, and schistosomiasis/rectum). The morphology of extracellular eosinophil sombrero vesicles was also compared with that of cytoplasmic eosinophil sombrero vesicles, isolated by subcellular fractionation from peripheral blood eosinophils. We demonstrated that (i) eosinophil cytolysis, releasing intact sombrero vesicles and membrane-bound granules, is a consistent event in all eosinophil-associated diseases; (ii) eosinophil sombrero vesicles persist intact even after complete disintegration of all cell organelles, except granules (late cytolysis); (iii) the eosinophil sombrero vesicle population, composed of elongated, curved, and typical sombreros, and the eosinophil sombrero vesicle 3-dimensional architecture, diameter, and density remain unchanged in the extracellular matrix; (iv) free eosinophil sombrero vesicles closely associate with extracellular granules; and (v) free eosinophil sombrero vesicles also associate with externalized chromatin during eosinophil ETosis. Remarkably, eosinophil sombrero vesicles appeared on the surface of other cells, such as plasma cells. Thus, eosinophil cytolysis/ETosis can secrete intact sombrero vesicles, alongside granules, in inflamed tissues of eosinophil-associated diseases, potentially serving as propagators of eosinophil immune responses after cell death.

Pulmonary antifibrotic mechanisms aspirin-triggered lipoxin A(4) synthetic analog.

No successful therapies are available for pulmonary fibrosis, indicating the need for new treatments. Lipoxins and their 15-epimers, aspirin-triggered lipoxins (ATL), present potent antiinflammatory and proresolution effects (Martins et al., J Immunol 2009;182:5374-5381). We show that ATLa, an ATL synthetic analog, therapeutically reversed a well-established pulmonary fibrotic process induced by bleomycin (BLM) in mice. We investigated the mechanisms involved in its effect and found that systemic treatment with ATLa 1 week after BLM instillation considerably reversed the inflammatory response, total collagen and collagen type 1 deposition, vascular endothelial growth factor, and transforming growth factor (TGF)-ß expression in the lung and restored surfactant protein C expression levels. ATLa also inhibited BLM-induced apoptosis and cellular accumulation in bronchoalveolar lavage fluid and in the lung parenchyma as evaluated by light microscopy and flow cytometry (Ly6G(+), F4/80(+), CD11c(+), CD4(+), and B220(+) cells) assays. Moreover, ATLa inhibited the lung production of IL-1ß, IL-17, TNF-α, and TGF-ß induced by BLM-challenged mice. ATLa restored the balance of inducible nitric oxide synthase-positive and arginase-positive cells in the lungs, suggesting a prevalence of M2 versus M1 macrophages. Together, these effects improved pulmonary mechanics because ATLa treatment brought to normal levels lung resistance and elastance, which were clearly altered at 7 days after BLM challenge. Our findings support ATLa as a promising therapeutic agent to treat lung fibrosis.

Functional extracellular eosinophil granules: a bomb caught in a trap.

Eosinophils store a wide range of preformed proteins, including cationic proteins and cytokines, within their morphologically unique granules. Recently, we have demonstrated that cell-free eosinophil granules are functional, independent, secretory organelles and that clusters of cell-free granules are commonly found at tissue sites associated with various pathologic conditions. Cytolytic release of intact eosinophil granules produces extracellular organelles that are fully capable of ligand-elicited, active, secretory responses and are hence able to act as 'cluster bombs' that amplify the differential secretory properties of eosinophils. Herein, we review recent progress in elucidating the molecular mechanisms involved in the cytolytical release of intact cell-free functional eosinophil granules in a process associated with the liberation of eosinophil DNA traps (nets), a known aspect of the innate response recognized in various immune cells and pathological conditions. We also discuss the importance of clusters of cell-free eosinophil granules trapped in eosinophil DNA nets in disease and speculate on their potential role(s) in immunity as well as compare available data on DNA-releasing neutrophils.

Eosinophils: Focus on DNA extracellular traps.

Eosinophils are multitasking granulocytes with implications for allergies, host response to helminths and, more recently, described roles in immunomodulation, homeostasis and tissue remodeling. Eosinophils secrete their preformed granule proteins by different pathways, especially piecemeal degranulation and cytolysis with granule release. Currently, there are different insights related to eosinophils' functional roles and biology that deserve to be highlighted. Cytolysis with granule release has also been associated with DNA extracellular trap formation, one of the most intriguing, recently described mechanisms of leukocyte activation. Focusing on DNA extracellular trap release, there are lessons to be learned from neutrophils considering the multitasking roles of these structures in inflammation, and the mechanisms involved in their release. This review explores a comparative analysis of the current knowledge considering DNA traps extrusion in neutrophils and eosinophils and update the major findings regarding the presence of these entities in eosinophilic-associated immune responses, inflammation and diseases.

Eosinophil granules function extracellularly as receptor-mediated secretory organelles.

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-gamma, and G protein-coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules.

BACKGROUND: Cysteinyl leukotrienes (cysLTs) are recognized to act via receptors (cysLTRs) expressed on cell surface plasma membranes. Agents that block cysLT(1) receptor (cysLT(1)R) are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis, and urticaria, but have unknown functional capabilities. OBJECTIVE: We evaluated the expression of cysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules. METHODS: We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysLTs, and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and Western blot. RESULTS: We report that receptors for cysLTs, cysLT(1)R, cysLT(2) receptor, and the purinergic P2Y12 receptor, are expressed on eosinophil granule membranes. Leukotriene (LT) C(4) and extracellularly generated LTD(4) and LTE(4) stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12 receptor antagonist, inhibited cysLT-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of cysLT(1)R, inhibited eosinophil cationic protein release elicited by LTC(4) and LTD(4) as well as by LTE(4). CONCLUSION: These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysLT-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly.

Structural and Signaling Events Driving Aspergillus fumigatus-Induced Human Eosinophil Extracellular Trap Release.

Eosinophils are granulocytes classically involved in allergic diseases and in the host immune responses to helminths, fungi, bacteria and viruses. The release of extracellular DNA traps by leukocytes is an important mechanism of the innate immune response to pathogens in various infectious conditions, including fungal infections. Aspergillus fumigatus is an opportunistic fungus responsible for allergic bronchopulmonary aspergillosis (ABPA), a pulmonary disease marked by prominent eosinophilic inflammation. Previously, we demonstrated that isolated human eosinophils release extracellular DNA traps (eosinophil extracellular traps; EETs) when stimulated by A. fumigatus in vitro. This release occurs through a lytic non-oxidative mechanism that involves CD11b and Syk tyrosine kinase. In this work, we unraveled different intracellular mechanisms that drive the release of extracellular DNA traps by A. fumigatus-stimulated eosinophils. Ultrastructurally, we originally observed that A. fumigatus-stimulated eosinophils present typical signs of extracellular DNA trap cell death (ETosis) with the nuclei losing both their shape (delobulation) and the euchromatin/heterochromatin distinction, followed by rupture of the nuclear envelope and EETs release. We also found that by targeting class I PI3K, and more specifically PI3Kδ, the release of extracellular DNA traps induced by A. fumigatus is inhibited. We also demonstrated that A. fumigatus-induced EETs release depends on the Src family, Akt, calcium and p38 MAPK signaling pathways in a process in which fungal viability is dispensable. Interestingly, we showed that A. fumigatus-induced EETs release occurs in a mechanism independent of PAD4 histone citrullination. These findings may contribute to a better understanding of the mechanisms that underlie EETs release in response to A. fumigatus, which may lead to better knowledge of ABPA pathophysiology and treatment.

Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially.

Eosinophils are innate immune leukocytes implicated in the initiation and maintenance of type 2 immune responses, including asthma and allergy. The ability to store and rapidly secrete preformed cytokines distinguishes eosinophils from most lymphocytes, which must synthesize cytokine proteins prior to secretion and may be a factor in the apparent Th2 bias of eosinophils. Multiple studies confirm that human eosinophils from atopic or hypereosinophilic donors can secrete over 30 cytokines with a varying and often opposing immune-polarizing potential. However, it remains unclear whether all of these cytokines are constitutively preformed and available for rapid secretion from eosinophils in the circulation of healthy individuals or are restricted to eosinophils from atopic donors. Likewise, the relative concentrations of cytokines stored within eosinophils have not been studied. Here, we demonstrate that human blood eosinophils are not singularly outfitted with Th2-associated cytokines but rather, constitutively store a cache of cytokines with nominal Th1, Th2, and regulatory capacities, including IL-4, IL-13, IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha. We demonstrate further rapid and differential release of each cytokine in response to specific stimuli. As agonists, strong Th1 and inflammatory cytokines elicited release of Th2-promoting IL-4 but not Th1-inducing IL-12. Moreover, a large quantity of IFN-gamma was secreted in response to Th1, Th2, and inflammatory stimuli. Delineations of the multifarious nature of preformed eosinophil cytokines and the varied stimulus-dependent profiles of rapid cytokine secretion provide insights into the functions of human eosinophils in mediating inflammation and initiation of specific immunity.

Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination.

Aspergillus fumigatus (A. fumigatus) is an environmental fungus and a human pathogen. Neutrophils are critical effector cells during the fungal infections, and neutropenia is a risk factor for the development of pulmonary aspergillosis. Neutrophil extracellular traps (NETs) are released by neutrophils in response to A. fumigatus and inhibit the conidial germination. In this work, we observed that the receptors TLR2, TLR4, and Dectin-1 were dispensable for the A. fumigatus induced NET release. In contrast CD11b/CD18 was critical for the NET release in response to A. fumigatus conidia, and this required the CD11b I-domain-mediated recognition, whereas the blockade of the CD11b lectin domain did not affect the A. fumigatus induced NET release. A. fumigatus induced NET release relied on the activity of spleen tyrosine kinase (Syk), Src family kinase(s), and class IA PI3 kinase δ. Although A. fumigatus promoted histone citrullination, this process was dispensable for the NET release in response to A. fumigatus conidia. The A. fumigatus induced NET release required the reactive oxygen species generation by the NOX2 complex, in a downstream pathway requiring CD11b/CD18, Src kinase family activity, Syk and PI3K class IA δ. Our findings thus reveal the signaling pathways involved in the formation of NETs in response to A. fumigatus.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein.

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Synthesis and antispasmodic activity of lidocaine derivatives endowed with reduced local anesthetic action.

The present structure-activity relationship (SAR) study focused on chemical modifications of the structure of the local anesthetic lidocaine, and indicated analogues having reduced anesthetic potency, but with superior potency relative to the prototype in preventing anaphylactic or histamine-evoked ileum contraction. From the SAR analysis, 2-(diethylamino)-N-(trifluoromethyl-phenyl) and 2-(diethylamino)-N-(dimethyl-phenyl) acetamides were selected as the most promising compounds. New insights into the applicability of non-anesthetic lidocaine derivatives as templates in drug discovery for allergic syndromes are provided.

Eosinophils in fungal diseases: An overview.

Eosinophils are the prominent cells in asthma, allergic bronchopulmonary mycosis (ABPMs), and fungal-sensitization-associated asthma, but their roles in the immunopathology of these disorders are not well understood. Moreover, the immunological mechanisms underlying the molecular direct effector interactions between fungi and eosinophils are rare and not fully known. Here, we provide an overview of eosinophil contributions to allergic asthma and ABPMs. We also revise the major general mechanisms of fungal recognition by eosinophils and consider past and recent advances in our understanding of the molecular mechanisms associated with eosinophil innate effector responses to different fungal species relevant to ABPMs (Alternaria alternata, Candida albicans, and Aspergillus fumigatus). We further examine and speculate about the therapeutic relevance of these findings in fungus-associated allergic pulmonary diseases.

Allergic Bronchopulmonary Aspergillosis-A Luminal Hypereosinophilic Disease With Extracellular Trap Cell Death.

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by an early allergic response and late-phase lung injury in response to repeated exposure to Aspergillus antigens, as a consequence of persistent fungal colonization of the airways. Here, we summarize the clinical and pathological features of ABPA, focusing on thick mucus plugging, a key observation in ABPA. Recent findings have indicated that luminal eosinophils undergo cytolytic extracellular trap cell death (ETosis) and release filamentous chromatin fibers (extracellular traps, ETs) by direct interaction with Aspergillus fumigatus. Production of ETs is considered to be an innate immune response against non-phagocytable pathogens using a "trap and kill" mechanism, although eosinophil ETs do not promote A. fumigatus damage or killing. Compared with neutrophils, eosinophil ETs are composed of stable and condensed chromatin fibers and thus might contribute to the higher viscosity of eosinophilic mucus. The major fate of massively accumulated eosinophils in the airways is ETosis, which potentially induces the release of toxic granule proteins and damage-associated molecular patterns, epithelial damage, and further decreases mucus clearance. This new perspective on ABPA as a luminal hypereosinophilic disease with ETosis/ETs could provide a better understanding of airway mucus plugging and contribute to future therapeutic strategies for this challenging disease.

Cysteinyl Leukotrienes in Eosinophil Biology: Functional Roles and Therapeutic Perspectives in Eosinophilic Disorders.

Cysteinyl leukotrienes (cysLTs), LTC4, and its extracellular metabolites, LTD4 and LTE4, have varied and multiple roles in mediating eosinophilic disorders including host defense against parasitic helminthes and allergic inflammation, especially in the lung and in asthma. CysLTs are known to act through at least 2 receptors termed cysLT1 receptor (CysLT1R) and cysLT2 receptor (CysLT2R). Eosinophils contain a dominant population of cytoplasmic crystalloid granules that store various preformed proteins. Human eosinophils are sources of cysLTs and are known to express the two known cysLTs receptors (CysLTRs). CysLTs can have varied functions on eosinophils, ranging from intracrine regulators of secretion of granule-derived proteins to paracrine/autocrine roles in eosinophil chemotaxis, differentiation, and survival. Lately, it has been recognized the expression of CysLTRs in the membranes of eosinophil granules. Moreover, cysLTs have been shown to evoke secretion from isolated cell-free eosinophil granules operating through their receptors expressed on granule membranes. In this work, we review the functional roles of cysLTs in eosinophil biology. We review cysLTs biosynthesis, their receptors, and argue the intracrine and paracrine/autocrine responses induced by cysLTs in eosinophils and in isolated free extracellular eosinophil granules. We also examine and speculate on the therapeutic relevance of targeting CysLTRs in the treatment of eosinophilic disorders.

CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils.

Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.

Evaluating the prophylactic potential of the phtalimide derivative LASSBio 552 on allergen-evoked inflammation in rats.

A previous study showed that the novel tetrazolephtalimide derivative LASSBio 552 (2-4-[3-(1H-1,2,3,4-tetraazol-5-yl)propoxy]phenethyl-1,3-isoindolinedione) prevents LTD(4)-evoked tracheal contraction. This led us to examine the putative anti-inflammatory effect of LASSBio 552 in comparison with the leukotriene CysLT(1) receptor antagonist zafirlukast using a model of allergic pleurisy in rats. Treatment with either LASSBio 552 (24-96 micromol/kg, i.p.) or zafirlukast (9-72 micromol/kg, i.p.), 1 h before challenge, inhibited eosinophil and mononuclear cell influx into the pleural cavity 24 h post-challenge, but failed to alter the increased levels of eotaxin, plasma leakage, mast cell degranulation and neutrophil infiltration noted 6 h post-challenge. CD4(+) T cell recruitment 24 h post-challenge was also sensitive to LASSBio 552. This treatment failed to alter cysteinyl leukotriene production at 6 h, but clearly inhibited the phenomenon 24 h and 48 h post-challenge. In in vitro settings LASSBio 552 inhibited allergen-evoked cysteinyl leukotriene generation from isolated mast cells, while histamine release remained unchanged. It also slightly inhibited cysteinyl leukotriene production by eosinophils and mononuclear cells triggered by Ca(+2) ionophore A23187. A leukotriene CysLT(1) receptor transfected cell-based assay revealed that LASSBio 552 did not prevent LTD(4)-evoked Ca(+2) influx, indicating that it was not a leukotriene CysLT(1) receptor antagonist. These findings indicate that LASSBio 552 is able to inhibit eosinophil influx triggered by allergen chalenge in a mechanism at least partially associated with suppression of CD4(+) T cell influx and cysteinyl leukotriene production.