Squillace, Diane L.

Previous studies have demonstrated that simvastatin possesses immunomodulatory effects in airways, but the precise mechanisms and therapeutic potential are poorly understood. The objective of the present study was to investigate the inhibitory effects of simvastatin and b‐methyl‐cyclodextrin (β‐MCD) on the innate immune response of the airway epithelium induced by Alternaria alternata (100 μg/ml) and house dust mite (200 μg/ml, HDM) allergens. Experiments were performed with cultured human bronchial epithelial cells transfected with the human IL‐33 gene to produce a robust and reproducible cell model for the study of IL‐33 secretion. Cells were pretreated with varying concentrations of simvastatin ranging from 0.1 to 10 μM for 24 hours prior to allergen exposure. IL‐33 release was quantified by ELISA (Quantikine ® ) following the procedure provided by the manufacturer. Simvastatin produced a concentration‐dependent inhibition of IL‐33 release that was nearly abolished at 10 μM (IC 50 ; 1.0 μM). In contrast, simvastatin was ineffective at blocking the effects of HDM on IL‐33 release (IC 50 ; >10 μM). Similarly, pretreatment with β‐MCD (5 mM) for 30 minutes also inhibited the Alternaria response, suggesting that depletion of cholesterol from the plasma membrane was responsible for the effects of simvastatin and β‐MCD. Subsequent experiments explored the effects of simvastatin and β‐MCD on Alternaria ‐evoked Ca 2+ uptake, which is necessary for activating IL‐33 release. Simvastatin (10 μM, 24 hrs) and β‐MCD (5 mM; 30 minutes) both abolished the increase in [Ca 2+ ] i induced by Alternaria . We next examined the possibility that abolition of Ca 2+ uptake resulted from inhibition of ATP release, since ATP activates P2X 7 receptors that mediate Ca 2+ influx in response to Alternaria exposure. ATP release was measured in real time using a luciferin/luciferase‐based photon counting assay. Both simvastatin and β‐MCD blocked ATP release. Moreover, addition of 100 μM cholesterol to the extracellular media rescued the effects of simvastatin. These results demonstrated that inhibition of IL‐33 secretion by simvastatin and β‐MCD following Alternaria exposure involved suppression of ATP release that was dependent on membrane cholesterol content. Thus, membrane cholesterol likely plays a pivotal role in innate immune function of airway epithelial cells. Support or Funding Information This study was supported by NIH R01 AI128729 to HK and SMO. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Key points Alternaria aeroallergens induce the release of ATP from human bronchial epithelial (HBE) cells by activating a conductive pathway involving voltage‐dependent anion channel‐1 (VDAC‐1) and by exocytosis of ATP localized within membrane vesicles. Inhibition of VDAC‐1 blocked Alternaria‐evoked Ca2+ uptake across the plasma membrane of HBE cells and interleukin (IL)‐33 release into the extracellular media. Reducing cholesterol content with a cholesterol scavenger (β‐methylcyclodextrin) or statin compound (simvastatin) blocked ATP and IL‐33 release by lowering the expression of VDAC‐1 in the plasma membrane. Pretreatment with simvastatin for 24 h also inhibited the increase in tight junction macromolecule permeability that occurs following Alternaria exposure. These results establish a novel role for VDAC‐1 as a mechanism underlying ATP release induced by fungal allergens and suggests a possible therapeutic use for cholesterol lowering compounds in reducing Alternaria‐stimulated allergic inflammation. AbstractHuman bronchial epithelial (HBE) cells exposed to allergens derived from the common saprophytic fungus, Alternaria alternata release ATP, which in turn stimulates P2X7 receptor‐mediated Ca2+ uptake across the plasma membrane. The subsequent increase in intracellular calcium concentration induces proteolytic processing and secretion of interleukin (IL)‐33, a critical cytokine involved in the initiation of allergic airway inflammation. A major objective of the present study was to identify the mechanism responsible for conductive ATP release. The results show that pretreatment of HBE cells with inhibitors of the voltage‐dependent anion channel‐1 (VDAC‐1) or treatment with a VDAC‐1 selective blocking antibody or silencing mRNA expression of the channel by RNA interference, inhibit Alternaria‐evoked ATP release. Moreover, inhibition of VDAC‐1 channel activity or reducing protein expression blocked the secretion of IL‐33. Similarly, reducing the cholesterol content of HBE cells with simvastatin or the cholesterol scavenger β‐methylcyclodextrin also blocked ATP release and IL‐33 secretion by decreasing the level of VDAC‐1 expression in the plasma membrane. In addition, simvastatin inhibited the increase in tight junction macromolecule permeability that was previously observed after Alternaria exposure. These results demonstrate a novel function for VDAC‐1 as the conductive mechanism responsible for Alternaria‐induced ATP release, an essential early step in the processing, mobilization and secretion of IL‐33 by the airway epithelium. Furthermore, the simvastatin‐evoked reduction of VDAC‐1 expression in the plasma membrane, suggests the possibility that cholesterol lowering compounds may be beneficial in alleviating allergic airway inflammation induced by fungal allergens.

Abstract IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33–mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an ∼3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.

AbstractBackgroundInterleukin (IL)‐33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL‐33 release is controlled. The transcription factor nuclear factor‐erythroid‐2‐related factor 2 (Nrf2) plays a key role in antioxidant response regulation.ObjectiveThe goal of this project was to investigate the role of cellular oxidative stress in controlling IL‐33 release in airway epithelium.MethodsComplementary approaches were used that included human bronchial epithelial cells and mouse models of airway type‐2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid methyl ester (CDDO‐Me) was used to evaluate the role of Nrf2‐induced antioxidant molecules.ResultsHuman bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N‐acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL‐33 release. Administration of CDDO‐Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO‐Me treatment reduced allergen‐induced ATP secretion and IL‐33 release by airway epithelial cells in vitro and protected mice from IL‐33 release and asthma‐like pathological changes in the lungs.ConclusionsThe balance between oxidative stress and antioxidant responses plays a key role in controlling IL‐33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.

Background Endotyping chronic rhinosinusitis (CRS) through simplified cytokine assays may help direct individualized therapy such as corticosteroids, antibiotics, or biologics. We performed an unsupervised network analysis to endotype CRS and control subjects using a commercially available cytokine‐chemokine immunoassay. Methods A 41‐plex cytokine‐chemokine array along with major basic protein (MBP) assay was performed on sinonasal surgical tissue of 32 adults. Subjects were defined as non‐CRS controls (n = 6), CRS with nasal polyps (CRSwNP; n = 13), and CRS without nasal polyps (CRSsNP; n = 13). Unsupervised network modeling was performed to reveal association cytokine‐chemokine (“analyte”) clusters and “subject” groups. Results Network mapping and unsupervised clustering revealed 3 analyte clusters and 3 subject groups. Analyte cluster‐1 was composed of T helper 1 (Th1)/Th17 type markers, analyte cluster‐2 Th2 markers, and analyte cluster‐3 chemokines (CC) and growth factors (GF). Subject group‐1 was devoid of CRSwNP, had fewer asthmatics, and was associated most strongly with analyte cluster‐3 (CC/GF) ( p < 0.001). Subject group‐2 was characterized with the most asthmatics (86%) and CRSwNP (100%) patients, and was associated with analyte cluster‐2 (Th2; p < 0.001). Subject group‐3 was associated with both analyte cluster‐1 (Th1/Th17) and analyte cluster‐3 (CC/GF) ( p < 0.001), and had the highest proportion of CRSsNP patients (62.5%). Tissue levels of MBP, eosinophilia, and computed tomography (CT) scores were significantly higher in subject group‐2 vs other groups ( p ≤ 0.05). Conclusion An unbiased network‐mapping approach using a commercially available immunoassay kit reveals 3 distinct tissue cytokine‐chemokine signatures that endotype CRS patients and controls. These signatures are prominent even in a limited number of patients, and may help formulate individualized therapy and optimize outcomes.

Abstract To establish a useful laboratory protocol to investigate possible cases of fatal anaphylaxis, we measured mast-cell-derived tryptase levels and allergen-specific immunoglobulin E (IgE) antibody levels in sera obtained prior to or within 24 h after death from 19 anaphylaxis victims. Elevated serum tryptase levels (range = 12 ng/mL to 150 μg/mL) were found in nine of nine Hymenoptera sting fatalities, six of eight food-induced fatalities, and two of two reactions to diagnostic/therapeutic agents. Tryptase levels were normal (&lt;10 ng/mL) in 57 sequential sera obtained postmortem from six control patients. Tryptase could not be measured by pleural or pericardial fluids for technical reasons. Serum IgE antibodies were elevated in five of the nine Hymenoptera sting fatalities and in eight of the eight fatal food reactions; assays were unavailable for the two diagnostic/therapeutic agents. If elevated, the victim's serum IgE antibodies to food could be used to identify allergens in uneaten portions of foods consumed shortly before the anaphylactic event. IgE antibodies were moderately stable during storage in a variety of anticoagulants at room temperature for up to 11 weeks. Elevated mast-cell-derived tryptase levels in postmortem sera reflect antemortem mast cell activation and may be used as a marker for fatal anaphylaxis. If assays are available for IgE antibodies to relevant allergens, such assays provide evidence for antemortem sensitization; these assays may be modified to identify allergens in foods consumed by victims of food-induced anaphylaxis.

BackgroundA histologic hallmark of chronic rhinosinusitis (CRS) is an eosinophilic inflammation, present with and without nasal polyposis and independent of atopy. Eosinophils migrate through nasal tissue including the epithelium into the nasal airway mucus, where they form clusters and degranulate, releasing granule proteins including the toxic major basic protein (MBP). Specific biomarkers for CRS, which could be used as a diagnostic test for CRS with a high sensitivity and specificity, are presently lacking. Recently, an enzyme‐linked immunosorbent assay (ELISA)‐based test for MBP in nasal airway mucus received regulatory approval.MethodsA new assay was specifically developed to detect released MBP in airway mucus. MBP levels in nasal mucus of 85 randomly selected CRS patients diagnosed by endoscopy, computed tomography (CT) scans and symptoms were compared to 13 healthy controls and 5 disease controls (allergic rhinitis).ResultsOverall, 92% (78/85) of CRS patients’ mucus were positive for MBP (mean 7722 ng/mL) vs none of 13 healthy controls and none of 5 allergic rhinitis patients (<7.8 ng/mL; p < 0.000000000002). In this study, the MBP ELISA had a 92% sensitivity and 100% specificity for CRS.ConclusionFree MBP in nasal mucus can be used as a biomarker to diagnose CRS. The MBP ELISA represents the first immunologically‐based test to potentially distinguish CRS from the eosinophilic inflammation in allergic rhinitis.