Kita, Hirohito

Abstract The impact of microbes on restraining the immune response to allergens has been extensively studied and is a key element of the hygiene hypothesis. Lung type 2 innate lymphoid cell responses to airway allergens can be inhibited by administration of a number of microbial products; however, it is unclear whether such an effect would be observed with natural infections and how sustained any observed inhibitory effects would be. To answer these questions, we used a murine model of physiological microbial exposures through cohousing SPF laboratory mice with pet store mice and examined the acute type 2 response to intranasally delivered fungal allergen extract Alternaria alternata. We found laboratory mice cohoused with pet store mice for two weeks display a suppressed ILC2 response to A. alternata which resulted in reduced eosinophilia. By comparison, mice cohoused for at least two months had ILC2 and eosinophil responses similar to SPF mice despite dramatic changes to the composition of the immune cell populations in the lungs. Lung ILC2 in two-month cohoused mice were still sensitive to subsequent inflammatory cues, as administration of poly(I:C) was able to suppress ILC2 activation and eosinophilia equally well in SPF and two-month cohoused animals. These findings suggest ILC2 dynamically respond to their environment and are not easily desensitized long-term.

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 μM), the VDAC-1 inhibitor, DIDS (100 μM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 μM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.

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 .

Alternaria aeroallergens stimulate allergic inflammation and exacerbation of respiratory distress in patients with asthma. Our previous studies have demonstrated that release of ATP and subsequent increases in intracellular [Ca 2+ ] was the result of Alternaria induced oxidative stress. Stimulation of Human Bronchial Epithelial Cells (HBECs) with Alternaria extract for 12 hours induced the expression of several phase II antioxidant enzymes, including cyclooxygenase 2 (COX2) which exhibited a 19 fold increase in mRNA expression. HBE cells loaded with CellRox™ Orange, a fluorogenic probe used for detecting oxidative stress, revealed that blocking COX2 inhibited resveratrol mediated inhibition of Alternaria induced oxidative stress. Furthermore induction of COX2 following Alternaria exposure was associated with an increase in expression of prostaglandin D 2 synthase and PGD 2 secretion into the extracellular media. Moreover, 15‐deoxy Δ 12,14 ‐protaglandin J 2 (15d‐PGJ 2 ) a metabolic product of PGD 2 was also shown to decrease Alternaria induced oxidative stress. These results suggest that upregulation of COX2 following sustained exposure to Alternaria induces PGD 2 synthesis and subsequent formation of 15d‐PGJ 2 , a known activator of Nrf2 signaling and ultimately, increased expression of phase II antioxidant enzymes.

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to Alternaria alternata extract evoked rapid and sustained ATP release with greater efficacy observed in cells from asthmatic subjects. Previously, Alternaria aeroallergens were shown to elicit sustained increases in intracellular [Ca 2+ ] i that depended on coordinated activation of P2Y 2 and P2X 7 receptors. In this study, pretreatment with BAPTA‐AM inhibited ATP release, indicating dependency on increases in [Ca 2+ ] i . Alternaria ‐evoked ATP release exhibited a greater peak response and higher EC 50 value in cells obtained from asthmatic subjects compared to normal controls. Furthermore, the maximum increase in [Ca 2+ ] i was greater in cells from asthmatic subjects. The vesicle transport inhibitor brefeldin A blocked Alternaria ‐stimulated incorporation of fluorescent lipid (FM1–43) labeled vesicles into the plasma membrane and ATP release. Additionally, inhibiting vesicular ATP uptake with bafilomycin also blocked ATP release similar to brefeldin A. These results indicated that a primary mechanism for Alternaria ‐induced ATP release involved Ca 2+ ‐dependent vesicular transport. Serine and cysteine protease inhibitors also reduced Alternaria ‐induced ATP release. This result was consistent with our previous data showing that Alternaria ‐evoked increases in [Ca 2+ ] i depend on purinoceptor signaling.

Alternaria aeroallergens have been previously shown to stimulate allergic inflammation and exacerbation of respiratory distress in patients with asthma. An early event following exposure to Alternaria extract is the sustained release of ATP and a persistent increase in intracellular [Ca 2+ ] ([Ca 2+ ] i ) resulting from uptake of Ca 2+ ions from the extracellular solution. In the present study we tested the hyopothesis that the release of ATP and the subsequent increase in [Ca 2+ ] i was due to oxidative stress induced by exposure to Alternaria . ATP release following apical stimulation with Alternaria was measured in human primary bronchial epithelial cells using a luciferase/luciferin ATP measurement kit in combination with a modified luminometer that allows for tracking ATP release in real time. Pretreatment with glutathione (GSH) produced a concentration‐dependent inhibition of ATP release with an EC 50 = 500 μM. Furthermore, GSH blocked the increase in [Ca 2+ ] i typically observed following apical exposure to Alternaria. Moreover an increase in the expression of specific antioxidant protein mRNAs was also observed after 12 hours of treatment with Alternaria extract. These proteins included peroxiredoxins 1‐6, keratin 1, cyclooxygenase 2 (COX2) and oxidative stress responsive 1 protein with COX2 having the highest level of expression (~20 fold). These results implicate oxidative stress as an important initiating event in Alternaria evoked airway inflammation. Grant Funding Source : Supported by NIH: HL110539

Aeroallergens produced by Alternaria alternata have been shown to induce life threatening exacerbations of airway inflammation in sensitized patients with severe asthma. Inflammation produced by Alternaria is associated with ATP release and purinergic receptor activation. In the present study the effects of Alternaria on signaling pathways and ion transport mechanisms associated with mucociliary clearance were investigated in an immortalized human bronchial epithelial cell line (16HBE14o ‐ cells). Cells were grown on Snapwell TM membrane filters until a stable transepithelial resistance (>500 Ω) was established. Cell monolayers were then voltage clamped at zero mV and apically stimulated with 50 μM Alternaria extract to induce a time‐dependent increase in short circuit current (Isc) consistent with anion secretion. Treatment with the CFTR channel blocker, CFTR inh ‐172 significantly reduced the Isc by ~25%. Furthermore, addition of the disulfonic stilbene compound DIDS (250 μM) abolished the Isc response. Measurements of the effect of Alternaria on intracellular Ca 2+ concentration ([Ca 2+ ] i ) showed that the extract caused a persistent increase in [Ca 2+ ] i that was blocked by chelating extracellular Ca 2+ with EGTA. Pretreatment with 250 μM DIDS also abrogated the effects of Alternaria on [Ca 2+ ] i but did not affect the increase in [Ca 2+ ] i produced by 4 μM ionomycin. Similarly, pretreatment of monolayers with glutathione also blocked the Alternaria induced increase in [Ca 2+ ] i . These results indicate that Alternaria aeroallergens are capable of stimulating CFTR and Ca 2+ ‐dependent anion secretion. Moreover, the increase in [Ca 2+ ] i appears to involve a DIDS‐sensitive uptake mechanism that is blocked when oxidant scavenger molecules are present in the extracellular solution. Grant Funding Source : Supported by NIH: HL110539

Abstract Background: Eosinophils play an important role in allergic inflammation. Glucocorticosteroids have been used as an anti-inflammatory medication for inflammatory diseases involving eosinophil infiltration. Some effect of nebulized lidocaine has been reported when treating certain patients with asthma, which is also an inflammatory disease. The goal of this study was to examine the effects of dexamethasone and lidocaine on eosinophil proliferation and differentiation using a model of human umbilical cord blood mononuclear cells (UCMC) cultured with IL-5. Methods: UCMC were cultured with IL-5 (5 ng/mL) for 4 weeks. The effects of dexamethasone and lidocaine on the number and morphology of eosinophilic cells were visualized with Wright-Giemsa and cyanide-resistant peroxidase stains. Moreover, the effect on eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPX) contents in cultured cells were evaluated using radioimmunoassay.Results: The number of eosinophilic cells and EDN and EPX content in cultured cells increased in a time-dependent manner in the presence of IL-5. Dexamethasone treatment slightly decreased the number of eosinophilic cells in one week, but this effect was lost in 2–4 weeks. Macrophages in cultured UCMC treated with dexamethasone contained more eosinophil granule proteins. Both EDN and EPX content in cultured cells were reduced by dexamethasone. Lidocaine decreased the number of eosinophilic cells and reduced both EDN and EPX contents in cultured cells.Conclusions: Dexamethasone suppressed the production of eosinophil granule proteins and may also induce apoptosis of eosinophils, while lidocaine suppresses eosinophilopoiesis.