Citations

Abstract Peanut allergy is a growing public concern; however, little is known about the immunological mechanism(s) that initiate the disease process. Our knowledge is also limited regarding the role of group 2 innate lymphoid cells (ILC2s) in regulating humoral immunity. To fill these major gaps in our knowledge, we investigated the immunological mechanisms involved in peanut allergen sensitization by using mouse models. To mimic environmental exposure in humans, naive BALB/c mice were exposed to peanut flour by inhalation without any exogenous adjuvants. When exposed to peanut flour, naive mice developed T follicular helper (Tfh) cells in their lung draining lymph nodes and produced IgE Abs to peanuts. Mice deficient in IL-13 showed decreased numbers of Tfh cells and germinal center B cells and produced significantly fewer IgE Abs. IL-13 was necessary and sufficient for induction of CD11c+ MHC class IIhi dendritic cells that are implicated in Tfh cell development. Importantly, lung ILC2s served as a predominant early source of IL-13 when naive mice were exposed to peanut flour. Furthermore, mice that are deficient in lung ILC2s by bone marrow transfer from Rorasg/sg mice or by genetic manipulation produced significantly fewer IgE Abs to peanuts compared with control mice. These findings suggest lung ILC2s that serve as a rapid source of IL-13 upon allergen exposure play a major role in Tfh cell development, IgE Ab production, and initiation of peanut allergy.

Allergens derived from the common fungus Alternaria alternata induce oxidative stress in human airway epithelial (HBE) cells that leads to the regulated release of ATP and subsequent activation of P2X 7 receptors that facilitate Ca 2+ uptake into the cell. Sustained increases in [Ca 2+ ] i are required for nuclear processing and secretion of IL‐33, which initiates allergic inflammation by activating ST2 receptors on immune cells to induce Th2 cytokine secretion. Pretreatment of HBE cells with the K2P channel blockers bupivacaine or quinidine produced a significant increase in ATP release following Alternaria stimulation. This response was blocked by inhibiting ATP loading into membrane vesicles by treatment with 200 nM bafilomycin A 1 before allergen exposure. Similarly, pretreatment with the PKC inhibitor Go6976 also blocked approximately 50% of Alternaria ‐induced ATP release and completely abolished the increase in ATP release produced by pretreatment with K2P channel blockers. The effects of bupivacaine and quinidine were comparable to the effect of phorbol 12‐myristate 13‐acetate (PMA, 2 mM), a well‐characterized PKC activating compound prior to Alternaria stimulation. These results support the conclusion that K2P channel inhibition is associated with PKC activation, which functions to potentiate the effects of Alternaria allergens on vesicular ATP release. We propose that enhanced PKC activity is dependent on membrane depolarization associated with inhibition of constitutive, K2P channel mediated K + efflux from these cells. Support or Funding Information This study was supported by grants from the NIH (R01‐AI128729), awarded to HK and SMO and from the Minnesota Agriculture Experiment Station (AES‐0016097) awarded to SMO.

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.

Introduction The lack of effective, consistent, reproducible and efficient asthma ascertainment methods results in inconsistent asthma cohorts and study results for clinical trials or other studies. We aimed to assess whether application of expert artificial intelligence (AI)-based natural language processing (NLP) algorithms for two existing asthma criteria to electronic health records of a paediatric population systematically identifies childhood asthma and its subgroups with distinctive characteristics. Methods Using the 1997–2007 Olmsted County Birth Cohort, we applied validated NLP algorithms for Predetermined Asthma Criteria (NLP-PAC) as well as Asthma Predictive Index (NLP-API). We categorised subjects into four groups (both criteria positive (NLP-PAC+/NLP-API+); PAC positive only (NLP-PAC+ only); API positive only (NLP-API+ only); and both criteria negative (NLP-PAC−/NLP-API−)) and characterised them. Results were replicated in unsupervised cluster analysis for asthmatics and a random sample of 300 children using laboratory and pulmonary function tests (PFTs). Results Of the 8196 subjects (51% male, 80% white), we identified 1614 (20%), NLP-PAC+/NLP-API+; 954 (12%), NLP-PAC+ only; 105 (1%), NLP-API+ only; and 5523 (67%), NLP-PAC−/NLP-API−. Asthmatic children classified as NLP-PAC+/NLP-API+ showed earlier onset asthma, more Th2-high profile, poorer lung function, higher asthma exacerbation and higher risk of asthma-associated comorbidities compared with other groups. These results were consistent with those based on unsupervised cluster analysis and lab and PFT data of a random sample of study subjects. Conclusion Expert AI-based NLP algorithms for two asthma criteria systematically identify childhood asthma with distinctive characteristics. This approach may improve precision, reproducibility, consistency and efficiency of large-scale clinical studies for asthma and enable population management.

AbstractBackgroundThe epithelial cell‐derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL‐33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, TSLP and IL‐33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown.MethodsBALB/c WT and TSLP receptor‐deficient (TSLPR−/−) mice were challenged intranasally with Alternaria extract (Alt‐Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre‐administered with rTSLP or vehicle, TSLPR−/− mice, and IL‐33 receptor‐deficient (ST2−/−) mice were challenged intranasally with Alt‐Ext or vehicle once or twice to evaluate IL‐33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL‐33, rTSLP + rIL‐33, or vehicle.ResultsThymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL‐5 and IL‐13 following Alt‐Ext‐challenge compared to WT mice. Further, eosinophilia, protein level of lung IL‐4, IL‐5, and IL‐13, and airway mucus score were also significantly decreased in TSLPR−/− mice compared to WT mice. Endogenous and exogenous TSLP increased Alt‐Ext‐induced IL‐33 release into BALF, and ST2 deficiency decreased Alt‐Ext‐induced TSLP expression in the lung. Further, rTSLP and rIL‐33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro.ConclusionThymic stromal lymphopoietin and IL‐33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt‐Ext‐challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.

Abstract Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.

Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.

Abstract Abstract 2917 Multiple myeloma (MM) is a cancer in the bone marrow (BM) characterized by the accumulation of transformed plasma cells (PCs). The pre-malignant form of the disease, monoclonal gammopathy of undetermined significance (MGUS), shares many of the genetic abnormalities found in MM, including chromosomal translocations, hyperdiploidy, and gene-specific mutations. Given this, we believe other factors within the tumor microenvironment must contribute to disease progression by influencing cell survival and/or proliferation. Eosinophils (Eos) are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states such as allergy and asthma. Recently, they were also shown to play a role in the regulation of murine BM PC homeostasis via their secretion of IL-6 and APRIL. Murine BM PCs and Eos both express CXCR4 and are believed to home to CXCL12 expressing stromal cells (SCs) in the BM. The goal of this study, therefore, was to investigate whether Eos, or soluble mediators released by Eos, have biological activity on MM cells. Thus, it is possible that this type of innate immune cell may be present in the tumor microenvironment in MM patients to support disease progression. To our knowledge, a potential role for Eos in MM has not been previously studied. We began our studies by assessing whether Eos are co-localized with normal BM PCs and/or MM PCs. Immunofluorescence analysis of BM core biopsies from normal subjects revealed occasional colocalization of PCs with Eos. Similarly, MM biopsies showed regions of MM cell clusters with increased Eos density, suggesting possible biological interactions. However, we also observed regions of MM cell clusters that lack Eos, which could indicate the liberation of these transformed cells from the requirement of Eos for survival/proliferation. Next, using Eos isolated from human BM aspirates and a panel of disease-relevant human MM cell lines (HMCLs) extensively characterized in our laboratory, we aimed to verify that MM cells and Eos could both migrate toward the chemokine CXCL12. Our data showed that indeed the KAS-6/1, ANBL-6, DP-6, and KP-6 HMCLs and human BM Eos all migrated toward this chemokine. We then determined if Eos had biological activity towards MM cells as revealed by enhanced DNA synthesis. Consistent with our interpretation of the immunofluorescence stained sections, the proliferation of some, but not all, HMCLs was enhanced when cocultured with Eos isolated from either human BM or peripheral blood. To address whether contact between Eos and HMCLs was required for this phenomenon, we assessed HMCL proliferation upon treatment with Eos culture supernatant (SN). Our data suggest that the effect of Eos on the Eos-inducible HMCLs can be contact-independent as treatment of these HMCLs with Eos SN increased their proliferation. Similarly, proliferation of primary CD138+ MM cells was also enhanced when treated with Eos SN. Because BM PCs reside in niches that include support cells such as SCs and that proliferation of MM PCs is enhanced in the presence of SCs, we next questioned whether Eos can substitute for SCs in this niche or if Eos and SCs support PC survival/proliferation through different mechanisms. We observed that Eos and BM SCs together stimulated more HMCL proliferation than either cell type did alone, indicating the presence of non-redundant roles for the two cell types. Finally, we began to investigate the mechanism by which Eos enhance HMCL proliferation. In contrast to prior reports that murine Eos express IL-6, mRNA transcripts of IL-6, a known proliferation-inducing cytokine for MM cells, were not detected in human Eos. Moreover, analysis of the Eos SN showed an absence of IL-6. Neutralization of IL-6 in the HMCL-Eos coculture did not abolish the induced proliferation. Altogether, these data suggest that human Eos utilize a yet to be identified, IL-6-independent mechanism to support malignant PC proliferation. Taken together, our data show that Eos and MM cells can colocalize in the BM via their migration toward a common source of CXCL12, i.e. BM SCs, to create a niche that promotes tumor cell growth. Eos can enhance malignant PC proliferation via soluble products, although additional contact-dependent effects may exist as well and have not been explored. Additional studies are currently underway to further characterize the mechanism by which Eos may influence the biology of MM in the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.