Hypoxia causes IL‐8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid‐induced apoptosis in human eosinophils

Abstract

Summary Background Inflamed environments are typically hypercellular, rich in pro‐inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. Objective We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. Methods Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F‐actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. Results Hypoxic incubation (3 kPa) caused (i) stabilization of HIF‐2α and up‐regulation of hypoxia‐regulated genes including BNIP3 (BCL2/adenovirus E1B 19‐kDa protein‐interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre‐formed IL‐8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F‐actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF‐κB and Class I PI3‐kinase‐dependent mechanism); and (v) complete abrogation of the normal pro‐apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS‐mediated gene transactivation under hypoxia. Conclusion and Clinical Relevance These data indicate that hypoxia promotes an eosinophil pro‐inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro‐apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.

Metabolic Profiling of Human Eosinophils

“…Inflamed lesions are often severely hypoxic due to increased cellular oxygen demand and reduced oxygen availability ( 38 – 40 ). Hypoxia is a potent pro-inflammatory stimulus for both neutrophils and eosinophils, contributing to an anti-apoptotic and a hyper-secretory profile in both cell types (notably enhanced release of neutrophil elastase and myeloperoxidase from neutrophils and Charcot–Leyden crystal/Galectin-10 production by eosinophils) ( 22 , 40 ). In our study, hypoxia profoundly suppressed mitochondrial oxidative phosphorylation in eosinophils, indicative of a metabolic pathway shift from oxidative phosphorylation to glycolysis.…”

TSLP promotes angiogenesis of human umbilical vein endothelial cells by strengthening the crosstalk between cervical cancer cells and eosinophils

“…EOS and tumor associated macrophages accumulated in the hypoxic and/or necrotic areas of mouse subcutaneous B16F10 melanoma, and this process was associated with EOS degranulation (22). These particles of EOS degranulation contain VEGF and other angiogenic factors, which may promote tumor angiogenesis in the hypoxic areas (38,39). In addition, EOS promotes angiogenesis in local cancer lesion by releasing a large number of vasoactive leukotrienes (40).…”

The Impact of Hypoxia on Neutrophil Degranulation and Consequences for the Host

“…In eosinophils, Porter et al showed that hypoxia increased the release of IL-8 but attenuated that of eosinophil-derived neurotoxin, with no change in mRNA levels even at 24 h [67]. The phenomenon of "piecemeal degranulation" has been described for both mast cells and eosinophils, whereby release of selected proteins or cytokines occurs via vesicle or vesiculo-tubular sequential emptying of granules [68]; it is possible that hypoxia may be able to access or modify this system of protein secretion.…”

“…How hypoxia might modulate PI3Kγ signalling in a HIF-independent fashion remains unclear. It is conceivable that hypoxic reorganisation of the actin cytoskeleton facilitates access of PI3Kγ to its phospholipid substrate; cytoskeletal disruption has been linked to enhanced neutrophil degranulation [85], and hypoxia has been shown to promote focal actin polymerisation in neutrophils, forming cap-like structures [24], similar to the actin reorganisation seen in hypoxic eosinophils [67]. Another possibility is that hypoxia is able to regulate PI3K expression.…”