Induction of eosinophil apoptosis by hydrogen peroxide promotes the resolution of allergic inflammation

Eosinophils are effector cells that have an important role in the pathogenesis of allergic disease. Defective removal of these cells likely leads to chronic inflammatory diseases such as asthma. Thus, there is great interest in understanding the mechanisms responsible for the elimination of eosinophils from inflammatory sites. Previous studies have demonstrated a role for certain mediators and molecular pathways responsible for the survival and death of leukocytes at sites of inflammation. Reactive oxygen species have been described as proinflammatory mediators but their role in the resolution phase of inflammation is poorly understood. The aim of this study was to investigate the effect of reactive oxygen species in the resolution of allergic inflammatory responses. An eosinophilic cell line (Eol-1) was treated with hydrogen peroxide and apoptosis was measured. Allergic inflammation was induced in ovalbumin sensitized and challenged mouse models and reactive oxygen species were administered at the peak of inflammatory cell infiltrate. Inflammatory cell numbers, cytokine and chemokine levels, mucus production, inflammatory cell apoptosis and peribronchiolar matrix deposition was quantified in the lungs. Resistance and elastance were measured at baseline and after aerosolized methacholine. Hydrogen peroxide accelerates resolution of airway inflammation by induction of caspase-dependent apoptosis of eosinophils and decrease remodeling, mucus deposition, inflammatory cytokine production and airway hyperreactivity. Moreover, the inhibition of reactive oxygen species production by apocynin or in gp91^phox−/− mice prolonged the inflammatory response. Hydrogen peroxide induces Eol-1 apoptosis in vitro and enhances the resolution of inflammation and improves lung function in vivo by inducing caspase-dependent apoptosis of eosinophils.Eosinophils express numerous receptors and secrete a wide variety of inflammatory mediators that influence many innate and adaptive immune responses. These multifunctional cells are important in the defense against helminth infection and are involved in the pathogenesis of many eosinophilic dominant allergic diseases.¹ High levels of eosinophil granule proteins (such as major basic protein (MBP)) have been found in bronchoalveolar lavage fluid from patients with asthma and evidence indicates that high-concentration granule products have contributed to the development of airway hyperreactivity (AHR), a cardinal feature of asthma.² Asthma is an inflammatory disease of the airways with participation of many cell types including leukocytes especially eosinophils and lymphocytes.^{3, 4} Activation of these cells (mainly lymphocytes) leads to the release of proinflammatory mediators and cytokines such as leukotriene B₄, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-9 (IL-9), interleukin-13 (IL-13) and colony-stimulating factor granulocyte-macrophage (GM-CSF).^{3, 5, 6, 7} Investigations using preclinical animal models of asthma and clinical studies in patients with asthma have demonstrated that the presence of eosinophils in the lungs are associated with epithelial damage, goblet cell hyperplasia, smooth muscle hypertrophy and airway hyperresponsiveness resulting in airflow limitation which can be fatal.^{3, 8, 9, 10} Recently, anti-IL-5 treatment has been shown to ameliorate lung function in patients with eosinophilic asthma.¹¹Apoptosis of leukocytes is regarded as an important process for the successful resolution of inflammatory responses. Reduced eosinophil apoptosis in bronchoalveolar lavage (BAL) fluid has been shown to correlate positively with severity of asthma.^{3, 12, 13, 14} Indeed, defective leukocyte apoptosis and subsequent removal of apoptotic cells by phagocytes is thought to be important for the initiation and propagation of chronic inflammatory diseases such as asthma.¹⁵ Therefore, a balance in the tissue microenvironment between pro- and antiapoptotic signals is likely to greatly influence the load of eosinophils in the asthmatic lung.¹⁶ Thus, there is a great interest in understanding the mechanisms responsible for the elimination of eosinophils and other leukocytes and inactivation of proinflammatory mediators in inflammatory sites.¹⁷Several molecular pathways have been shown to modulate the survival and death of leukocytes at sites of inflammation, including reactive oxygen species (ROS).¹⁸ ROS are a family of molecules containing oxygen and includes hydrogen peroxide (H₂O₂), superoxide O₂⁻, hydroxyl radical (OH) and nitric oxide (NO).¹⁹ In inflammatory conditions, ROS are increased as they help in neutralizing invading organisms during infection either directly or indirectly by formation of extracellular traps (ETs).²⁰ ROS have traditionally been regarded as quintessentially proinflammatory. However, evidence for ROS-mediated anti-inflammatory actions has been described.²¹ The importance for ROS production in the context of infection can be exemplified in patients with chronic granulomatous disease (CGD) where defective production in ROS results in multiple infections and often early death.^{22, 23} Furthermore, studies in mouse models have shown that NADPH oxidase is key for regulating lung inflammation and injury as well as NF-κB activation and downstream cytokine production in response to LPS.²⁴ More recently, our group has demonstrated that NADPH oxidase-derived H₂O₂ is directly linked to induction of apoptosis of neutrophils and resolution of inflammation in a model of antigen-induced arthritis.¹⁸ However, the role of ROS in the context of the resolution of allergic inflammation is still unknown.Here, we evaluated whether H₂O₂ drives apoptosis of eosinophils and thereby influences the resolution of established eosinophilic inflammation and reduction of airflow obstruction. Our study provides evidence that H₂O₂ is released during allergic inflammation in a gp91^phox−/−-dependent manner and induces a caspase-dependent proapoptotic effect in eosinophils, thus having a crucial role in the resolution of allergic inflammation.