Oxidative stress and chronic inflammation are key elements of human airway and lung diseases. Harmful gases, such as cigarette smoke or noxious particles, stimulate neutrophils and macrophages to produce inflammatory cytokines and mediators, leading to inflammation and a subsequent increase in reactive oxygen species (ROS). As a result, oxidative stress escalates, promoting a broad spectrum of immune disorders and pathophysiological conditions, including chronic obstructive pulmonary disease (COPD). Heme oxygenase (HO), a family of enzymes that manage the cellular response to oxidative stress, is known to degrade heme (the precursor to hemoglobin) to reduce oxidative stress, restore intracellular redox balance, and maintain cellular homeostasis. HO enzymes, particularly the inducible isozyme HO-1, produce carbon monoxide (CO), biliverdin, and iron in mammalian cells. HO-1 activity is upregulated by cellular stress in all tissues, including the brain. However, the precise mechanisms by which HO-1 exerts these anti-inflammatory and anti-oxidative effects in human bronchial epithelia remain unclear. The purpose of this study is to investigate how HO-1/CO modulates oxidative stress and inflammation in human bronchial epithelium.
In this study, we used three different human bronchial epithelial (16HBE14o-) cell models: wildtype (WT), H25A (HO-1 inactive) mutant, and HO-1 knockdown (KD). Intracellular ROS was measured using the cell-permeable probe, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), by fluorescence microscopy. A cytokine array analysis was used to detect the expression levels of 80 human cytokines semi-quantitatively. The data show that ROS fluorescent intensity increased significantly in KD (p < 0.01) and H25A mutant cells (p < 0.001) after stimulation with hemin, a well-known oxidative inducer (n = 4; one-way ANOVA followed by Tukey’s multiple comparison test). Therefore, in the presence of a previous oxidative factor or stimulus, a decrease in HO-1 expression or mutation of HO-1 resulted in greater levels of ROS, indicating high oxidative stress. Cytokine array analysis data show that in HO-1 KD cells, pro-inflammatory mediators MCP-1 (CCL2), IL-8, IL-6, TIMP-1 and BDNF were increased , while anti-inflammatory mediators IGFBP-3 and RANTES (CCL5) were decreased (n = 3). In H25A mutant cells, pro-inflammatory cytokines TNF-α, IL-8, and IL-6 were reduced (n = 3). Our findings suggest that HO-1 regulates cellular oxidative stress and exerts an anti-inflammatory effect in human bronchial epithelial cells. These findings offer insight into some of the mechanisms that underpin HO-1 activity as a therapeutic strategy for oxidative stress-related respiratory diseases.