We previously showed that Th1/type 1 inflammation marked by increased IFN-γ levels in the airways can be appreciated in 50% of patients with severe asthma, despite high dose corticosteroid (CS) treatment. We hypothesized that a downstream target of IFN-γ, CXCL10, which recruits Th1 cells via the cognate receptor CXCR3, is an important contributor to Th1highasthma and CS unresponsiveness. We show high levels of CXCL10 mRNA closely associated with IFNG levels in the BAL cells of 50% of severe asthmatics and also in the airways of mice subjected to a severe asthma model, both in the context of high-dose CS treatment. The inability of CS to dampen IFNG or CXCL10 expression was not because of impaired nuclear translocation of the glucocorticoid receptor (GR) or its transactivational functions. Rather, in the presence of CS and IFN-γ, STAT1 and GR were recruited on critical regulatory elements in the endogenous CXCL10 promoter in monocytes, albeit without any abatement of CXCL10 gene expression. High CXCL10 gene expression was also associated with a mast cell signature in both humans and mice, CXCR3 being also expressed by mast cells. These findings suggest that the IFN-γ–CXCL10 axis plays a central role in persistent type 1 inflammation that may be facilitated by CS therapy through GR-STAT1 cooperation converging on the CXCL10 promoter.
Asthma is a disease with significant prevalence and morbidity throughout the developed world, affecting nearly 5%–10% of populations (1). It is increasingly recognized that asthma is a disease with multiple phenotypes, each with its own unique molecular mechanisms, natural history, and response to therapy (2, 3). Corticosteroids (CS) have remained the mainstay of therapy, but for many patients, these therapies are ineffective (4), and research of underlying mechanisms may give insight into the etiologies of CS resistance and identify future therapeutic targets.
Our previous studies have shown that the number of Th1 cells, which produce IFN-γ, is elevated in approximately 50% of severe asthma (SA) patients and in our mouse model of SA (5), and increased IFNG mRNA levels are also evident in the airways of these subjects (6). Furthermore, IFN-γ was associated with increased airway hyperreactivity (AHR) and poor CS response (5). A high Th1/IFN-γ response in any tissue is typically induced during infections by bacteria and viruses (7). Infections by viruses (rhinovirus being the most common) and bacteria have been observed in patients with SA and can trigger asthma exacerbations (7). Several bacterial species have also been associated with severe disease (8). Once generated in lung-draining lymph nodes, Th1 cells need to be recruited to the site of infection, and the best known chemoattractant for Th1 cells is CXCL10 (9), initially cloned as an IFN-γ–induced molecule from monocytes (10). Additional chemokines that belong to the same family induced by IFN-γ include CXCL9 and CXCL11, although CXCL10 is the most studied (11). While there is significant redundancy in their effects, the expression of the 3 family members is not uniform in disease settings, including allergic disease (11, 12).
In this study, we sought to better understand the possible etiologies of this resistance to CS therapy, and as such, we focused on CXCL10, given its role in recruiting Th1 cells to reinforce type 1 inflammation to combat and eliminate viral and bacterial pathogens, a function that when uncontrolled can lead to significant pathology (13). The expression of CXCL10 can be induced not only by IFN-γ, but also by additional stimuli, including LPS, which can lead to differential levels of CXCL10 production and response to therapies (11, 14–16). In addition to its expression on Th1 cells, the CXCR3 receptor — which mediates chemoattraction by CXCL10 and its family members — is also present on mast cells (17), neutrophils (18), and eosinophils (19). Elevated CXCL10 levels have been detected in multiple compartments, including blood and bronchoalveolar lavage (BAL) in mild and atopic asthma (20–22) and may be increased during asthma exacerbations (23). CXCL10 can be secreted by multiple cell types, including airway epithelial cells, smooth muscle cells, monocytes, and macrophages (11). However, several studies have implicated monocytes/macrophages as possible drivers of CXCL10 expression in asthma (24, 25).
Given its association with IFN-γ, we asked whether CXCL10 may be a contributor to steroid resistance in SA. Here, we show high levels of CXCL10 mRNA closely associated with IFNGlevels in the airways of 50% of SA subjects and in mice subjected to our SA model, both in the context of high-dose CS treatment. Our investigation of possible impairment of glucocorticoid receptor (GR) function in the presence of IFN-γ showed no such impairment with preservation of nuclear translocation and transactivational functions of GR. However, as revealed using ChIP assay, in the presence of CS and IFN-γ, we observed simultaneous enrichment of STAT1 and GR on critical regulatory elements in the endogenous CXCL10 promoter in monocytes; importantly, this did not cause inhibition of CXCL10 expression at either mRNA or protein levels. In contrast, CS inhibited LPS-induced binding of NF-κB to the CXCL10 promoter and inhibited LPS-induced CXCL10 gene expression, showing selective impairment of CS-mediated suppression in the presence of IFN-γ. High CXCL10 gene expression was also associated with markers of mast cells in the airways of severe asthmatics, consistent with known human mast cell expression of CXCR3. Taken together, these findings suggest that the IFN-γ/CXCL10 axis is prominent in CS-refractory disease. Increased expression of both IFN-γ and the chemokine that recruits IFN-γ–producing Th1 cells may establish a persistent type 1 inflammation that may actually worsen with CS therapy through GR-STAT1 cooperation in promoting CXCL10 gene expression.