Asthma is a chronic airway inflammatory disease which shows infiltration of immune cells into the airways, airway obstruction, airway hyperresponsiveness (AHR), and mucus hypersecretion (1, 2). A risk factor for developing asthma is exposure to substances that provoke allergic responses, including environmental allergens such as pollen and house dust mites (HDMs) (2, 3). HDMs are the most prevalent allergen and a risk factor for asthmatic allergies (4, 5). In total, 45-85% of asthma patients are allergic to HDMs, with geographical differences (2, 6). Previous studies using mouse models reported that chronic exposure to HDMs induces eosinophilic airway inflammation and elevation of TH2-associated cytokines in the bronchoalveolar lavage fluid (BALF) (3, 4, 7).
Leukotriene B4 (LTB4) is a major lipid mediator of inflammatory processes and immune responses. LTB4 is derived via the arachidonic-acid pathway and works as a chemoattractant molecule for leukocytes, such as granulocytes, monocytes, and T lymphocytes, to sites of acute inflammation (8-13). This proinflammatory molecule has two receptors, BLT1 and BLT2 (14). These receptors are members of the G protein-coupled receptor (GPCR) protein and are expressed on cell surface (14-16). BLT1, a high-affinity receptor of LTB4, is exclusively expressed on the surface of leukocytes. On the other hand, BLT2 shows a low-affinity for LTB4, and it is expressed in various tissues, including the spleen, lung, and liver (14, 17). BLT2 was shown to interact with various arachidonic acid-derived metabolites, such as 12(
In this study, we examined the mediatory role of the BLT1/2-linked cascade in HDM-driven airway inflammation. Inhibition of BLT1/2 markedly reduced the production of TH2 cytokines, IL-4, IL-5, and IL-13, in the BALF of HDM-induced model mice. We also observed that BLT1/2 inhibition reduced lung inflammation and mucus secretion. In addition, the production of TH2 cytokines, lung inflammation, and mucus secretion are inhibited by the blockade of 5-/12-LO, which are enzymes that catalyze the production of ligands for BLT1/2. Collectively, these results suggest that the 5-/12-LO-BLT1/2-linked cascade contributes to the development of HDM-induced eosinophilic airway inflammation via TH2 cytokine production. Thus, we propose that BLT1/2 may be a potential therapeutic target for HDM-sensitive asthmatic patients.
We established HDM-induced eosinophilic airway inflammation as previously described (3) with some modifications. Mice were intranasally sensitized with 25 µg of HDMs on days 0, 1 and 2 and then challenged with 6.25 µg HDMs on days 14, 15, 18, and 19. Mice were sacrificed on day 20. Inhibitors were intraperitoneally injected 1 h before every challenge (Fig. 1A). To study the roles of BLT1/2 signaling in HDM-induced airway inflammation, we measured the levels of their ligands (LTB4 and 12(
To examine whether BLT1/2 have roles in HDM-induced eosinophilic airway inflammation, we investigated lung histology and cell populations. Histopathological analysis and quantitative analysis of inflammation scores with H&E staining showed that lung inflammation induced by HDM administration was markedly attenuated by BLT1 antagonist U75302 or BLT2 antagonist LY255283 (Fig. 2A). In addition, increased mucus secretion induced by HDMs was also attenuated by U75302 and LY255283 (Fig. 2A). HDM administration markedly increased the influx of total cells in BALF. The increased total cell influx in BALF by HDM administration was markedly abolished by pretreatment with U75302 or LY255283 (Fig. 2B). However, the numbers of neutrophils, macrophages, and lymphocytes were not affected by HDMs (Fig. 2B). Taken together, these results suggest that BLT1 and BLT2 contribute to HDM-induced eosinophilic airway inflammation.
To further elucidate the contributory roles of BLT1 and BLT2 in the HDM-induced eosinophilic airway inflammation model, we investigated whether 5-LO and 12-LO, the enzymes catalyzing the synthesis of BLT1/2 ligands, are also involved. The levels of LTB4 and 12(
Finally, we investigated the effect of 5-/12-LO inhibition on HDM-induced eosinophilic airway inflammation. MK886 or baicalein treatment clearly attenuated alveolar hemorrhage and the influx of immune cells into the airway (Fig. 4A). Additionally, the administration of MK886 or baicalein significantly reduced mucus secretion (Fig. 4A). The numbers of total cells and eosinophils were also decreased by treatment with these inhibitors (Fig. 4B). Together, these results suggest that the 5-/12-LO-BLT1/2 cascade contributes to HDM-induced eosinophilic airway inflammation.
In this study, we found that the levels of LTB4 and 12(
Previous studies reported that repeated exposure to HDMs induces eosinophilic airway inflammation with greater TH2-associated humoral immune responses and airway remodeling (4, 7). The critical roles of TH2 cytokines in the HDM-induced allergic asthma mouse model are already known (2, 7, 22-24). These cytokines have been reported to induce eosinophil influx, airway smooth muscle hyperplasia, and mucus secretion (25). Recent studies also revealed that HDM treatment induces the release of lipoxygenase-derived lipid mediators in BALF (26). In addition, HDM-induced airway inflammation showed increased levels of cysteinyl leukotrienes and 12/15-LO metabolites (27). However, the role of BLT1/2, receptors for LTB4 or 12(
Eosinophilia is one characteristic of HDM-induced allergic airway inflammation (Fig. 2). Nonetheless, we cannot rule out the contribution of other types of immune cells including macrophages, mast cells, and T lymphocytes (31). For example, the mediatory role of mast cells in the exacerbation of asthma has been well reported (32-36), and we previously demonstrated the role of BLT2 in the mast-cell activation and secretion of TH2 cytokines
In summary, our results suggest that the 5-/12-LO-BLT1/2 cascade clearly contributes to the development of HDM-induced eosinophilic airway inflammation via TH2 cytokine production. This is the first report on the role of BLT1/2 in an HDM-induced asthmatic mouse model, and our results may provide a potential therapeutic target for HDM allergic asthma.
Dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO).
MK886 from Calbiochem (La Jolla, CA, USA).
U75302 and baicalein from Enzo Life Sciences (Farmingdale, NY, USA).
LY255283 was obtained from Cayman Chemical (Ann Arbor, MI).
We obtained female C57BL/6 mice (8-9 weeks old) from Young-Bio (Seongnam, Korea). HDM extract (Greer Laboratories, Lenoir, NC), derived from
We quantified the levels of IL-4, IL-5, IL-13, LTB4 and 12(
Inflammatory cells collected from BALF by centrifugation (1,000 × g for 3 min) were washed with PBS. Next, cytocentrifuge slides of BAL cells were fixed and stained with Diff-Quik. We harvested lung tissues, fixed them in 10% formaldehyde for 3 weeks, and embedded them in paraffin. We mounted Lung sections (4.5 µm thickness) onto SuperfrostTM Plus glass slides (Fisher Scientific, Pittsburgh, PA, USA) and deparaffinized and stained them with H&E and periodic acid-schiff (PAS). We did a quantitative histological analysis by five blinded investigators. We evaluated the degree of peribronchial and perivascular lung inflammation on a subjective scale from 0 to 3, as previously described (8). For the quantification of goblet cells in the airway, we used a five-point grading system: 0 < 0.5% PAS positive cells; 1 < 25%; 2, 25-50%; 3, 50-75%; and 4 > 75% (39). All images were acquired using a BX51 microscope (Olympus, Tokyo, Japan) equipped with a DP71 digital camera (Olympus).
We did all statistical analyses with one way analysis of variance, followed by Tukey’s
This work was supported by a Bio & Medical Technology Development Program Grant (2017M3A9D8063317) and a Mid-Career Researcher Program Grant (2020R1A2B5B01002046) through the National Research Foundation funded by the Ministry of Science, Information, and Communication Technologies (ICT) and Future Planning, Republic of Korea. This work was also supported by Korea University grant.
The authors have no conflicting interests.