Initial sensitization and subsequent repeated exposure to antigens are known to compose the pathogenesis of asthma development (Khalaf et al., 2019). During the antigen sensitization process, antigen-presenting dendritic cells are known to play a pivotal role (Van Rijt and Lambrecht, 2005). The activation of eosinophils and mast cells leads to inflammatory reactions in the airways during the antigen exposures process (Gilfillan et al., 2009). The importance of sphingolipid synthesis and sphingosine 1-phosphate (S1P) signaling cascade has been reported in bronchial asthma and allergic diseases (Moffatt et al., 2007; Saluja et al., 2017; Worgall, 2017). Reportedly, S1P levels are elevated after antigen exposure in patients with asthma (Ammit et al., 2001). Antigen-induced crosslinking of the immunoglobulin E (IgE) antibody on mast cells induces sphingosine kinase activation, generating S1P in mast cells (Choi et al., 1996; Prieschl et al., 1999; Jolly et al., 2004). The secreted S1P, the ligand of specific five S1P receptors, S1P_1-5, induces a variety of pathophysiological responses (Graler et al., 1998; Park and Im, 2017).

In asthma, S1P₁ and S1P₂ has been extensively studied (Park and Im, 2019). In particular, S1P-induced degranulation of mast cells is mediated through S1P₂ receptors (Jolly et al., 2004; Oskeritzian et al., 2010). Previously, the involvement of S1P₂ in allergic asthma and atopic dermatitis has been studied in murine models (Park and Im, 2019, 2020). In contrast, S1P₄ has been found in mast cells, but its functions have been poorly investigated in an allergic asthma model in vivo (Kulinski et al., 2018). Therefore, first, RBL-2H3 mast cells were used to determine whether S1P₄ is involved in antigen-induced degranulation by treatment with CYM50358, a selective antagonist of S1P₄ (Guerrero et al., 2012). Second, using an ovalbumin (OVA)-induced allergic asthma model, the effects of S1P₄ suppression was assessed.

Materials

CYM50358 was purchased from Tocris (Bristol, UK). Other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

Rat RBL-2H3 mast cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). RBL-2H3 cells were cultured at 37°C in a 5% CO₂-humidified incubator, and maintained in 10% (v/v) heat-inactivated fetal bovine serum containing high-glucose Dulbecco’s modified Eagle medium (DMEM) with 2 mM glutamine, 100 U/mL penicillin, 1 mM sodium pyruvate, and 50 μg/mL streptomycin (Huang et al., 2018).

Animals

Female five-week-old BALB/c mice were purchased from Daehan Biolink (Seoul, Korea). They were housed in the laboratory animal facility at Kyung Hee University (Seoul, Korea) and provided ad libitum water and food. The Kyung Hee University Institutional Animal Care Committee reviewed and approved the protocol with respect to ethical issues and scientific care (Approval Number, KHSASP-20-197).

Assessment of degranulation

By measuring β-hexosaminidase activity in the medium, degranulation of RBL-2H3 cells was assessed. Monoclonal anti-dinitrophenyl mouse immunoglobulin E and human dinitrophenyl albumin were used to induce degranulation (Huang et al., 2018).

Asthma induction in mice and administration of CYM50358

Following a simple randomization procedure, 6-week-old female BALB/c mice (22 g) were randomly assigned to one of four treatment groups (n=5): phosphate-buffered saline (PBS)-injected control group, OVA-injected asthma group, CYM50358-treatment before sensitization plus OVA-injected group, and CYM50358-treatment before challenge plus OVA-injected group. Asthma was induced by intraperitoneal injection of 50 μg OVA and 1 mg aluminum hydroxide on D0 and D14 (sensitization). Mice were challenged by exposing to nebulized OVA for D28, D29, and D30 (challenge) (Kim and Im, 2019; Park and Im, 2019). CYM50358 was administered via intraperitoneal injection 30 min before OVA sensitization or OVA challenge. Bronchoalveolar lavage fluids (BALF) were collected from the lungs on D32, and cell population of BALF cells was analyzed after staining.

Cell counting and analysis in BALF

Using a Cellspin^® centrifuge (Hanil Electric, Seoul, Korea), immune cells in BALF were adhered to a glass slide and fixed in methanol for 30 s. Staining with May-Grünwald solution was conducted in the cells on slides for 8 min and subsequently by Giemsa solution for 12 min.

Histological examination of the lungs

Tissue sections of lungs from mice of each group were prepared. Hematoxylin and Eosin (H&E) staining and periodic acid-Schiff (PAS) staining were conducted to find mucus-secreting goblet cells and eosinophil infiltration, respectively. For PAS staining, Schiff’s regent was used and for H&E staining, hematoxylin and eosin regents were used (Heo and Im, 2019).

Degree of lung inflammation was measured using a subjective scale of 0-3 by a treatment-blind observer. Mucin-secreting cells stained with PAS in the airways were counted from two lung sections per mouse. At the same time we also measured the length of the bronchi basal lamina using ImageJ software (National Institutes of Health, Bethesta, MD, USA). Mucous production was expressed by the number of PAS-positive cells per mm of bronchiole (Kim and Im, 2019).

Measurement of total serum IgE levels and IL-4 cytokine levels

Mouse IgE levels in the serum were determined using ELISA kits (eBioscience, San Diego, CA, USA). IL-4 and IL-13 levels in BALF were quantitated using ELISA kits (eBioscience). Capture antibodies and biotinylated detection antibodies specific for IL-4 and IL-13 were obtained from eBioscience (IL-4: cat. 14-7041-68 and cat. 33-7042-68C or IL-13: cat no. 14-7043-68 and 33-7135-68B). Avidin-horseradish peroxidase was used and the absorbance was measured at 450 nm (Lee et al., 2018).

Statistical analysis

Results are expressed as means ± standard errors (SEs). For statistical significance analysis of variance (ANOVA) was used, and followed by Turkey’s post hoc test using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). p values<0.05 indicated statistical significance.

CYM50358 repressed degranulation of mast cells

Mast cells play a pivotal role in asthma episodes (Prussin and Metcalfe, 2003). Antigen exposure induces cross-linking of IgEs on mast cell membranes, resulting in degranulation (Gilfillan et al., 2009). The degranulation of the mast cells releases mediators of allergic responses such as histamine, leukotrienes, and prostaglandins (Brown et al., 2008). RBL-2H3 rat basophilic leukemia cells were used to measure degranulation responses. The β-hexosaminidase activity in the medium was increased following antigen exposure (Fig. 1). Treatment of CYM50358 suppressed the release of β-hexosaminidase in a concentration-dependent manner (Fig. 1). The inhibition induced by CYM50358 was significant at a concentration of 10 μM (Fig. 1).

Figure 1. CYM50358 represses antigen-induced degranulation in RBL-2H3 mast cells. After sensitization with anti-DNP IgE for 18 h, RBL-2H3 cells were challenged with DNP human serum albumin (HSA). CYM50358 was treated at the indicated concentrations 30 min before antigen challenge. Basal degranulation shows samples without IgE and HSA, and the positive control of antigen-induced degranulation is shown in the samples with IgE and HSA. The results are presented as the means ± standard error (SE) of three independent experiments. ***p<0.001 vs. the HSA-untreated group. ^###p<0.001 vs. the HSA-treated group.

CYM50358 repressed the increase of eosinophils and lymphocytes in the bronchoalveolar lavage fluid (BALF)

Next, an OVA-induced mouse model of asthma was employed to verify the inhibitory effect of CYM50358. The total cell number was assessed in the BALF, and the distribution of immune cell populations was calculated. In the BALF, the total cell number increased to 293.1% in the OVA-induced asthma group when compared with that in the PBS-treated control group (Fig. 2). CYM50358 treatment before antigen sensitization or before antigen challenge significantly inhibited the OVA-induced increase in the total cell number by 56.6 and 41.7%, respectively (Fig. 2B). Immune cell populations in the BALF were also assessed, with the eosinophil number increased by the OVA treatment and significantly decreased by CYM50358 treatment before sensitization or before challenge, by 69.3 and 48.5%, respectively (Fig. 2B). Although the lymphocyte number was lower than the eosinophil number, OVA induced an increase and CYM50358 treatment decreased the lymphocyte counts by both treatments (Fig. 2C). Macrophage numbers were not significantly altered by OVA or CYM50358 (Fig. 2B).

Figure 2. CYM50358 represses OVA-induced immune cell accumulation in BALF. (A) Mice were sensitized with OVA twice by i.p. injection on day 0 (D0) and D14, and later challenged on D28, D29, and D30 with nebulized OVA. CYM50358 was administrated intraperitoneally at the dose of 10 mg/kg, 30 min before OVA sensitization or before OVA challenge. BALF cells were stained using May-Grünwald stain and counted. (B) Total cell counts, eosinophils, and macrophages in BALF. (C) Lymphocytes counts in the BALF. The results are presented as the mean ± SE cell count values (n=5). ***p<0.001 vs. the PBS-treated group, ^##p<0.01, ^###p<0.001 vs. the OVA-treated group.

CYM50358 repressed the mucin secretion and inflammation in the lungs

Additionally, histological analysis of the lung samples was performed. In the H&E staining, eosinophils in the lung sections were present as small, navy-blue dots (Fig. 3). Although substantially few eosinophils were detected in the PBS control group, numerous eosinophils densely surrounded bronchioles in the OVA group (Fig. 3). CYM50358 treatment before sensitization or before challenge reduced eosinophil numbers (Fig. 3). On using a subjective scale of 0-3, semi-quantitative evaluation of lung inflammation indicated an average inflammation score of 2.2 in the OVA-treated group, and CYM50358 treatment before sensitization or before challenge significantly reduced the score (Fig. 3).

Figure 3. CYM50358 protects against airway inflammation. (A) Panels show H&E-stained sections of lung tissues from the PBS group, OVA group, and CYM50358-treated OVA groups (before sensitization or before challenge). Small navy blue dots around the bronchioles indicates eosinophils. Eosinophils are scarcely observed in the PBS group, whereas they are densely accumulated around bronchioles in the OVA group (green arrows). However, eosinophil accumulation was less obvious in the OVA+CYM50358 groups than in the OVA group. (B) Lung inflammation was semi-quantitatively evaluated; histological findings were scored as described in the Materials and methods section. Values represent the means ± SEs (n=5). ***p<0.001 vs. the PBS-treated group, ^###p<0.001 vs. the OVA-treated group.

PAS staining was also performed to reveal mucins and mucous glycoproteins produced by goblet cells. As shown in Fig. 4, secreted or stored mucins appeared as dark violet. Mucins were stained in cells surrounding bronchioles in the OVA group. However, mucin production was suppressed following CYM50358 treatment before sensitization or before challenge (Fig. 4). Furthermore, a semi-quantitative analysis of mucin production was performed by counting PAS-positive cells in bronchioles (Fig. 4). Stained cells were scarce in the PBS-treated group. However, in the OVA-treated group, approximately 100 PAS-positive cells/mm were detected, and CYM50358 treatment before sensitization or before challenge significantly suppressed the number of PAS-positive cells (Fig. 4).

Figure 4. CYM50358 protects against mucin production. (A) Panels show PAS/hematoxylin-stained sections of lung tissues from the PBS group, OVA group, and CYM50358-treated OVA groups (before sensitization or before challenge). In PAS staining, mucin is stained as purple color. Darker and thicker purple color is observed surrounding the bronchiole in the OVA group compared to the PBS group. (B) Mucous production was measured by counting the number of PAS-positive cells (red arrows) per mm of bronchiole (n=5 per group). ***p<0.001 vs. the PBS-treated group, ^###p<0.001 vs. the OVA-treated group.

CYM50358 suppressed OVA-induced increase in serum IgE and BALF IL-4 levels

Serum IgE levels were assessed to confirm the immunological effects of OVA and CYM50358. IgE production was increased in the sera of OVA-treated mice (Fig. 5A). An OVA-induced increase in serum IgE levels was significantly repressed by CYM50358 treatment, both before antigen sensitization and challenge.

Figure 5. Effect of CYM50358 on IgE levels in serum and IL-4 levels in BALF. (A) Serum IgE levels. Results are presented as means ± SEM (n=5). ***p<0.001 vs. the PBS-treated group, ^#p<0.05, ^###p<0.001 vs. the OVA-treated group. (B) ELISA was used to measure the protein levels of IL-4 in BALF. The results represent the mean ± SE of protein levels (n=5). ***p<0.001 vs. the PBS-treated group, ^###p<0.001 vs. the OVA-treated group.

Th2 cytokines, such as IL-4, play major roles in the progression of allergic asthma (Romagnani, 2002). Th2 cytokines induce eosinophil recruitment and activation, hypersecretion of mucus in epithelial cells, metaplasia of goblet cells, and proliferation of smooth muscle cells (Tagaya and Tamaoki, 2007). The protein levels of Th2 cytokines, IL-4 and IL-13 in BALF were measured by ELISA. The IL-4 levels were increased in the OVA-induced group compared to the vehicle-treated control group, and the increase in IL-4 levels was significantly suppressed by both treatments of CYM50358 (Fig. 5B). We were not able to detect IL-13 levels in the BALF, because they were below the detectable ranges.

In the present study, two new findings were revealed using the S1P₄ antagonist, CYM50358. First, the S1P₄ receptor was involved in mast cell degranulation in RBL-2H3 cells and OVA-induced allergic asthma. Following the administration of CYM50358 before antigen challenges, immunological responses such as increase of IgE levels, immune cell accumulation in BALF, and increased mucin-secreting cells in the lungs were significantly suppressed. CYM50358-mediated inhibition of mast cell degranulation might contribute to the suppressive actions of the CYM50358 in vivo. In particular, effect of CYM50358 treatment before antigen challenge imply that suppression of mast cell degranulation plays an important role in the in vivo efficacy. Second, blockage of the S1P₄ receptor suppressed exacerbation of allergic asthma responses in vivo, which was demonstrated by both CYM50358 treatments, that is, before sensitization and before antigen challenge. The effect of CYM50358 treatment before sensitization may imply that S1P₄ functions in dendritic cells. Reportedly, S1P₄ expression is found to be high in lymphoid tissues and hematopoietic cells, including dendritic cells and neutrophils (Graler et al., 1998; Schulze et al., 2011; Kulinski et al., 2018). Previously, S1P₄ deficiency was found to affect dendritic cell migration and cytokine secretion and reduce Th17 differentiation of T cells in a murine model (Schulze et al., 2011). Also S1P₄ is suggested to be required for plasmacytoid dendritic cell differentiation and CYM50358 prevented S1P₄-dependent reduction of IFN-α production in human plasmacytoid dendritic cells (Dillmann et al., 2015, 2016). S1P accumulation in S1P lyase deficient mice caused neutrophilia and deletion of S1P₄ partially rescued the neutrophil recruitment (Allende et al., 2011). Therefore, further researches on other cell types expressing S1P₄ need to be conducted to elucidate their contribution on anti-inflammatory and anti-allergic effects.

The results with S1P₄ deficient mice are in contrast with the present results. In the present study, inhibition of S1P₄ using CYM50358 suppressed allergic responses, while S1P₄ gene deficiency increased the magnitude of Th2-dominated immune responses, including the aggravation of passive systemic anaphylaxis to IgE/anti-IgE in mice (Schulze et al., 2011; Kulinski et al., 2018). Conversely, Th1-dominated mechanisms were diminished in S1P₄ deficient mice (Schulze et al., 2011). S1P suppresses collagen-induced activation of human platelets and induces anti-inflammatory effects in vitro and in vivo via the S1P₄ receptor (Onuma et al., 2017; Fettel et al., 2019). The discrepancy of phenotypes between S1P₄ deficient mice and CYM50358-treated mice may be attributed to the compensatory adaptation in S1P₄ deficient mice as S1P₄ gene deficiency was maintained from the embryonic stage. Instead, the present study temporarily suppressed the S1P₄ functions during antigen sensitization or during antigen challenge. Therefore, the phenotypes observed with CYM50358 treatments could be the more relevant outcomes when S1P₄ is temporarily suppressed.

An abundant expression of S1P₄ has been observed in cultured mouse mast cells (Kulinski et al., 2018). However, S1P₄ gene deficiency did not affect mast cell proliferation in culture or the differentiation of bone marrow progenitors into mast cells (Kulinski et al., 2018). Therefore, S1P₄ was suggested to be dispensable for cytokine/chemokine production, degranulation, and FcεRI-mediated chemotaxis in mast cells in vitro (Kulinski et al., 2018). However, in the present study, CYM50358 suppressed antigen-induced degranulation of RBL-2H3 mast cells as well as OVA-induced allergic responses. Additionally, enhancement of IgE-induced degranulation by IL-33 is reportedly suppressed in peritoneal mast cells from S1P₄ deficient mice, suggesting a negative regulatory role of S1P₄ in mast cells (Kulinski et al., 2018). Therefore, the functions of S1P₄ in mast cells might be more complicated depending on the circumstances.

Previously, the significance of S1P and its receptors has been reported in animal models, such as antigen-induced allergic asthma and airway inflammation (Roviezzo et al., 2007; Chiba et al., 2010; Park and Im, 2019). S1P₄ expression and functions in mast cells and dendritic cells have been evaluated in S1P₄ deficient mice (Schulze et al., 2011; Kulinski et al., 2018). However, direct chemical modification of S1P₄ has not been attempted. In the present study, the in vitro efficacy of CYM50358 was tested in the RBL-2H3 cells for the first time, revealing the stimulatory role of S1P₄ on mast cell degranulation. Additionally, the in vivo efficacy of CYM50358 was investigated for the first time using an OVA-induced asthma model, demonstrating significant suppression of allergic asthma in both CYM50358 treatments, before sensitization and before challenge. Significant suppression was observed in immune cell accumulation, lung inflammation, IL-4 secretion, and mucin production. In summary, in the present study, the efficacy of CYM50358, a chemical antagonist of S1P₄, was revealed on allergic responses, demonstrating the suppression of mast cell degranulation, as well as reduced OVA-induced allergic responses, and thus providing evidence for the potential therapeutic applications of S1P₄ in allergic asthma.

This research was supported by the Basic Research Laboratory Program (BRL) and the Basic Science Research Program of the Korean National Research Foundation funded by the Korean Ministry of Science, ICT and Future Planning (NRF-2020R1A4A1016142 and NRF-2019R1A2C1005523).

The authors declare that there is no conflict of interest.