Sphingosine 1-phopshate (S1P) is an intercellular lipid mediator, which acts by binding to its specific G protein-coupled receptors in the plasma membrane, now referred to as S1P1-5 (Park and Im, 2017). Sphingosine kinases 1 and 2 are responsible for S1P synthesis from sphingosine, while S1P lyase degrades S1P irreversibly to hexadecenal and ethanolamine phosphate (Spiegel and Milstien, 2007; Leong and Saba, 2010). S1P regulates cell differentiation, migration, survival and complex physiological processes (Proia and Hla, 2015; Park and Im, 2017). S1P and its receptors have also been implicated in the development of atopic dermatitis-like skin lesions in NC/Nga mice (Kohno
Previously, S1P was found to act as a pro-allergic factor via S1P2 in murine ovalbumin-induced allergic asthma model (Park and Im, 2019a). In addition, S1P modulates antigen capture by murine and human Langerhans cells via S1P2 in the skin (Japtok
JTE-013 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). 1-Chloro-2, 4-dinitrobenzene (DNCB) and olive oil were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Seven-week-old male BALB/c mice were purchased from Daehan Biolink (DBL; Seoul, Korea) and housed in the laboratory animal facility at Pusan National University. They were provided food and water
Following simple randomization procedure, 7-week old male BALB/c mice were divided into three groups (n=5): a vehicle-treated control group, a DNCB-treated mice group, and a JTE-013/DNCB-treated group. To induce experimental atopic dermatitis, ventral skin was shaved, and 300 μL of 1% DNCB in acetone/olive oil (3:1) was applied to the ventral skin on day 0. On day 7, mice were challenged again by the application of 200 μL of 0.3% DNCB to the ears every other day for up to 42 days. From day 20 until the completion of the experiment, the JTE-013/DNCB-treated group was topically treated with JTE-013 (200 μg/20 μL for each ear) every other day, which is an alternative to DNCB treatment days for up to 42 days. JTE-013 was dissolved in a mixture of acetone/olive oil (3:1). The mice were sacrificed on day 49.
After sacrifice on day 49, ear tissues were steeped in 10% formalin, dehydrated in 30% sucrose solution, embedded in O.C.T. compound, and sectioned in 8 μm thick segments. Sections were stained with hematoxylin and eosin (H&E) or with toluidine blue O (TBO). For H&E staining, sections were washed in distilled water and stained with hematoxylin solution for 15 s. After being rinsed with warm running tap water, sections were stained with eosin reagents for 10 s, followed by rinsing, dehydration, and coversliped with Permount.
For the detection of mast cells by TBO staining, sections were washed in distilled water and stained with toluidine blue solution for 2 min, then rinsed in distilled water, dehydrated, and coversliped with Permount.
Mouse IgE levels in the serum were determined by the use of ELISA kits (eBioscience, San Diego, CA, USA). Briefly, 96-well plates (Thermo FIsher Scientific, Waltham, MA, USA) were coated overnight at 4°C with capture antibodies for IgE. Following washing, plates were blocked for 2 h at room temperature with a blocking buffer. Serum was added to each well, and plates were incubated for 2 h at room temperature. Pre-titrated, biotin-conjugated detection antibodies for IgE were then added and incubated for 1 h at room temperature, while pre-titrated streptavidin-HRP was added for 30 min at room temperature, and plates were incubated with substrate solution for 15 min at room temperature. Stop solution was then added and absorbance was read at 450 nm.
To assess the expression levels of inflammatory markers in the lymph nodes of mice by RT-PCR, first-strand cDNA was first synthesized from total RNA isolated using TRIzol reagent (Invitrogen, Waltham, MA, USA). Total RNAs were isolated from draining lymph nodes. Synthesized cDNA products, primers for each gene, and Promega Go-Taq DNA polymerase (Madison, WI, USA) were used for PCR. Specific primers and PCR conditions were previously described (Park and Im, 2019a). Aliquots (5 μL) were electrophoresed in 1.2% agarose gels and stained with StaySafeTM Nucleic Acid Gel Stain (Real Biotech Corporation, Taipei, Taiwan). Intensities of each PCR product were quantified by means of ImageJ software (NIH, Bethesda, MD, USA) and normalized with GAPDH levels.
Results are expressed as the means ± standard error of the mean (SEM) of six evaluations for animal experiments. Statistical significances of differences were determined by the analysis of variance (ANOVA) and Tukey’s multiple comparison test. Statistical significance was accepted for
In order to identify the therapeutic potential of JTE-013 topical application, BALB/c mice were treated with JTE-013 after the induction of atopic dermatitis (Fig. 1A). To induce experimental atopic dermatitis, 300 μL of 1% DNCB in acetone/olive oil (3:1) was applied to shaved ventral skin on day 0, and mice were challenged again through the application of 200 μL of 0.3% DNCB to the ears from day 7 up to day 49 every other day (Fig. 1A). JTE-013 (200 μg/20 μL for each ear) was topically applied to the ears from day 20 up to day 49 every other day, which is an alternative to DNCB treatment days. Symptoms of DNCB-induced atopic dermatitis, such as edema, erythema, and cracking of the skin, were observed in the exposed ears (Fig. 1B). Topical JTE-013 treatment reduced these symptoms as shown in Fig. 1B. In addition, H&E staining confirmed that DNCB induced extensive hypertrophy of epidermis due to hyperkeratosis, as well as infiltration of immune cells was increased in DNCB-treated group compared to the control group (Fig. 1C). Topical JTE-013 treatment significantly reduced the DNCB-induced hypertrophy of the epidermis, as well as DNCB-induced immune cell infiltration to the ears (Fig. 1C).
TBO staining was employed to confirm increased infiltration of mast cells into the dermis (Fig. 2A). Mast cells were shown as small, red-purple dots (Fig. 2A). Not only increased infiltration of mast cells, but also hypertrophy, was observed in the DNCB-treated atopic group (Fig. 2A). In sections from mice treated with DNCB plus topical JTE-013, however, fewer stained mast cells were observed than in sections from mice treated only with DNCB (Fig. 2A). The numbers of mast cells were counted semi-quantitatively. As shown in Fig. 2B, the number of mast cells in the dermis was increased by 628% in the DNCB group than in the control group, while topical JTE-013 treatment significantly reduced DNCB-induced increase of mast cell infiltration into the dermis by 55% (Fig. 2B).
Serum IgE levels were measured to determine the immunological effects of DNCB and topical JTE-013 treatment. Hyper-production of IgE was observed in the sera of DNCB-treated mice, while topical JTE-013 treatment significantly reduced the elevated serum IgE level (Fig. 3). The IgE level in DNCB-treated mice was increased by 2750% compared to the control mice, while topical JTE-013 treatment significantly reduced the DNCB-induced increase of IgE level by 65% (Fig. 3).
Lymph node sizes were evaluated in drained lymph nodes. Lymph nodes are important components of the lymphatic system, and are filled with B cells and T cells. When an infection or an allergic reaction occurs, lymph nodes undergo swelling. The lymph nodes from DNCB-treated mice were swollen compared to those from the control mice, and lymph node weight was increased by 600% (Fig. 4). Topical JTE-013 treatment significantly reduced the DNCB-induced increase in lymph node weight by 32% (Fig. 4).
Atopic dermatitis is believed to be regulated not only by Th2 but also by Th17 and Th1 responses (Koga
Based on the pro-allergic functions of S1P2 in allergic asthma, topical JTE-013 treatment in this study was investigated in the murine DNCB-induced atopic dermatitis model. Topical treatment of JTE-013 reduced not only atopic responses in the skin but also immune responses in the lymph nodes and serum. This study shows a therapeutic possibility of JTE-013 S1P2 antagonist. Because topical JTE-013 treatment was undertaken after the induction of atopic dermatitis, JTE-013 or S1P2 antagonists have strong therapeutic significance. Furthermore, topical application could avoid the possible side effects of systemic JTE-013 administration. In addition, this study supports the previous finding of pro-allergic functions of S1P2 (Park and Im, 2019a) and also pro-inflammatory functions of S1P2 (Park and Im, 2019b). Therefore, this finding proves a new direction for S1P2 receptor therapeutics.
Previously, the topical administration of S1P diminished imiquimod-induced psoriasis symptoms, such as, ear swelling and epidermal thickness in the ear (Schaper
This research was supported by the Basic Science Research Program of the Korean National Research Foundation funded by the Korean Ministry of Education, Science and Technology (NRF-2019R1A2C1005523).
The authors declare that there is no conflict of interest.