CYSLTR1 antagonist inhibits Th17 cell differentiation by regulating the NF-κB signaling for the treatment of psoriasis

Cysteinyl leukotriene receptor 1 (CYSLTR1) is observed to increase in psoriatic skin lesions. Montelukast, a CYSLTR1 antagonist, effectively treats inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis, and atopic dermatitis. Thus, blocking CYSLTR1 may be a promising strategy for psoriasis immunotherapy. We prepared a montelukast sodium cream and solution and investigated their effects on psoriasis-like skin lesions induced by imiquimod (IMQ). After the treatment, serum, skin, and spleen samples were collected for evaluation. We treated human T helper (Th) 17 cells with montelukast in vitro to study its effect on Th17 differentiation and nuclear factor kappa-B (NF-κB) signaling. We also created a keratinocyte proliferation model induced by M5 cytokines and assessed the influence of montelukast on key psoriasis-related genes. We induced psoriasis in CYSLTR1 knockout (KO) mice using IMQ to explore the role of CYSLTR1 in psoriasis development. Montelukast sodium cream and solution effectively reduced the psoriasis area and severity index (PASI) and alleviated disease symptoms in IMQ-induced mice. Furthermore, reduced infiltration of inflammatory cells (Th1, Th17, and T follicular helper [Tfh] cells), decreased mRNA expression of cytokines in the skin (interleukin [IL]-17/F and IL-23), and lower serum concentrations of various cytokines (IL-2, IL-6, IL-13, and IL-17A/F) were observed. Montelukast cream and solution also decreased spleen size and the proportion of Th17 and Tfh cells, and significantly inhibited NF-κB signaling-related genes after application. Moreover, montelukast inhibited Th17 cell differentiation and suppressed NF-κB signaling in vitro. CYSLTR1 KO mice induced with IMQ showed improvement in PASI scores, serum IL-17A/F levels, and lower Th1 and Th17 cells in the spleen and skin compared to wild-type mice. Montelukast also suppressed the proliferation and inflammatory response of keratinocytes by regulating NF-κB signaling. Collectively, our results strongly indicate that inhibition of CYSLTR1 signaling to target the Th17 response holds significant promise as a therapeutic approach to manage psoriasis.


Preparation of montelukast sodium cream
To formulate montelukast sodium cream, we performed a multifactor orthogonal test to optimize the preparation procedure.We considered several factors, including triethanolamine content (factor A), glyceryl monostearate content (factor B), emulsification temperature (factor C), and emulsification time (factor D), as the variables for investigation.The optimization process involved evaluating various parameters related to cream preparation quality, including appearance, pH, cold resistance, heat resistance, and centrifugal stability (Table S3).Subsequently, we successfully developed an optimized montelukast sodium cream.
The cream formulation comprised two distinct phases, an aqueous and an oil phase.The ingredients in the aqueous phase included triethanolamine (5.2%), glycerol (6.3%), DMSO (3.5%), montelukast sodium (3%), ethyl 4-hydroxybenzoate (0.1%), and purified water.The following constituents were present in the oil phase: octadecanoic acid (10.5%), glyceryl monostearate (5.2%), and vaseline (4.2%).Montelukast sodium was initially dissolved in DMSO under sulfoxide by heating.Subsequently, the montelukast sodium solution was blended with other components of the aqueous phase.Finally, the cream formulation was prepared by adding purified water.
To create an oil-in-water (O/W) emulsion for cream preparation, the oil phase was introduced into the aqueous phase to create an oil-in-water emulsion for cream preparation.Initially, the oil phase components were combined and heated to 80 ℃ until complete melting occurred.Subsequently, the heated oil phase was carefully incorporated into the aqueous phase, maintained at 80 °C.The mixture was continuously stirred for 15 min.Afterward, the mixture was allowed to cool down to 40 °C and continued to be stirred for an additional 15 min to achieve emulsification.
The O/W cream containing 3% montelukast sodium was considered complete after the emulsion was cooled to 25 ℃.The same technique was used to create the vehicle cream without montelukast.
Deionized water was purified using a Milli-Q water purification system (Millipore, Billerica, MA,

In vitro permeation test (IVPT)
Skin samples used for in vitro permeation test (IVPT) were obtained from normal BALB/c mice.On the day of the experiment, the skin (2 cm × 3 cm) was collected and the subcutaneous fat was removed after euthanasia.A LOGAN DHC-6TD transdermal diffusion instrument with an automated fraction collector was used for the IVPT experiments.The volume and permeation area of the diffusion cell were 12 mL and 1.767 cm 2 .The receiver solution was phosphate-buffered saline (pH 7.4), and ultrasound was used for 30 min to remove bubbles.The speed of magnetic stirring was 600 r/min, and the temperature was maintained at 32 ± 0.2 ℃.The receiving cells were stirred, and the pretreated skin was fixed between the diffusion and receiving cells.The prepared montelukast sodium cream was taken and closely applied to the pretreated skin in a diffusion cell.Timing: 6 mL of the receiving solution was collected at time points of 5 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h.Fresh receiving solutions of the same volume were added simultaneously.
The resulting receiver solution samples were analyzed by HPLC.The rate of montelukast sodium permeation through the skin and the cumulative total permeation of montelukast sodium at different sampling time points were chosen as two key parameters to evaluate the skin permeation of montelukast sodium.

HPLC analysis of samples
The solvent of the resulting receiver solution samples was first removed using a Termovap Sample Concentrator, and then the samples were dissolved in methanol (1 mL).After passing it over 0.22 μM microporous filter membrane, the IVPT samples for 20 μl were injected and analyzed using a SHIMADZU LC-2050C HPLC system (Kyoto, Japan).A HPLC method was used to quantify the IVPT samples.BDS Hypersil TM C18 (2.4 μm, 4.6×100 mm) column was used to elute montelukast sodium.The mobile phase comprised 0.1% trifluoroacetic acid (A, 55%) and acetonitrile (B, 45%), with equal elution for 20 min.The flow rate was 1.0 ml/min.Montelukast sodium peak was detected at 220 nm.

Determination of the level of investigation factors in the preparation of montelukast sodium cream
The role of emulsifiers in cream preparation is well established, with triethanolamine and glyceryl monostearate being common choices.Triethanolamine serves as both an emulsifier and an alkalizing agent, contributing to the formation of a uniform and stable cream for topical applications [1].On the other hand, to make the cream base, glyceryl monostearate self-emulsifies and esterifies with stearic acid [2].Consequently, the quantities of triethanolamine and glyceryl monostearate were identified as critical variables in formulating the montelukast sodium cream.Furthermore, the emulsification temperature and duration have been recognized as pivotal factors in the preparation process.
As presented in Table S5, the R values for the four factors followed the order D > C > A > B.
An analysis of the extreme differences revealed that the order of influence of the four factors on the experimental outcomes was D > C > A > B. The values K1', K2', and K3' denoted the average comprehensive scores associated with levels 1, 2, and 3 for each factor, respectively.The highest average comprehensive score for Factor A was 94.30, corresponding to Level 3. Regarding Factor B, the highest average comprehensive score was 94.07, which was attributed to Level 2. For Factor C, the highest average comprehensive score was 94.23 at level 2. Finally, the average comprehensive score for Factor D was 94.33, representing Level 1.In conclusion, the optimal preparation process for montelukast sodium cream was identified as A3B2C2D1, which corresponds to a triethanolamine content of 5.2%, glyceryl monostearate content of 5.2%, an emulsification temperature of 80 ℃, and an emulsification time of 15 min.As shown in Table S6, all four factors significantly influenced the experimental outcomes.Consequently, A3B2C2D1 was established as the optimal protocol for the preparation of montelukast sodium cream.

In vitro permeation test of montelukast sodium cream
As presented in Figure S1A and B, montelukast sodium was detected at 14.742 min.The flux profile delineates the permeation rate of montelukast sodium through the skin at various sampling intervals, including 5 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h (Figure S1C).While the permeation rate of montelukast sodium remained constant at 0 from 5 min to 2 h, it subsequently increased to 38.4524, 66.2457, 184.5740, and 92.3390 ng/cm 2 from 4 h to 10 h, respectively.Figure S1D illustrates the cumulative total permeation of montelukast sodium at different time points, registering 0, 0, 0, 153.8094, 551.2837, 2027.8756, and 2951.2651ng/cm 2 , correspondingly.
These observations suggest that montelukast sodium can permeate the skin following a 2-hour application of montelukast sodium cream.Furthermore, the rate of montelukast sodium permeation through the skin peaks at 8 h before gradually declining.

FiguresFigure S1.
Figures Figure S1.In vitro permeation test of montelukast sodium cream.(A) The chemical structure of montelukast sodium.(B) Representative HPLC chromatograms of the montelukast sodium standard solution and the resulting receiver solution sample.(C) The flux profile showing the rate of montelukast sodium permeating through the skin at different sampling time points, including 5 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h.(D) The cumulative total permeation of montelukast sodium at different sampling time points, including 5 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h.

Figure S3 .
Figure S3.Montelukast's function in regulating mRNA levels of pro-inflammatory cytokines, and genes associated with keratogenesis of keratinocyte, as well as angiogenesis, autoimmunity, and chemokines in the skin lesions of IMQ-induced psoriasis-liked mice.Horizontal bars represent the mean ± SEM. *p < 0.05.

Figure S4 .
Figure S4.The effect of CYSLTR1 knockout on the NF-κB signaling pathway after IMQ application.qPCR screening of NF-κB signaling-related genes in the (A) skin lesions and (B) spleen tissues.(C) Western blotting of NF-κB signaling pathway in the spleens from WT and CYSLTR1 KO mice.Horizontal bars represent the mean ± SEM. *p < 0.05, **p < 0.01.