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A novel S1P1 modulator IMMH002 ameliorates psoriasis in multiple animal models

Psoriasis is characterized by abnormal proliferation of keratinocytes, as well as infiltration of immune cells into the dermis and epidermis, causing itchy, scaly and erythematous plaques of skin. The understanding of this chronic inflammatory skin disease remains unclear and all available treatments have their limitations currently. Here, we showed that IMMH002, a novel orally active S1P1 modulator, desensitized peripheral pathogenic lymphocytes to egress signal from secondary lymphoid organs and thymus. Using different psoriasis animal models, we demonstrated that IMMH002 could significantly relieve skin damage as revealed by PASI score and pathological injure evaluation. Mechanistically, IMMH002 regulated CD3+ T lymphocytes re-distribution by inducing lymphocytes’ homing, thus decreased T lymphocytes allocation in the peripheral blood and skin but increased in the thymus. Our results suggest that the novel S1P1 agonist, IMMH002, exert extraordinary capacity to rapidly modulate T lymphocytes distribution, representing a promising drug candidate for psoriasis treatment.


Introduction
Psoriasis is a chronic immune system disorder with an approximately 2%e3% prevalence worldwide. The pathophysiology of psoriasis is characterized by abnormal proliferation of keratinocytes and infiltration of immune cells into the dermis and epidermis, inducing itchy, scaly, erythematous plaques of skin 1 . Various comorbidities, including psoriatic arthritis, cardiovascular and psychiatric complications, have been associated with psoriasis, which severely impact the quality of life 2 . Currently, most of clinical available drugs against psoriasis can only relieve the symptoms with different limitations or side effects, thus development of new generation of anti-psoriasis drugs with sustained efficacy and improved safety is utmost demanded 3e5 .
Sphingosine-1-phosphate receptor subtype 1 (S1P 1 ) is the most well-defined subtype among five sphingosine-1-phosphate receptors (sphingosine-1-phosphate receptor subtypes 1e5, S1P 1e5 ) 6,7 . It has been widely recognized that S1P 1 is essential for lymphocyte recirculation and responsible for lymphocyte egress from secondary lymphatic organ (SLO) and thymus. Deletion of S1P 1 in mouse hematopoietic cells results in absence of T cells in the periphery blood as the essential signal in regulating mature T cells to exit from SLO and thymus is missing 6,8,9 . Since S1P 1 is indispensable for lymphocyte trafficking and immune system regulation, it has been actively studied as a promising therapeutic target for autoimmune diseases which are usually correlated with lymphocyte dysfunction and infiltration.
Till now, the molecular mechanisms underlying psoriasis progression are still unclear despite recent evidences highlighted that the infiltration of lymphocytes, particularly Th17 cells, as well as elevated cytokines, triggered epidermal hyperplasia, and then induce psoriatic phenotype 10e12 . It is widely recognized that inhibiting the accumulation of pathogenic lymphocytes in the circulation and infiltration into the skin legion by targeting S1P 1 represents an emerging therapeutic avenue for psoriasis treatment. For example, fingolimod (FTY720/Gilenya, Novartis), a multifunctional S1P receptors modulator (S1P 1 , S1P 3 , S1P 4 and S1P 5 ), has been proved effective in many animal models of autoimmune diseases, such as experimental autoimmune encephalitis, rheumatoid arthritis, and systemic lupus erythematosus 13e15 . It was approved by the U.S. Food and Drug Administration (FDA) for treating relapsing-remitting multiple sclerosis (RMS) since 2010 16 . Another similar compound, ponesimod, has successfully completed phaseIIclinical trials for the treatment of moderate-to-severe chronic plaque psoriasis and showed great efficacy, safety and tolerability for psoriasis patients 17,18 . As a more specific S1P receptor modulator, Syl930 alleviates inflammation in sodium dodecyl sulfate (SDS)-induced mouse skin irritation 19 .
A promising drug candidate of S1P 1 agonist with high efficacy and good safety, IMMH002, was previously reported in our group 20 . IMMH002 retained the key polar head group of FTY720 with inserted aromatic substituent in order to increase its structural rigidity, thus increasing the selectivity to S1P 1 receptor. It is a novel S1P 1 modulator which can be further converted to its active phosphorylated form (IMMH002-P) in vivo, and exhibits improved receptor selectivity and optimized pharmacokinetic properties compared to FTY720 21 . In this study, IMMH002 was demonstrated to induce significant S1P 1 receptor internalization and a rapidly reversible reduction of periphery blood lymphocytes (PBLs) in rats without any side effects such as bradycardia, a complication usually occurred in preclinical and clinical studies of FTY720. Furthermore, IMMH002 were shown relieving SDSinduced mouse skin irritation and alleviating propranololinduced damage in a guinea pig model of psoriasis. Moreover, IMMH002 effectively decreased T-lymphocyte infiltration in skin legion in the imiquimod (IMQ)-induced mouse psoriasis model.

Animals
Age matched, 6e8 weeks old male SpragueeDawley (SD) rats, female/male Kunming (KM) mice and male BALB/c mice were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Female Hartley guinea pigs (4e6 weeks old) were obtained from the National Institute for Food and Drug Control (Beijing, China). All rodents used in the study were housed under standardized light-and temperature-controlled conditions with free access to food and water. All experiments were approved by the ethical committee and carried out in accordance with the guidelines of the Committee on Animals of the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China).

Agents
IMMH002, IMMH002-P, FTY720 and FTY720-P were synthesized as previously described methods 22 . Methotrexate (MTX) was obtained from Shanghai XinYi Parmaceutical Co., Ltd. (Shanghai, China). All the compounds were dissolved in dimethyl sulfoxide (DMSO) for in vitro analysis. For in vivo studies, IMMH002, FTY720 and MTX were dissolved in distilled water and given orally. Control animals received the vehicle only.

b-Arrestin assay
The b-arrestin assay was performed with DiscoveRx using enzyme fragment complementation with b-galactosidase as the functional reporter. Briefly, PathHunter CHO-K1-EDG1(S1P 1 ) cells, EDG3(S1P 3 ) cells, EDG5(S1P 2 ) cells, EDG6(S1P 4 ) cells and EDG8(S1P 5 ) cells were separately seeded in a total volume of 20 mL into white walled, 384-well microplates and incubated at 37 C overnight. Five mL of 5 Â samples with different concentrations of compounds were added and incubated at 37 C for 2 or 4 h. Assay signal was generated by a single addition of 12.5 mL of PathHunter detection reagent cocktail, followed by 1 h of incubation at room temperature. Microplates were then read with EnVision (PerkinElmer Company, Shelton, CT, USA) instrument for chemiluminescent signal detection and compound activity was determined using the CBIS data analysis suite (Chem Innovation, San Diego, CA, USA).

HTRF-IP1 assay
The homogeneous time resolved fluorescence-IP one (HTRF-IP1) assay was performed by IP-One Tb kit purchased from Cisbio (France). According to the manufacturer's protocols, hS1P 1 -CHO cells and hS1P 3 -CHO cells were prepared in 1 Â stimulation buffer (containing LiCl) and distributed in 7 mL of suspension with a density of 7 Â 10 4 cells/well in 384-well plates. Then, 7 mL test compounds in different concentrations were added into the assay plate and incubated at 37 C and 5% CO 2 for 2 h. Meanwhile, a standard curve was generated according to the manufacture's instruction. Next, 3 mL IP1-d2 diluted by 1 Â lysis buffer was added into each well followed by 3 mL diluted Ab-Cryp. The plates were incubated at room temperature for 1 h and then recorded with EnVision (PerkinElmer) reader for the detection of absorbance at 665 and 615 nm. The ratio of absorbance 665 nm / absorbance 615 nm (A 665 /A 615 ) emissions was then calculated. A standard curve was plotted of ratio (A 665 /A 615 ) versus IP-1 concentrations using non-linear least squares fit. The values were calculated with the standard curve and presented as concentration (nmol/L) of IP-1. The A 665 /A 615 value of tested compounds should fall into the linear portion of the standard curve. EC 50 values were calculated by Graphpad software 5.0 (GraphPad Software Inc, La Jolla, CA, USA).

Flow cytometry analysis
For the S1P 1 internalization assay, hS1P 1 -CHO cells were incubated for 1 h with 10, 100 and 400 nmol/L FTY720-P and IMMH002-P at 37 C. Then, cells were washed with ice-cold phosphate-buffered saline and blocked with human IgG for 15 min at room temperature followed by incubation with either 25 mg/mL Phycoerythrin (PE)-conjugated mouse anti-human EDG1/S1P 1 IgG2B (R&D systems, Minneapolis, MN, USA) or with PE-conjugated isotype control mouse IgG2B (R&D systems) for 1 h at room temperature. Cells were washed and subjected to flow cytometry measurements using 10,000 viable cells per sample. The percentage of internalization of S1P 1 (%) was calculated as Eq. (1): Percentage of internalization of S1P 1 (%)Z(Fluorescent intensity of solvent controleFluorescent intensity of compounds)/(Fluorescent intensity of solvent controleFluorescent intensity of isotype control) Â 100 (1) For the in vivo assay, single-cell suspensions of spleen and thymus, as well as peripheral blood, were prepared as previously described 23 . Single-cell suspensions were blocked with nonspecific Fc binding with anti-mouse CD16/32 (clone 2.4G2, eBiosciences, San Diego, CA, USA) and labeled with anti-CD3 for T cells; anti-CD11c, anti-CD11b for conventional dendritic cells; anti-CD11c, anti-CD11b and anti-IA/IE for mature conventional dendritic cells; anti-CD11c, anti-CD45R for plasmacytoid dendritic cells; anti-CD11c, anti-CD45R and anti-IA/IE for mature plasmacytoid dendritic cells. Data were acquired on a FACS Calibur cytometer (Becton Dickinson, East Rutherford, NJ, USA) and analyzed with FlowJo software 9.0 (TreeStar Inc., Ashland, OR, USA).

Peripheral blood lymphocytes (PBLs) determination after drug treatment
The rats were distributed randomly. Twenty mL blood samples were collected from the tail vein and measured by the MEK-7222K type blood cell analyzer (Nihon Kohden, Japan) with the baseline PBLs level prior to administration (0 h). Then, rats were administrated orally with compounds (0.3, 1, 3 mg/kg IMMH002, and 3 mg/kg FTY720) and vehicle, respectively. The PBLs levels were monitored at 2, 4, 8, 12, 24, 72, 96, 144 and 168 h post administration (n Z 5).

Heart rate measurement
The basic heart rates were measured using the intelligent noninvasive blood pressure measurement meter-mice instrument (Softron, Japan). The rats were then administrated with 10 mg/kg IMMH002, 10 mg/kg FTY720 and vehicle, respectively. The heart rates were measured at 2, 4, 8, 12, 24 and 48 h post administration (n Z 5).

SDS-induced acute skin irritation and inflammation mouse model
A total of 88 female KM mice were involved in this study. The back hair was removed and 200 mL 15% SDS solution were added onto the hairless area, once a day for 7 days. On Day 8, mice were orally administered with vehicle, 0.3, 0.6, 1.2 and 2.4 mg/kg IMMH002, 1 mg/kg FTY720 or 1.5 mg/kg MTX for 10 days. The mice in control group received vehicle only. On Day 18, all mice were sacrificed and the treated area of back skin was dissected to assess the thickness using a tape measure and fixed in 4% paraformaldehyde followed by histological analysis (n Z 11).

Propranolol-induced guinea pig psoriasis like skin lesion
The auricle dorsal surfaces of guinea pigs (female, 250e400 g) were treated with 5% propranolol in emulsifying ointment, twice per day for 3 weeks. The treated animals were then administered with vehicle, IMMH002 (0.3, 0.6, 1.2 and 2.4 mg/kg) or FTY720 (1 mg/kg) once a day and 1 mg/kg MTX twice a week for 2 weeks. The control animals were challenged with emulsifying ointment, twice per day for 3 weeks and received vehicle once a day for 2 weeks. Ear specimens of animals were randomly obtained at the end of the experiment, fixed in 4% paraformaldehyde, and embedded in paraffin. Five mm thick sections were prepared and followed by hematoxylin/eosin analysis. The epidermal thickness was measured (n Z 8) by Image-Pro Plus 6.0 (Media Cybernetics, Buckinghamshire, UK).

IMQ-induced mouse psoriasis-like disease model
The mouse model of IMQ-induced psoriasis-like lesions was established as previously reported. A total of 80 female BALB/c mice were used in this study. Except for 10 mice in control group, the left 70 mice were treated with 50 mg of Aldara (5% IMQ cream; 3M Pharmaceuticals, Saint Paul, MN, USA) for 7 consecutive days in a daily fashion (Days 0e6) on the shaved back skin. One hour before IMQ treatment, mice were orally administered with vehicle, 0.3, 0.6, 1.2 and 2.4 mg/kg IMMH002, 1 mg/kg FTY720 or 1.5 mg/kg MTX from Day 1 to Day 6, except on Day 0. To evaluate the severity of inflammation of the back skin, a psoriasis area and severity index (PASI) was analyzed from Day 1 to Day 6. Mice were assessed individually by evaluating erythema, scaling and thickening on a scale from 0 (no alteration) to 4 (very distinct alteration), respectively. These three individual assessments were added to generate accumulative score from 0 to 12.24 h after the last IMQ treatment, mice were sacrificed by cervical dislocation and back skin samples were isolated and fixed in 4% paraformaldehyde followed by histological assessment (n Z 10).
The immunohistochemistry staining was carried out as previously described 24 . Briefly, sections were de-paraffinized and dehydrated followed by antigen retrieval using hot citrate buffer. Sections were then blocked in 5% normal horse serum and 1% normal goat serum, and then incubated with the anti-proliferating cell nuclear antigen (PCNA) antibody (1:100, Bioss, Woburn, MA, USA) overnight. The ABC Elite Kit and the DAB Kit (Vector, Burlingame, CA, USA) were used to visualize PCNApositive cells according to the manufacturer's instructions. After immune-peroxidase labeling, the expression of PCNA was assessed by calculating the portion of PCNA-positive cells among every 200 random cells. The images were acquired with an Olympus BX43 microscope and the analysis was performed using Image-Pro Plus 7.2 AMS software (Media Cybernetics Inc., Buckinghamshire, UK).

Western blot analysis
The skin tissues of mice were lysised with radio immunoprecipitation assay (RIPA) lysis buffer in addition to protein degradation inhibitors. Equal amounts of total protein, determined by BCA protein quantitative reagent (CWBIO), were separated by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDSePAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes. Membranes were first blocked with 5% skimmed milk at room temperature for 2 h then incubated with the anti-CD3 antibody (1:100, Abcam, Cambridge, UK) at 4 C overnight. Membranes were detected with anti-rabbit IgGeHRP in a standard ECL reaction (Image Quant LAS 4000 mini, GE, Boston, MA, USA) according to the manufacturer's instructions. Densitometry analysis of the band intensity was carried out using Image J software (NIH, Bethesda, MD, USA).

Targeted protein-chip assay
The targeted protein chip assay was carried out by RayBio R (Beijing, China). In brief, mice skin samples were isolated and lysed by tissue lysate to get the protein samples. Meanwhile, the multi-protein-chips were pretreated by blocking with 30 mL blocking solution/well at room temperature for 1 h. Next, the prepared samples were incubated in different wells for 45 min followed by incubation with detection antibody for 1 h at room temperature. Subsequently, diluted streptavidin-Cy3 was added and incubated for 1 h at room temperature. After several washes, the protein-chip images were detected by LuxScanTM 10 K Microarray Scanner.

ELISA assay
Fifty mg back skin tissue of each mouse was isolated and lysed (10 mg tissue per 100 mL tissue lysate, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 4 C for 30 min. The obtained protein solutions were quantified by BCA protein quantitative kit (Beijing Lablead Biotech Co., Ltd., Beijing, China). Subsequently, the contents of IL-17A of each sample were detected by IL-17A ELISA kit (Neobioscience, Shenzhen, China) according to the manufacturer's protocols. Briefly, 100 mL sample was added to the reaction well and incubated for 90 min at 37 C. The plate was washed for 5 times and biotinylated antibody working solution was added to the reaction well and incubated for 1 h at 37 C. Then, the plate was washed for 5 times and 100 mL enzyme conjugate working solution was added to the reaction well, and incubated for 30 min at 37 C in the dark. One hundred mL of chromogenic substrate was added to each well and incubated for 15 min at 37 C in the dark. Finally, 100 mL of reaction stop solution was added to each well and detected at OD 450 by microplate reader (Synergy H1, BioTek, Winooski, VT, USA) immediately.

Statistical analysis
All values were presented as mean AE standard error. Analysis was performed using unpaired t-test (for 2 group analysis) or one-way ANOVA test (for analysis of 3 or more groups).

IMMH002-P activates S1P 1/4/5 in vitro
Like FTY720, IMMH002 can be transformed to IMMH002-P in vivo. The structure of IMMH002 and IMMH002-P are shown in Fig. 1A. The effect of IMMH002 and IMMH002-P on 5 different S1P receptors was first tested using b-arrestin assay. As shown in Table 1, S1P 1 , S1P 4 , and S1P 5 were significantly activated by IMMH002-P with EC 50 of 12.4, 19.8 and 29.4 nmol/L, respectively. However, IMMH002-P only showed mild activation effect on S1P 2 and S1P 3 with EC 50 more than 5000 nmol/L. The prodrug IMMH002 also showed weak activity on S1P receptors.

IMMH002-P induces activation and internalization of S1P 1
In order to further investigate the agonistic activity of IMMH002-P on S1P 1 , both HTRF-IP1 assay and flow cytometry analysis were performed. As previously reported 25 , the HTRF-IP1 assay was an ideal cellular evaluation system to analyze the S1P 1 receptor's activation using a GPCR coupled Gq signal transduction. As shown in Fig. 1B, in line with the result of b-arrestin assay, S1P 1 was fully activated by IMMH002-P with an EC 50 of 2.2 nmol/L, which was similar to FTY720-P (EC 50 Z 4.4 nmol/L). Currently, most reported S1P 1 agonists are functional inhibitors which decrease the expression of membrane S1P 1 by inducing S1P 1 internalization 16,26,27 . Therefore, we incubated human S1P 1 receptors overexpressed hS1P 1 -CHO cells with IMMH002-P and determined surface receptor expression levels by flow cytometry analysis. Our data showed that 34%, 53% and 59% of total S1P 1 receptors were internalized after 1 h incubation of 10, 100 and 500 nmol/L IMMH002-P, respectively (Fig. 1C). Similarly, FTY720-P induced 28%, 73% and 78% internalization of S1P 1 receptors at same concentrations, indicating the capacity and mechanism to activate S1P 1 of IMMH002-P was comparable to FTY720-P.

3.3.
A single oral administration of IMMH002 dramatically decreases the number of peripheral blood lymphocytes in SD rats S1P 1 agonists, such as FYT720, KRP203 and SEW2871, can significantly inhibit S1P 1 -dependent lymphocyte egress from SLO and decrease PBLs, which contribute to their therapeutic effects against many autoimmune diseases 28 . To investigate the effect of IMMH002 on the lymphocytes' circulation, we examined PBLs of SD rats at different time points with a single oral administration of different concentrations IMMH002. PBLs of SD rats decreased rapidly followed by IMMH002 administration and reached the minimum level 12 h after the treatment (Fig. 2), which was similar to FTY720. However, although FTY720 showed a similar lymphopenia effect as IMMH002, the decreased PBLs induced by FTY720 lasted for a much longer period; as one administration of FTY720 could still reduce 26% lymphocyte 1 week later. Moreover, the maximal inhibition ratio of PBLs in SD rats treated with  0.3, 1 and 3 mg/kg IMMH002 was 25%, 50% and 71% respectively, suggesting a good dose-dependent response. Importantly, the maximal capacity of IMMH002 to decrease PBLs was very close to FTY720 at the same condition ( Fig. 2A).

3.4.
A single oral administration of IMMH002 has no effect on heart rate of SD rats Bradycardia is one of the most serious side effects induced by FTY720. To explore whether IMMH002 displayed such similar side effect, the heart rate of SD rats was monitored at 0, 2, 4, 8, 12 and 24 h after a single dose of 10 mg/kg IMMH002. Intriguingly, the heart rate of rats was not affected by 10 mg/kg IMMH002 treatment (Fig. 2B). In contrast, the heart rate of rats administrated with equal dose of FTY720 decreased 22% as early as 2 h after treatment and lasted for 12 h. In conclusion, IMMH002 had no significant effect on heart rate as displayed by FTY720 in SD rats.

IMMH002 displays significant therapeutic effect on the SDS-induced skin irritation in mice
SDS-induced mouse skin irritation model is a well-established skin irritation model. When the skin is exposed to the aqueous solution of the surfactant SDS, it penetrates into the skin and disrupts this barrier. We therefore used this model to assess whether IMMH002 had therapeutic effect on skin irritation. Normal control KM mice received vehicle treatment (normal control), while animals challenged with 15% SDS for 7 days received vehicle (skin irritation control), 1 mg/kg FTY720, 1.5 mg/kg MTX, and different concentrations of IMMH002 treatment once a day from Day 8 to Day 18. On Day 18, mean skin thickness was determined and pathological assessment was performed. All the treatment groups showed significant improvement compared to the irritation control treated with vehicle. IMMH002 remarkably decreased the skin thickness of mice exposed to SDS and was slightly more effective than FTY720 or MTX (Fig. 3). Importantly, only mice treated with IMMH002 (0.6, 1.2 and 2.4 mg/kg) improved the pathological status despite all compounds ameliorated the skin thickness, indicating that IMMH002 had a potent inhibitory effect on skin inflammation. As the first-line drug used for psoriasis treatment in the clinic, MTX efficiently decreased the thickness of irritated skin, but the body weight started to decline from Day 10, similar effect was also observed in thymus and spleen, which was considered as the adverse effect of MTX treatment (data not shown). Surprisingly, there was no such effect of IMMH002 on the mice body weight, suggesting a safer profile of IMMH002 than MTX.

IMMH002 has therapeutic effect on psoriasis form skin of guinea pig
We next generated a guinea pig model of psoriasiform skin lesions to evaluate the effect of IMMH002 in a psoriasis model more mimic the human condition. Briefly, a period of 3-week 5% propranolol challenge generated the psoriasiform pathological changes on the ears of guinea pigs. Animals were then orally administered with 0.3, 0.6, 1.2 and 2.4 mg/kg IMMH002, 1.5 mg/kg MTX and 1 mg/kg FTY720 once a day for 2 weeks. Normal control and psoriasis model groups received vehicle treatment only. Intriguingly, IMMH002 attenuated the psoriasiform epidermal changes by decreasing acanthosis, hyperkeratosis, dermal inflammatory infiltration and basilar papilla as well as the thickness of epidermal (Fig. 4A, C, and D). Compared to the psoriasis model group, significant improvements were only observed in the 1.2 or 2.4 mg/kg IMMH002, FTY720 and MTX groups. Moreover, IMMH002 could also decrease epidermal PCNA in a dose dependent manner ( Fig. 4B and E). Collectively, IMMH002 has a potent therapeutic effect on both skin irritation in mice and psoriasiform skin lesions in guinea pigs with great advantages compared to FTY720 or MTX, indicating IMMH002 represents a potential candidate for the treatment of inflammatory skin disease and psoriasis.

IMMH002 relieves skin damage in IMQ-treated mice
IMQ is a toll-like receptor (TLR) 7/8 agonist which is used for the treatment of actinic keratosis and superficial basal cell carcinoma 29 . However, it also provokes psoriasis-like flares in predisposed patients as an adverse effect. In line with previous reports, we observed that topical application of IMQ on the skin of BALB/c mice induced inflammation with similar features found in psoriatic skin. The PASI score system was applied to evaluate the skin damage, including skin erythema, scaling and thickening. In IMQ-treated mice (IMQ model group), the erythema became detectable on the 3rd day after IMQ treatment (indicated as Day 2 after drug treatment in Fig. 5A). The erythema symptoms developed more severely between Day 3 and Day 5, and then started to recover automatically. The S1P 1 /S1P 3 /S1P 4 /S1P 5 agonist FTY720 relieved the erythema only on Day 2 and Day 5, which was similar to MTX. Of note, the more selective S1P 1 agonist, IMMH002 (S1P 1 /S1P 4 /S1P 5 agonist), rescued the erythema from Day 2 till the end, especially at the doses of 1.2 and 2.4 mg/kg. The therapeutic effect of IMMH002 on erythema appeared to be superior compared with both FTY720 and MTX. Interestingly, all the treatments could ameliorate the early stage of scaling and skin thickening similarly. As revealed by PASI score analysis, all compounds showed their capacity to improve IMQ-induced skin damage to some extent, while IMMH002 exhibited slight advantages compared to FTY720 and MTX (Fig. 5A).

IMMH002 rescues the IMQ-induced pathological skin damage
IMQ treatment induces different significant pathological change in the skin. For keratose, it causes microabscess, hyperkeratosis and parakeratosis. For epidermis, it mainly induces acanthosis and basilar papilla. And for dermis, inflammatory cells infiltrations, vasodilatation, and thinning above papillae are all observed after IMQ challenge. Treatment of IMMH002 at higher doses (1.2 and 2.4 mg/kg) substantially decreased the pathological damage of all skin layers, and the therapeutic effects were much significant than MTX and FTY720. Acanthosis is the major damage found in psoriasis, so we measured the thickness of epidermis. Our results suggested that all these compounds could relieve the thickening of epidermis, particularly IMMH002 ( Fig. 5B and C).

IMMH002 decreases T lymphocyte numbers in both the circulating system and psoriasis region
In order to check whether IMMH002 affected lymphocyte homing in IMQ-treated mice, flow cytometry was performed to investigate T cell numbers in the blood, spleen and thymus. The expression of the T cell marker CD3 in skin was also determined by Western blot. In contrast to the IMQ-treated model group, CD3 þ T cells were markedly decreased in the blood and spleen, but highly increased in the thymus after IMMH002 treatment, further supporting the essential role of S1P 1 in lymphocyte homing ( Fig. 6A  and B). Moreover, IMQ challenge increased the expression of CD3 protein levels in skin legions, whereas IMMH002 could significantly decrease this effect ( Fig. 6C and D). Furthermore, protein chip analysis showed that IMMH002 suppressed the expression of the pro-inflammatory cytokines, such as IL17A and IL12p40/p70 in IMQ-dependent psoriasis skin legions (Fig. 7), which might further explain its therapeutic effect in this psoriasis model.

Discussion
Psoriasis is a chronic immune system disorder with cutaneous and systemic manifestations, which substantially impairs patients' life qualify. Similar to other autoimmune diseases, lymphocyte infiltration and activation is involved in psoriasis development 30,31 . S1P 1 modulators show great beneficial effects on many autoimmune diseases by inducing lymphopenia and preventing tissue inflammation, leading to the development of a new class of targeted therapies for psoriasis. For instance, ponesimod, a selective agonist on S1P 1 /S1P 4 /S1P 5 , has successfully entered a phase-II clinical trial for the treatment of moderate-to-severe chronic plaque psoriasis. It turned out to be of great efficacy, safety and tolerability for psoriasis patients 17 . IMMH002 was first synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (IMM, CAMS & PUMC, Beijing, China) and the previous study revealed that structural rigidity of the lipophilic chain was required to increase S1P 1 agonistic potency and subtype selectivity. Therefore, the chemical structure of IMMH002 was modified according to FTY720 in order to avoid undesired S1P3 activity. The b-arrestin assay indicated that the active form of IMMH002, IMMH002-P, had great agonist activity on S1P 1 , S1P 4 and S1P 5 , but not S1P 2 or S1P 3 . This specificity excluded its potential side effects such as reduction of heart rate and an increase of blood pressure as reported with FTY720 32 . Moreover, modulation of S1P 2 and S1P 3 on myofibroblasts by FTY720 also triggered extracellular matrix synthesis, which might contribute to its adverse side effects in the lung 33e36 . Thus, with higher selectivity, as proved by the in vivo studies, IMMH002 might induce less adverse effects than FTY720.
We then investigated the downstream action after S1P 1 activation by IMMH002-P. Flow cytometry analysis showed that IMMH002-P decreased the expression of S1P 1 on hS1P 1 -CHO cell membrane. Therefore, after binding and activating S1P 1 , IMMH002-P caused a temporary S1P 1 -deficient state, which might render lymphocytes insensitive to the S1P signal necessary for egress from SLO and the thymus. These results were consistent with the mechanisms of other reported S1P 1 agonists, such as FTY720 and BAF312 37,38 . In addition, the effect of IMMH002-P on the polyubiquitination and degradation of S1P 1 receptors was tested. Interestingly, in contrast to FTY720-P, IMMH002-P did not induce degradation of S1P 1 receptors, suggesting different mechanisms to modulate S1P 1 turnover between these two compounds (data not shown).
Driven by the exciting in vitro data, we investigated the effect of IMMH002 on PBLs. In SD rats, IMMH002 administration resulted in significant lymphopenia in a dose-dependent manner. The maximal lymphopenia efficacy of IMMH002 was similar to FTY720, and both of their maximal efficacies were observed at 12 h post-administration. However, IMMH002-treated rats exhibited a quicker recovery of PBLs suppression, whereas the immunosuppressive effects lasted much longer in the FTY720treated group. This might be partially explained due to the relatively shorter half-life (t 1/2 ) of IMMH002-P (about 11 h) compared to FTY720-P (25 h). The relatively short t 1/2 of IMMH002 might allow a rapid recovery of blood lymphocyte counts upon deprivation of the treatment and would require daily oral administration in clinical.
The influence of IMMH002 on heart rate in rats was also investigated since bradycardia was one of the most severe adverse effects observed in clinic induced by FTY720 treatment. Our data suggested that single oral administration of SD rats with 10 mg/kg IMMH002 did not affect the heart rate but single oral administration with equal dose of FTY720 indeed caused an immediate 20% heart rate decrease which lasted for 12 h. Previously, studies in rodents revealed that the activation of S1P 3 on cardiac myocytes by FTY720 was associated to the reduction of heart rate by stimulating G-protein-coupled inwardly rectifying potassium channels (GIRK), a key player that regulated pacemaker frequency 39 . Intriguingly, Hamada et al. 40 Reported that simple removal of the S1P 3 agonist was not sufficient to avoid such cardiovascular side effect in rats. Furthermore, Fryer et al. 41 Suggested that in rats, bradycardia was caused by S1P 1 receptor agonist while the increased arterial blood pressure was triggered by S1P 3 activation. Importantly, bradycardia in humans was also observed in the recent clinical trials of the S1P 1 /S1P 5 dual agonist, BAF312. Therefore, whether IMMH002 would cause bradycardia in humans remains unclear. Nevertheless, sparing the S1P 3 activation has been still actively proposed as a practical approach to avoid potential side effects. Several more selective S1P 1 agonists, such as KRP203, BAF312, CS-0777, ACT128800, ONO-4641, RPC-1063 and MT-1303, have been tested in clinical trials for the treatment of different autoimmune diseases 26,27,42e45 .
The etiology of psoriasis is closely correlated with lymphocyte dysregulation. Inspired by the results of ponesimod for psoriasis therapy, the effect of IMMH002 was tested on psoriasis in different animal models.
Skin inflammation is one of the most common and basic characteristics of psoriasis. In order to assess whether IMMH002 could relieve skin inflammation, the SDS-induced skin irritation model was employed. Our data indicate that IMMH002 decreased the pathological score in a dose-dependent manner with a corresponding reduction in thickness of irritated skin. Although FTY720 and MTX decreased the pathological damage to some extent, it seemed their effect was not as significant as IMMH002. We then used propranolol-induced psoriasiform skin lesions in guinea pig to evaluate the therapeutic effect of IMMH002 on psoriasis. This model mimics the characteristics of epidermal damage in human psoriasis, especially hyperkeratosis, parakeratosis and acanthosis. In addition to the improvement of pathological damage in this model, IMMH002 also decreased the percentage of PCNA in the epidermis, which was closely associated with a reduction of pathological acanthosis.
Consistently, we found that topical application of IMQ on the skin of BALB/c mice induced inflammation with similar features found in psoriatic skin, including formation of microabscesses, skin thickening, hyperkeratosis, acanthosis, scaling and erythema. Orally administration of IMMH002 significantly improved these symptoms and dramatically reduced the PASI score. Notably, T cells, especially T helper (Th) cells producing interleukin (IL)-17, IL-22, and tumor necrosis factor (TNF), form the major T cell population that drive psoriasis pathogenesis. They orchestrate the inflammation in the skin which further induces the proliferation of keratinocytes and endothelial cells. In the IMQ-induced psoriasis model, IMMH002 significantly reduced CD3 þ T cells in the skin and blood but increased them in the thymus; furthermore, the key cytokines in psoriasis skin, IL17A and the well-known T cell stimulating factor IL12p40/p70, were also markedly reduced in the IMMH002-treatment group. Previous studies have shown that dendritic cell-induced T cells accumulation is closely related to IMQ-induced mouse psoriasis 46,47 . Thus, dendritic cells (DCs) in the blood were also measured (Supporting Information Fig. S1). There were no significant changes of conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) in the blood, but mature DCs were decreased, which were consistent with the reduction of T cells in blood treated with IMMH002. It is indicated that FTY720 could inhibit migration of DCs and function in many inflammatory diseases 48e50 . Similarly, we also found that IMMH002 reduced blood mature DCs. However, whether it could modulate the migration of DCs requires further study.

Conclusions
Our study demonstrated that with high selectivity against S1P 1, the novel S1P 1 modulator IMMH002 could bind and regulate S1P 1 expressed on the cell membrane. The active form of IMMH002, IMMH002-P, could effectively decrease the expression of S1P 1 by inducing internalization of these receptors, acting as a functional antagonist. MMH002 could remarkably decrease PBLs without obvious adverse effects on heart rate in SD rats and had a good pharmacokinetic profile, especially a relatively short half-life, allowing a quick recover of lymphopenia after the treatment. IMMH002 exhibited significant therapeutic effect on many psoriasis animal models because of its effect on lymphocytes redistribution and infiltration. Based on these above findings, we propose IMMH002 may represent a potential drug candidate for the treatment of psoriasis.