CB2R Deficiency Exacerbates Imiquimod-Induced Psoriasiform Dermatitis and Itch Through the Neuro-Immune Pathway

Human Sample Collection

Skin sample were obtained through surgical biopsy from Union Hospital, Tongji Medical College of Huazhong University of Science and Technology in Wuhan, China. All patients involved signed informed consent. The research protocols conformed to the declaration of Helsinki Principles and were approved by the medical ethics committee of Tongji Medical College of Huazhong University of Science and Technology.

Animals

All the animal experimental protocols in this study were conducted in accordance with the ARRIVE guidelines and were approved by the Institutional Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China). Male CB2R^−/- mice on C57BL/6 background and C57BL/6 wild-type mice aged 6–8 weeks were purchased from Jackson Laboratories. Experimental groups consisted of five mice each and the studies were repeated three times.

Induction of Psoriasiform Skin Inflammation by Imiquimod

A daily topical dose of 62.5 mg of commercially available imiquimod cream (5%) (Mingxin Pharmaceuticals, Sichuan, China) was applied to the shaved back skin (2.5 cm × 2 cm) of mice for seven consecutive days (days 0–6) (Moos et al., 2019). Control mice were treated similarly with vaseline jelly as the control vehicle cream. Disease severity was assessed by using a scoring system based on the clinical Psoriasis Area and Severity Index (PASI). To be precise, erythema, scaling, and thickening were scored independently on a scale from 0 to 4 (0, none; 1, slight; 2, moderate; 3, marker; 4, very marked), and the cumulative score was used as a total score (scale 0–12). Disease severity was assessed by two researchers in a blinded manner. On day 8, serum samples and skin tissues were taken from the sacrificed mice for subsequent studies.

Behavioral Scratching Testing

The behavioral scratching was recorded by video. Mice were habituated individually to an observation chamber for 30 min before testing for five consecutive days. Twenty to 22 hours after each topical application of IMQ, mice were videotaped from below for 60 min. The number of videotaped scratch bouts was counted by two trained observers blinded to the treatment condition. A scratch bout was defined as one or more rapid back-and-forth hind paw motions directed toward and contacting the treated area or scratched directly at the area around the injection site continuously for any length of time and lasted, ending with licking or biting of the toes or placement of the hind paw on the floor (Sakai et al., 2016). The use of each fore paw was classified as grooming behavior and was not considered scratching.

Histology and Immunohistochemistry

Animals were euthanized under sodium pentobarbital anesthesia, and the skin was acutely dissected on the ice. Skin was fixed in 4% paraformaldehyde, embedded in paraffin and cut in 15-μm sections on a microtome. The sections were partly stained with hematoxylin and eosin (H&E) or toluidine blue, or prepared for rabbit monoclonal anti-CD4 (1:1000, Abcam) for IHC. Means of epidermal thickness, the number of mast cells and the degranulation mast cell from toluidine blue staining were calculated based on five randomly selected fields of view per mouse as previously described (Liu et al., 2017). And stained cells were counted under high original magnification (×200) power fields of a light microscope by ImageJ software. Each section was examined independently by two investigators in a blinded manner.

Immunofluorescence Analysis

Skin was fixed in 4% paraformaldehyde for 24 h and dehydrated with 30% sucrose, frozen in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA), and cut in 15-μm sections on a cryostat. The sections were incubated with 5% donkey serum and then immunofluorescent staining with a rabbit CB2R (1:200, Abcam, Cambrige, United Kingdom), PGP 9.5 (1:500, ABclonal), c-kit (1:500, RD system) at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with Alexa Fluor 594 (1:500; Jackson Laboratory) at 37°C for an hour. All sections were counterstained with 4′,6-diamino-2-phenylindole (DAPI) at room temperature for 5 min. Images were captured from 3-4 skin sections from each animal and were imaged at 20× magnification.

The PGP 9.5-immunoreactive nerve fibers crossing the epidermis from the dermis were quantified by IENFD (expressed as fibers/linear mm) as described previously (Gylfadottir et al., 2022).

Western Blotting

Total proteins samples from the mouse tissues were extracted using lysis buffer for 30 min and the lysates were centrifuged at 12,000 rpm for 15 min at 4°C. The concentrations of proteins in the supernatants were measured using a BCA protein assay kit. Equal amounts of protein were separated by SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. After blocking with 5% skim milk powder for 1 h at room temperature, the membranes were incubated with primary antibodies against CB2R (1:500, ABclonal), NGF (1:500, Affinity) and β-actin (1:20,000, ABclonal) at 4°C overnight. Secondary antibodies were labeled with horseradish peroxidase and incubated for 1 h at room temperature. The antigen-antibody complexes formed were detected by enhanced chemiluminescence in accordance with the manufacturer’s instructions. Band signal intensities of the western blot films were assessed using the ImageJ software. Relative protein expression levels were normalized to that of β-actin (internal control).

RNA Isolation and Quantitative Real-Time PCR

RNA isolation was performed using Trizol reagent (Vazyme Biotech Co. Ltd, Nanjing, China). Conversion of total RNA to cDNA was performed with cDNA Reverse Transcription Kit (Vazyme Biotech Co. Ltd, Nanjing, China). All real-time PCR reactions were performed using the Real Time PCR System and the quantitative reverse-transcription PCR assay was carried out using SYBR Green PCR Master Mix (Vazyme Biotech Co. Ltd, Nanjing, China) on StepOne v2.3 detection system (Life Technologies, CA, United States). Forty cycles of amplification were performed involving sequential denaturation at 95°C for 30 s, annealing at 60°C for 5 s, and extension at 72°C for 30 s. The messenger RNA (mRNA) levels were normalized to those of the β-actin gene. Real-time PCR analysis was performed using the 2^-△△CT method. All primer pairs are listed in Table 1.

Flow Cytometry Analysis

Spleens from the individual mice were freshly isolated and mechanically dissociated. RBCs were lysed with lysis buffer (Biosharp, Beijing Labgic Technology Co. Ltd). Single cell suspensions of spleen were passed through a 40-μm sterile wire screen. Cell suspensions were washed twice in RPMI 1640 (Gibco, ThermoFisher Biochemical Products Co. Ltd) and stored in media containing 10% FBS on ice until used within 2 h. Splenocytes were stimulated with 50 μL phorbol 12-myeistate 13-acetate (PMA) (1 μg/ml), 40 μL ionomycin (50 μg/ml), and 20 μL monensin (0.1 mg/ml) for 5 h at 37°C humidified incubator. For cell surface antigen staining, cells were pre-blocked for Fc receptors (BD, Pharmingen) for 15 min at 4°C. The cells were then washed with staining buffer following by staining with Abs for 1 h at 37°C. The cells were washed with staining buffer, then resuspended in 300 μL of staining buffer. Flow cytometry was carried out using a FACS Canto Ⅱ flow cytometer (BD) and the ratio of Th17 cells and Treg cells were analyzed. All the antibodies information were listed in Supplementary Table S1.

Cytometric Beads Array for Detecting Mouse Th1/Th2/Th17 Cytokines

The content of Th1/Th2/Th17 cytokines including IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17A concentrations was measured by the CBA kits (BD, Pharmingen, United States) according to the manufacturer’s protocols. Data were acquired on FACS Canto-II flow cytometer (BD Bioscience, United States) and analyzed using FCAP Array software (BD Bioscience).

CB2R Deficiency Increased CD4⁺ T Cells Infiltration and Proinflammatory Cytokines Expression in IMQ Mice

In both psoriasis and relevant mouse models, the IL-23/Th17 axis and Th1/Th2/Th17 balance play a major role in disease (Gauld et al., 2019). To appraise the effects of CB2R on IMQ-induced local and systemic inflammation, we used IHC to observe the infiltration of CD4⁺ T cells, which are the main source that induce differentiation into various subtypes of T cells in psoriasis. The numbers of CD4⁺ T cells in IMQ-treated CB2R KO mice were significantly increased compared with those in IMQ-treated WT mice (Figure 2A and Supplementary Figure S2A). We also examined the mRNA expression of key inflammation genes that are known to regulate psoriatic skin inflammation. As shown in Figure 2B, relatively increased mRNA levels of IL-17A, IL-23A, TNF-α, IL-1β, and IL-17C were noted in the psoriasiform skin lesions of CB2R KO mice as compared with WT mice. Although the expression of IL-17F, CXCL-1, and CCL-20 in IMQ-treated CB2R KO PsD mice was higher than that in CB2R KO vehicle mice, there was no significant difference in PsD between CB2R KO mice and WT mice, and CCL-20 mRNA levels were somewhat lower in the two groups. In addition, the CBA assay showed that the levels of Th1 (IL-2, IFN-γ, TNF-α) and Th17 (IL-17A) cell-related cytokines in the serum were markedly increased, whereas the levels of Th2 cell-related cytokines (IL-4, IL-10) were significantly decreased in PsD mice compared with normal mice in both the WT and CB2R KO groups. Interestingly, CB2R deficiency exhibited the greatest impact on the expression of IL-2 in the serum after applying IMQ, given that the changes in several other cytokines were not statistically significant (Figure 2C). These results indicated that CB2R deficiency exacerbated the IMQ-induced skin inflammation by regulating inflammatory cell infiltration and the release of local and systemic inflammatory cytokines.

CB2R Deficiency Promoted the Differentiation Proportion of Th17/Treg Cells Among CD4⁺ T Cells

It is well known that psoriasis may result from massive inflammatory cellular infiltrates and/or an imbalance in T cells, especially in Th17/Treg cells (Shi et al., 2019). Furthermore, it has been reported that CB2R activation may change the balance of Th17/Treg cells in lung tissues (Wei et al., 2021), but it remains unclear whether it plays the same role in psoriasis. To probe the effect of CB2R in regulating the Th17/Treg cell balance on IMQ-induced systemic inflammation, we next performed flow cytometric analysis of splenic single cells. We found that the proportion of Th17/Treg cells was increased in IMQ-induced PsD mice compared with vehicle-induced control mice. However, CB2R deficiency slightly increased the ratio of Th17/Treg cells in the spleen compared to that of WT mice after IMQ application. However, this effect was more obvious in vehicle mice (Figures 3A–C). We also verified the mRNA expression levels of ROR-γt, TGF-β, IL-6, Foxp3 and IL-10, which play a critical role in the differentiation of Th17 and Treg cells, respectively. Importantly, the mRNA levels of Treg-related transcription factors and cytokines, including Foxp3, IL-10, and TGF-β, were significantly decreased in CB2R KO-PsD mice compared with CB2R KO-vehicle mice (Figures 3D–F). In contrast, Th17-related transcription factors and cytokines, including ROR-γt and IL-6, were significantly increased in the two groups (Figures 3G,H). Compared with WT mice, CB2R deficiency had a more obvious effect on IL-10 and ROR-γt expression after applying IMQ. In summary, the experiments demonstrated that CB2R deficiency promoted the proportion of Th17/Treg cells among CD4⁺ T cells.

The CB2R Agonist JWH-133, was Effective Against IMQ-Induced PsD

We further applied the CB2R agonist JWH-133 and the CB2R antagonist AM-630 to observe their effects on PsD. JWH-133, AM-630 or solvent (10% DMSO+20% Tween-80 + 70% saline) was intraperitoneally (i.p.) injected into WT mice during IMQ treatment. We first found that 5 mg/kg was the optimum dose because 1 and 3 mg/kg were not effective. Intraperitoneal treatment with 5 mg/kg JWH-133 improved the PASI score. Because CB2R was mainly located on the epidermis, then we observed that mice received 2.5 mg/kg JWH-133 intracutaneously (i.c.) showed a significant attenuation of psoriasis inflammation compared with those that received an intraperitoneal injection. Thus, an IMQ-induced PsD mouse model and simultaneous intracutaneous injection pretreatment with AM-630 or JWH-133 were conducted to verify the role of CB2R in vivo (Figure 4A). Compared with the IMQ group, JWH-133 visibly alleviated the clinical phenotype of IMQ-induced PsD but did not completely eliminate the macroscopic clinical symptoms, which included redness and thickening of the skin reduction. Histological results showed that JWH-133 significantly improved the histologic changes, which showing less parakeratosis and less inflammatory cell infiltration than the IMQ group. In contrast, AM-630 had the opposite effect (Figure 4B). Similarly, the i.c. JWH-133 group showed lower PASI scores (Figure 4C) and less epidermal thickening (Figure 4D). Consistently, JWH-133 also significantly reduced the mRNA expression levels of proinflammatory cytokines, including IL-17A, IL-23A, TNF-α, IL-1β, CXCL-1 and CCL-20, in skin lesions (Figure 4E). Of note, the expression levels of proinflammatory cytokines also decreased after i.c. administration of AM-630. It is possible that the dose was insufficient or the pharmacological effect of the antagonist was not as thorough as knocking out the receptor. These results demonstrated that CB2R activation alleviated inflammation in IMQ-induced PsD.

CB2R Deficiency Intensified the Scratching Behaviour of IMQ-Induced Psoriatic Mice

It has been reported that CB2R agonists significantly suppress scratching behaviour induced by compound 48/80, histamine, substance P or serotonin in mice or rats (Odan et al., 2012; Haruna et al., 2015), but all of those compounds are the pruritogens of acute itch. Spontaneous scratching and psoriasiform skin lesions can be induced by topical stimulation of IMQ (Sakai et al., 2016), which simulates the chronic itch of psoriasis. To investigate the effect of CB2R on the chronic itch in psoriasis, we followed the schematic experimental protocol (Figure 4A). The results showed that mice treated with IMQ indeed exhibited more scratching bouts than the control group. Both CB2R deficiency and i.c. AM-630 injection intensified the scratching behaviour, whereas i.c. JWH-133 alleviated the scratching behaviour compared with that in the IMQ model group (Figures 5A,B). We next examined the sensory nerve fibers in lesion skin and untreated skin from WT mice and CB2R KO mice. Nerve fibers density was analyzed using immunofluorescence to visualize protein gene product 9.5 (PGP 9.5)-positive nerve fibers, which are known as peripheral sensory nerve markers (Feld et al., 2016). As shown in Figure 5C and Supplementary Figure S2B, CB2R KO showed that dermal nerve fibers extended to the epidermis, and a significant increase in the density of PGP 9.5-positive nerve fibers at the junction of the dermis and epidermis was noted in IMQ-induced PsD skin compared with WT mice or vehicle-treated skin. To further explore the mechanism of itch, we hypothesized that the changes in nerve fibers involved NGF-induced neuronal growth. Western blot and qRT-PCR revealed an increase in NGF expression levels in the IMQ group compared with the normal control group. However, CB2R KO only affected NGF gene expression level but did not significantly influence NGF protein expression (Figures 5D,E). Taken together, these results suggested that the extension of nerve fibers into the epidermis could be involved in the itch-related scratching of mice, and CB2R deficiency intensified the scratching behaviour of IMQ-induced psoriatic mice by regulating the expression of nerve fibers. Moreover, all of these effects could be reversed by JWH-133, but the effects of the antagonists AM-630 were not as significant as those of the knockout mice.

The authors thank all patients and healthy individuals who participated in this study.