Patients
The skin biopsies used in this project were from psoriasis patients and healthy volunteers in the dermatology clinic. The collection and processing experiments were approved by the Institutional Review Board of the Second Affiliated Hospital, Zhejiang University School of Medicine. Written and informed consent was obtained from all psoriasis patients and healthy controls.
Mice
Female C57BL/6 wild-type mice at 6 weeks old were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., and bred in the Animal Experiment Center of the Second Affiliated Hospital, Zhejiang University School of Medicine. All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee.
IMQ induced psoriasis-like mouse model
Imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd.) was used to induce psoriasis-like dermatitis in mice. The back of the mice was depilated and 62.5 mg 5% imiquimod cream was topically applied for 6 days.
Co-culture of mouse DRG neurons and keratinocytes
The method of culturing keratinocytes is the same as described before [14]. Mouse L4-L6 DRG was taken, digested with 2ml collagenase and 2ml trypsin for 30 minutes, neutralized with 5ml FBS, centrifuged at 1000rpm/min for 10 minutes. The precipitate was mixed with keratinocyte culture medium, and added to the keratinocyte culture flask. Nerve growth factor was added for neuron incubation. After 4-5 days, cells were used for immunofluorescence detection.
Spinal cord hemisection model
The mouse was anesthetized with 1% sodium pentobarbital. The T10 thoracic spinous process was used as surface landmark. Under microscope observation and with the spinal cord anterior and posterior veins as the boundary, microsurgery scissors were used to cut the left half of the spinal cord. Then the muscle and skin were sutured layer by layer. The effectiveness of the mouse model of spinal cord injury was evaluated by runk imbalance and paralysis of the ipsilesional hind limb and pain and temperature sensory disturbance on the contralesional side of the injury.
BTX-A injection
Normal saline was applied to formulate BTX-A (Allergen) dry powder into 0.005U/μl solution. The back of the mice was depilated. Four evenly distributed injection points were selected on the back, and 50μl of BTX-A of working concentration was injected subcutaneously, and each mouse was injected with 1 unit in total. The control group was injected with 50μl of normal saline.
Immunofluorescence and Immunohistochemistry
Skin biopsy was fixed in OCT, sectioned by freezing microtome (Leica) and blocked with 10% BSA for 1 h. Tissues were stained with anti-βIII-tubulin antibody (1:200, Abcam), anti-CGRP antibody (1:200, CST), anti-CD103 antibody (1:400, Abcam), anti-CD11b antibody (1:200, Abcam) or anti-Nav1.8 antibody (1:200, Abcam) in PBS with 5% FBS for 1 h, and then incubated with Alexa Fluro 488 or 555-conjugated secondary antibody (1:2000, Invitrogen) for 1 h. Nuclei were counterstained with DAPI. Images were pictured by Leica DM5500B.
For IHC of Ki67 and PGP9.5, skin sections from mice were blocked with 10% BSA 1 h, incubated with anti-Ki67 antibody (1:500, Abcam) or anti-PGP9.5 antibody (1:500, Abcam), washed, then incubated with secondary antibody and stained with DAB (Vector Laboratories). Images were scanned by Nano Zoomer (Hamamatsu) and captured by NDP.view software.
Immunoelectron microscopy
The psoriatic lesional skin from psoriasis patients was placed in 4% paraformaldehyde-0.5% glutaraldehyde fixative (PG) for fixation overnight and was cut into 50μm sections. Then the tissue pieces were fixed in 4% paraformaldehyde. Skin slices were treated with 1% hydrogen peroxide for 15 minutes and then incubated with goat serum at room temperature for 1 h. Then the sample slice was incubated with the anti-βIII-tubulin antibody (1:200, Abcam) for 24 hours at 4°C. After washed with PBS for 3 times, the sample was incubated with colloidal gold-labeled antibody solution for 1 hour at room temperature and washed with ddH2O. Picture was taken with Tecnai G2 Spirit 120kV cryo-electron microscope.
qRT-PCR
Tissue preparation and qRT-PCR was carried out as described before [15]. CT values were analyzed by qBase Plus 2 software.
The following primer sets were used: β-actin, forward 5’-GTCATTCCAAATATGAGATGCGT-3’ and reverse 5’-GCTATCACCTCCCCTGTGTG-3’, IFN-αR1 forward 5’-TCCACATGGTATGAGGTTGA -3’ and reverse 5’-AGCTTGAACGATCCATAGCC-3’, IFN-αR2, forward 5’-GTCTTGACACCCTACAAACC -3’ and reverse 5’-TCAGGCCACTTTGACTGCAA-3’, IL-17RC, forward 5’-ATGCCTGTGTCCTGGTTCCT-3’ and reverse 5’-TTCTAGTGTAGTGCAGGGTC-3’.
ELISA
A 6mm skin punch was taken from each mouse and quickly transferred to a 12-well cell culture plate containing 500μl DMEM. The skin was incubated on a 32°C shaker for 30 minutes. Supernatant was used for CGRP ELISA. The protein expression levels of CGRP secreted into the supernatants of cultured skin sheets from mice were quantified via ELISA kits (Cusabio) following the manufacturer’s instructions.
RNA Sequence Reanalysis from GEO
The transcriptome data was obtained from the GSE121212 and GSE53552 in Gene Expression Omnibus (GEO) database [16, 17]. Read counts were normalized by TMM, and the VOOM transformation was used to model the mean-variance relationship. Bayes linear model in the limma package was used to analyze DEGs.
Statistical analysis
Statistical analyses were performed using GraphPad Prism6. Analyses were carried out by using One-way ANOVA. P value < 0.05 was considered statistically significant.