Furthermore, the frequency of Th17 cells in the DLNs of the mice in the DS + NTN-1 Ab group was higher than that in the DS + IgG group (Fig.?7F). Open in a separate window Figure 7. NTN-1 neutralizing antibody exacerbated inflammation in DED. addition, NTN-1 decreased Vinflunine Tartrate the number of DCs, inhibited the activation of the DCs and Th17 cells, and reduced the expression of inflammatory factors in DED mice. In contrast, blocking endogenous NTN-1 activity with an antiCNTN-1 antibody aggravated the disease, enhanced DC activation, and upregulated the inflammatory factors in the conjunctivae of DED mice. Conclusions We identified decreased NTN-1 expression in the corneal epithelium of DED mice. Our findings elucidate the role of NTN-1 in alleviating DED Vinflunine Tartrate and impeding DC activation, thereby indicating its therapeutic potential in suppressing ocular inflammation in DED. 0.05 were defined as differentially expressed genes (DEGs). Volcano plots of DEGs were obtained using the ggplot2 R package. Cluster analysis was performed using the Pheatmap R package. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of DEGs were performed using a clusterProfiler. Western Blot Analysis The total protein was extracted from corneal epithelium as described previously.32 The quantified and denatured total proteins were separated by 10% SDS-PAGE and then transferred electronically to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Thereafter, the blots were blocked in 5% milk-Tris-buffered saline containing 0.1% Tween-20 for 1 hour and then incubated with primary antibodies (antiCNTN-1 antibody [1:1000; ab126729, Abcam, Cambridge, MA, USA] and anti-GAPDH antibody [1:3000; KC-5G5, Kangcheng, Shanghai, China]) at 4C overnight. The following day, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5000, ZB-2301; ZSGB-BIO, Beijing, China) for 1 hour at room temperature. An ECL kit (P1050-250; Applygen, Beijing, China) was used for detection. Immunofluorescent Staining Samples were collected and cut into frozen sections as described previously.33 The frozen corneal sections (7 m thick) were fixed in 4% paraformaldehyde (PFA) for 20 minutes, rehydrated with PBS, and permeabilized in 0.1% Triton X-100 (Solarbio, Beijing, China) for 2 minutes. Then, the sections were blocked with 5% BSA (AR0004; Boster, Wuhan, China) for 60 minutes at room temperature and then stained with antiCNTN-1 antibody at 4C overnight. Negative controls were incubated with the rabbit IgG isotype control antibody (1:200, ab172730; Abcam). Thereafter, the sections were incubated with fluorescein-conjugated secondary antibody for 60 minutes. All the sections were photographed using a Vinflunine Tartrate fluorescence microscope (Olympus BX50) after counterstaining with DAPI (H-1200; Vector, Burlingame, CA, USA). Periodic AcidCSchiff Staining The eyeballs were fixed in 4% PFA, embedded in paraffin, and then cut into 5-m-thick paraffin sections. The sections were then stained using a periodic acidCSchiff (PAS) staining kit (G1008-20ML; Servicebio, Wuhan, China) as per the manufacturer’s instructions. The upper and lower eyelids were observed under a light microscope and photographed. Next, three sections from the central part of the eye of each mouse were stained. The number of PAS-positive cells was manually counted and averaged for each eyelid. Sections from five mice from each of the aforementioned groups were used. Phenol Red Thread Test Tear secretion was measured using the phenol red thread test at day 5 after intraperitoneal injection of 5% pentobarbital sodium (0.1 mL/10 g) under general Vinflunine Tartrate anesthesia. A phenol redCimpregnated cotton thread (AYUMI Pharmaceutical Corporation, Tokyo, Japan) was gently placed on the lateral canthus for 20 seconds for each eye with the eyes closed. The length of wetting was recorded in millimeters under the stereomicroscope. Corneal Fluorescein Staining One microliter of 0.25% fluorescein sodium (Jingming, Tianjin, China) was applied topically on the cornea of each eye of mice 2 hours after the last administration. After the dye was instilled, the eyes were allowed to blink several times and then were rinsed with normal saline. Corneal fluorescein staining was observed and photographed using Prkwnk1 a slit-lamp microscope (66 Vision-Tech Co. Ltd., Suzhou, China) under cobalt blue light. Corneal fluorescein staining scores were evaluated in a masked manner according to the scoring system, as described in our previous study.31 Briefly, the cornea was separated into four quadrants, which were correspondingly scored. The four scores were summed and analyzed for each eye (minimum = 0; maximum = 16). The fluorescein score was analyzed as described previously and also as follows: positive fluorescein plaque, 4; severe diffuse staining but no positive plaque, 3; dense punctate staining with more than 30 spots, 2; slightly punctate staining with fewer than 30 spots, 1; and absent, 0. Immunostaining of Whole-Mount Corneal Vinflunine Tartrate Tissues Whole-mount corneal staining was performed as described previously.34 In brief,.