Microarray Gene Expression Analysis of Lesional Skin in Canine Pemphigus Foliaceus.

Canine pemphigus foliaceus (PF) is considered the most common autoimmune skin disease in dogs; the mechanism of PF disease development is currently poorly understood. Therefore, this study aimed to characterize the molecular mechanisms and altered biological pathways in the skin lesions of canine PF patients. Using an RNA microarray on formalin-fixed, paraffin-embedded samples, we analyzed the transcriptome of canine PF lesional skin (n = 7) compared to healthy skin (n = 5). Of the 800 genes analyzed, 420 differentially expressed genes (DEGs) (p < 0.05) were found. Of those, 338 genes were significantly upregulated, including pro-inflammatory and Th17-related genes. Cell type profiling found enhancement of several cell types, such as neutrophils, T-cells, and macrophages, in PF skin compared to healthy skin. Enrichment analyses of the upregulated DEGs resulted in 78 statistically significant process networks (FDR < 0.05), including the Janus kinase signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein kinase (MAPK) signaling. In conclusion, canine PF lesional immune signature resembles previously published changes in human pemphigus skin lesions. Further studies with canine PF lesional skin using next-generation sequencing (e.g., RNA sequencing, spatial transcriptomics, etc.) and the development of canine keratinocyte/skin explant PF models are needed to elucidate the pathogenesis of this debilitating disease.

Characterization of the serum and skin inflammatory profile in canine pemphigus foliaceus using multiplex assay and quantitative real-time polymerase chain reaction (qRT-PCR).

Canine pemphigus foliaceus (PF) is a common autoimmune skin disease characterized by autoantibodies binding to epithelial adhesion molecules resulting inflammatory response. The immune network of cytokine and chemokine abnormalities that characterize the immune response in canine PF are poorly explored. This study evaluated serum and lesional skin cytokine and chemokine profiles of dogs diagnosed with PF compared to healthy control dogs. Serum samples obtained from 11 PF dogs and 16 healthy control dogs were analyzed using commercially available canine multiplex assay for 13 biomarkers (Canine Milliplex assay). Eight lesional skin samples from seven PF dogs and five healthy site-matched samples from five healthy dogs were evaluated for 20 immune markers using quantitative real-time PCR. Immunomodulating medications were suspended for at least four weeks in all dogs before obtaining serum and skin samples. PF patients showed significantly higher serum concentrations of tumor necrosis factor-α, interleukin (IL)- 6, IL-8, IL-18, CCL2, KC-like, and granulocyte-macrophages colony-stimulating factor when compared to healthy controls (Mann-Whitney U test; p < 0.05 for all). Lesional PF skin exhibited significant expression and upregulation of pro-inflammatory/T helper (Th1) 1 markers IL-1ß, MX1, GZMB, OAS1, and IFN-γ as well as Th2 cytokines IL-13, IL-33, TSLP, IL-31 and Th17/22 markers IL-17A and IL-22 (Mann-Whitney U test; p < 0.05 for all). Taken together, the findings from this study describe the role of numerous cytokines and chemokines associated with immune response in the skin and serum of canine PF patients. Further larger-sample proteomics and RNA-sequencing transcriptomics studies are needed to understand the immune pathogenesis of canine PF skin lesions.

Establishment of an Intradermal Canine IL-31-Induced Pruritus Model to Evaluate Therapeutic Candidates in Atopic Dermatitis.

Pruritic models in healthy dogs utilizing intravenous administration of interleukin 31 (IL-31) bypass the "natural" itch sensation in AD, which is initiated by pruriceptive primary afferent neurons in the skin. This study aimed to evaluate the immediate/delayed pruritus responses and the pruritic behaviors observed in an intradermal IL-31-induced pruritic model of healthy dogs and the anti-pruritic effect of oclacitinib on said model. In Phase 1, all the dogs were randomized and video-recorded for 300 min after intradermal canine recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline) injections. In Phase 2, all the dogs received oral oclacitinib (0.4-0.6 mg/kg, twice daily for 4 consecutive days and once daily on day 5), with the intradermal IL-31 injection performed on day 5. Two blinded investigators reviewed the pruritic behaviors in all the video recordings. Intradermal IL-31 administration to healthy dogs caused a significant increase in the total (p = 0.0052) and local (p = 0.0003) seconds of pruritic behavior compared to the vehicle control. Oral oclacitinib administration significantly reduced the total (p = 0.0011) and local (p = 0.0156) intradermal IL-31-induced pruritic seconds; there was no significant difference in pruritic seconds between the vehicle and oclacitinib within the IL-31 groups. Significant delayed pruritic responses at 150-300 min after IL-31 injections were observed, and intradermal IL-31 failed to induce acute itch (first 30 min). Intradermal injection of IL-31 induces delayed itch responses in dogs that are diminished by the effect of oclacitinib, an oral JAK inhibitor.