Cytokine induced 3-D organotypic psoriasis skin model demonstrates distinct roles for NF-κB and JAK pathways in disease pathophysiology.

Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.

Optimization of decalcification techniques for histologic examination of the rat maxillary and mandibular incisors for toxicity studies.

The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.