IL31 Canine, HEK

What is canine IL-31 and what cellular sources produce it in vivo?

Canine interleukin-31 (IL-31) is a T helper 2 cytokine that plays a significant role in pruritus (itch sensation) pathways, particularly in canine atopic dermatitis. In vivo, IL-31 is primarily produced by activated helper T cells (particularly Th2 cells), mast cells, macrophages, and dendritic cells . Research has confirmed that the majority of IL-31-positive cells in canine skin are CD3-positive (91-100%) and CD4-positive (63-100%), indicating they are primarily helper T cells . Interestingly, sebaceous glands also show strong IL-31 immunolabeling, which has been validated through immunoabsorption testing with IL-31 .

How does the HEK293 expression system benefit canine IL-31 research?

The HEK293 (Human Embryonic Kidney) expression system has become a preferred platform for producing recombinant canine IL-31 for research purposes because it provides proper protein folding, post-translational modifications, and glycosylation patterns that more closely mimic native canine IL-31 . This expression system yields a protein with a predicted molecular weight of 16.63 kDa, though due to glycosylation, the protein typically migrates to 26-36 kDa when analyzed by Bis-Tris PAGE . The HEK293 system enables researchers to produce high-purity (>95%) recombinant canine IL-31 with proper biological activity, which is crucial for experimental models examining pruritus and atopic dermatitis in dogs .

What is the IL-31 signaling pathway and how does it relate to pruritus in canines?

Canine IL-31 produces signals by activating a receptor complex composed of IL-31 receptor A (IL-31RA) and the oncostatin M receptor (OSMR) . This activation leads to downstream phosphorylation of several kinase signaling pathways, including the Janus kinase (JAK) signal transducer and activator of transcription (STAT) and phosphatidylinositol-3 kinase (PI3K/AKT) . IL-31 induces pruritus through direct activation of its heterodimeric receptor complex on free nerve endings and/or by promoting other cell types (e.g., keratinocytes, fibroblasts) to release additional pruritogens that then trigger the itch signal . Research has demonstrated that IL-31RA is expressed on keratinocytes and a small proportion of dermal nerves in canine skin, providing the anatomical basis for IL-31-induced pruritus .

How should researchers design an intradermal IL-31-induced pruritus model in dogs?

When designing an intradermal IL-31-induced pruritus model in dogs, researchers should consider the following methodological approaches:

• Animal selection: Utilize healthy laboratory dogs (e.g., beagles) without pre-existing skin conditions.

• Dose determination: Administer recombinant canine IL-31 diluted in sterile phosphate-buffered saline (PBS) at approximately 1.75 μg/kg for intradermal injection .

• Control procedures: Include vehicle control (PBS) injections for comparison.

• Preparation: Clip the skin areas for injections (e.g., right lateral thoracic region) at least 72 hours before injection to avoid potential skin irritation from clipping .

• Observation period: Video-record the dogs for an extended period (e.g., 300 consecutive minutes) following injection to capture both immediate and delayed responses .

• Behavior assessment: Establish clear criteria for evaluating pruritic behaviors (scratching, biting/chewing, licking, head shaking) using categorical scoring systems or total seconds of observed behavior .

• Randomization: Employ a randomized, controlled crossover design with appropriate wash-out periods (e.g., 4 weeks) between interventions .

This model allows for studying both spontaneous pruritus and the efficacy of anti-pruritic compounds in a controlled setting .

What are the key temporal considerations when studying IL-31 expression in canine atopic dermatitis?

When investigating IL-31 expression in canine atopic dermatitis, several critical temporal factors should be considered:

• Time course sampling: Collect skin and blood samples at multiple time points (e.g., 0h, 24h, 48h, and 96h) after allergen provocation to capture the dynamic changes in IL-31 expression .

• Peak expression window: Research indicates that peak IL-31 expression typically occurs at 24h or 48h post-allergen challenge, and starts to decrease by 96h .

• Delayed pruritic response: When using intradermal IL-31 models, significant delayed pruritic responses occur at 150-300 minutes after injection, with minimal acute responses in the first 30 minutes .

• Specific time intervals for analysis: When analyzing pruritic behaviors, segment observations into discrete time periods (e.g., 30-minute intervals from 0-300 minutes) to identify patterns in response intensity .

• Correlation timing: Note that there may not be significant correlations between IL-31 expression scores and macroscopic skin lesion scores or serum IL-31 concentrations, suggesting complex temporal dynamics in the pathophysiology .

Understanding these temporal patterns is crucial for designing intervention studies, particularly when testing anti-pruritic therapies targeting the IL-31 pathway .

How should researchers optimize immunofluorescence protocols for detecting IL-31 and IL-31RA in canine skin samples?

Optimizing immunofluorescence (IF) protocols for IL-31 and IL-31RA detection in canine skin samples requires several methodological considerations:

• Single-staining approach: Establish baseline protocols for IL-31 and IL-31RA single-staining IF to identify positive cells and structures .

• Double-staining techniques: Implement double-staining IF (e.g., IL-31/CD3, IL-31/CD4, IL-31RA/β3-tubulin) to identify the cellular sources of IL-31 and locations of IL-31RA .

• Validation controls: Include immunoabsorption controls using recombinant IL-31 protein to verify staining specificity, especially for unexpected positive structures like sebaceous glands .

• Quantification method: Develop a systematic approach for counting IL-31-positive cells, potentially using digital image analysis to enhance objectivity .

• Tissue preparation: Ensure consistent fixation and processing of skin samples to minimize artifacts and optimize antigen preservation.

• Antibody selection: Use antibodies validated for canine tissues, with appropriate isotype controls.

These optimized protocols allow researchers to accurately assess the spatial and temporal expression patterns of IL-31 and its receptor in canine skin during atopic dermatitis and other pruritic conditions .

What statistical approaches are most appropriate for analyzing IL-31-induced pruritic behaviors in canine models?

When analyzing IL-31-induced pruritic behaviors in canine models, researchers should consider these statistical approaches:

• Two-way mixed effects models: Use these for analyzing time series data, particularly when examining acute (first 30 min) and delayed (after 30 min) itch effects of recombinant canine IL-31 .

• Multiple comparison adjustments: Apply the Holm–Šídák correction test when making multiple comparisons across different time intervals .

• Non-parametric testing: Use non-parametric tests like the Mann-Whitney U test for comparing total pruritic seconds between treatment groups when data does not follow normal distribution .

• Correlation analysis: Employ appropriate correlation tests to examine relationships between IL-31 expression, serum IL-31 levels, and macroscopic skin lesion scores .

• Power analysis: Conduct a priori power analysis to determine adequate sample size; research suggests 10 dogs provides 90% power to detect a 2-fold difference in pruritic scores between treatment groups .

• Interobserver reliability assessment: Evaluate agreement between multiple blinded investigators reviewing pruritic behaviors to ensure consistent scoring .

Setting statistical significance at p < 0.05 is standard practice, with clear reporting of actual p-values for significant findings (e.g., p = 0.0052 for total pruritic behavior comparisons) .

How do IL-31RA expression patterns in canine skin inform therapeutic targeting strategies?

The expression patterns of IL-31 receptor A (IL-31RA) in canine skin provide crucial insights for developing targeted therapeutic approaches:

• Cellular localization: IL-31RA is expressed primarily on keratinocytes and a smaller proportion of dermal nerves in canine skin . This dual localization suggests multiple cellular targets for IL-31 signaling inhibition.

• Temporal dynamics: Understanding when IL-31RA is most highly expressed during the course of atopic dermatitis can inform optimal timing for therapeutic intervention.

• JAK inhibition rationale: The expression of IL-31RA on target cells provides the biological basis for using JAK inhibitors like oclacitinib, which block the downstream signaling cascade initiated by IL-31 binding to its receptor .

• Proactive therapy concept: The early and transient nature of IL-31 production by T helper cells in atopic dermatitis supports using IL-31 inhibiting strategies as proactive therapy to prevent flares rather than treating established lesions .

• Dual-targeting opportunities: The expression of IL-31RA alongside OSMR in the heterodimeric receptor complex suggests potential benefits from therapies targeting both components simultaneously .

These expression patterns explain why both direct IL-31 neutralization and JAK inhibition approaches have proven effective in managing canine atopic dermatitis .

What are the methodological considerations for evaluating JAK inhibitor efficacy in IL-31-induced pruritus models?

When evaluating JAK inhibitor efficacy in IL-31-induced pruritus models, researchers should implement these methodological considerations:

• Dosing regimen: Establish a consistent dosing protocol, such as oral oclacitinib at 0.4–0.6 mg/kg, twice daily for 4 consecutive days and once daily on day 5, with the intradermal IL-31 challenge performed on day 5 .

• Blinded assessment: Ensure investigators reviewing pruritic behaviors are blinded to treatment allocation to prevent bias .

• Comprehensive behavior recording: Video-record dogs for extended periods (e.g., 300 minutes) following IL-31 challenge to capture the full temporal profile of pruritic responses .

• Differentiation of behaviors: Distinguish between total (generalized) pruritic behaviors and local pruritic behaviors at the injection site .

• Control groups: Include both a vehicle control group and an IL-31-only group to fully assess the anti-pruritic effect of the JAK inhibitor .

• Temporal analysis: Analyze efficacy across different time intervals, paying particular attention to the 150-300 minute period when delayed pruritic responses to IL-31 are most pronounced .

Using this systematic approach, researchers can quantitatively demonstrate that JAK inhibitors like oclacitinib significantly reduce both total and local intradermal IL-31-induced pruritic behaviors in canine models .

How should researchers address the discrepancy between tissue IL-31 expression and serum IL-31 levels in canine atopic dermatitis?

The lack of significant correlation between tissue IL-31 expression and serum IL-31 levels in canine atopic dermatitis presents an important research challenge . To address this discrepancy, researchers should consider:

• Multi-compartment sampling: Simultaneously collect skin biopsies, serum samples, and potentially interstitial fluid to track IL-31 dynamics across different compartments.

• Temporal profiling: Implement more frequent sampling timepoints to better capture potential transient correlations between tissue and serum IL-31 levels.

• Protein degradation assessment: Investigate whether differential protein stability or degradation rates in tissue versus serum explain the discrepancy.

• Local versus systemic production: Examine whether local skin production of IL-31 may not significantly contribute to systemic levels, or whether serum IL-31 derives from multiple tissue sources.

• Receptor binding dynamics: Assess whether receptor-bound IL-31 in tissues reduces detectable free IL-31 in circulation.

• Sensitivity of detection methods: Compare and optimize detection methods for both tissue expression (immunofluorescence) and serum levels (ELISA) to ensure comparable sensitivity.

Understanding this discrepancy has important implications for using serum IL-31 as a biomarker for disease activity and for monitoring response to targeted therapies in canine atopic dermatitis .

What experimental refinements could improve the translational value of intradermal IL-31 canine models for human pruritus research?

To enhance the translational value of intradermal IL-31 canine models for human pruritus research, several experimental refinements should be considered:

• Comparative receptor studies: Directly compare IL-31RA and OSMR expression patterns between canine and human skin to validate cross-species similarities and differences.

• Dose-response refinement: Establish detailed dose-response relationships for intradermal IL-31 to identify optimal doses that mirror pathophysiological conditions in both species.

• Combined allergen-IL-31 models: Develop models that incorporate both allergen sensitization and IL-31 administration to better reflect the complexity of atopic dermatitis.

• Objective measurement tools: Implement wearable sensors or automated video analysis systems to quantify scratching behaviors more objectively and continuously.

• Neurophysiological assessment: Integrate electrophysiological recordings from sensory neurons to directly measure neuronal responses to IL-31 stimulation alongside behavioral outcomes.

• Chronic models: Extend the traditional acute IL-31 challenge model to create chronic or repeated challenge models that better mimic the persistent nature of clinical atopic dermatitis.

• Barrier function assessment: Incorporate measurements of skin barrier function alongside pruritus assessments to examine the relationship between IL-31, pruritus, and barrier disruption.

These refinements would help address current limitations of intradermal IL-31 models, including the observation that they fail to induce acute itch in the first 30 minutes post-injection despite the clinical reality of rapid-onset pruritus in many patients .