IL31 Canine, His

What is IL-31 and what role does it play in canine atopic dermatitis?

Interleukin-31 (IL-31) is a member of the gp130/interleukin-6 cytokine family produced primarily by T helper 2 lymphocytes and cutaneous lymphocyte antigen-positive skin homing T cells . In canine atopic dermatitis (AD), IL-31 serves as a key mediator of pruritus, as evidenced by its detection in 57% of dogs with naturally occurring AD at levels ≥13 pg/mL, while remaining below quantification limits in normal, non-diseased animals . IL-31 belongs to the IL-6 cytokine family but uniquely does not signal through receptor complexes containing gp130 . Instead, it utilizes a heterodimeric receptor complex composed of IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMRβ) . The cytokine initiates signaling cascades through several pathways, including Janus kinase (JAK) signal transducer and activator of transcription (STAT) and phosphatidylinositol-3 kinase (PI3K/AKT), ultimately leading to pruritic sensations in affected animals .

How is recombinant canine IL-31 produced for experimental studies?

Recombinant canine IL-31 is primarily produced using mammalian expression systems to ensure proper post-translational modifications. According to available research, the production typically follows this methodology:

• The canine IL-31 gene sequence is inserted into an appropriate expression vector.

• The mammalian HEK293 expression system is commonly employed to produce the recombinant protein .

• Following expression, the protein is harvested and purified using standard chromatography techniques.

• Quality control measures include mass spectrometry (tryptic digest and mapping) and N-terminal sequencing to confirm the correct canine IL-31 sequence .

• Biological activity verification is performed using IL-31-induced STAT3 phosphorylation assays in canine DH82 cells .

This methodology ensures the production of functional recombinant canine IL-31 that maintains the biological activities observed in naturally occurring IL-31.

What experimental models exist for studying IL-31-induced pruritus in dogs?

Several experimental models have been developed to study IL-31-induced pruritus in dogs:

• Intravenous administration model: This was the first established model where recombinant canine IL-31 is administered intravenously to healthy dogs. This model has been extensively used to evaluate anti-pruritic drugs including oclacitinib, lokivetmab, and glucocorticoids .

• Intradermal administration model: More recently developed, this model involves intradermal injection of recombinant canine IL-31 (typically at 1.75 μg/kg) in the lateral thoracic region of healthy dogs . This model more closely mimics the natural disease process by directly targeting cutaneous tissues.

• House dust mite (HDM) sensitization model: This model uses HDM-sensitized atopic Maltese-beagle dogs to study natural allergen-induced inflammatory responses, with or without IL-31 inhibition .

The intradermal model is particularly valuable as it allows for the study of both immediate and delayed pruritic responses, with significant delayed responses observed 150-300 minutes after IL-31 injection, while interestingly failing to induce acute itch within the first 30 minutes post-injection .

What is the binding affinity of canine IL-31 to its receptors compared to human and feline IL-31?

Recent research has revealed significant species-specific differences in IL-31 receptor binding patterns:

• Human IL-31 (hIL-31) directly interacts with both IL-31RA and OSMRβ independently, with stronger binding affinity to IL-31RA than to OSMRβ .

• Feline IL-31 (fIL-31) demonstrates a higher binding affinity for feline OSMRβ .

• Canine IL-31 (cIL-31) has been found to bind directly to OSMRβ with higher binding affinity than to IL-31RA . This represents a distinct binding pattern compared to the human ortholog.

The binding constants for canine IL-31 are in the range of 10^-8 M, indicating relatively high affinity for its receptors . These species-specific binding differences are crucial to consider when developing targeted therapies and when extrapolating findings across species.

How effective are IL-31 inhibitors in treating canine atopic dermatitis?

IL-31 inhibitors, particularly lokivetmab (LKV), a caninised monoclonal antibody directed against IL-31, have shown varied effectiveness in treating canine atopic dermatitis:

These findings suggest that while IL-31 inhibition effectively controls pruritus, it does not fully address the underlying inflammatory process in atopic dermatitis, explaining why this treatment approach may be less efficacious in some cases.

What other cytokines are involved in canine atopic dermatitis that are not affected by IL-31 inhibition?

Research using RNA sequencing analysis has identified several key cytokines and chemokines that remain active despite IL-31 inhibition in canine atopic dermatitis:

These findings from a cross-over study comparing cytokine profiling of acute atopic dermatitis skin lesions with or without lokivetmab-induced inhibition of IL-31 suggest that multiple inflammatory pathways operate independently of IL-31 . This cytokine network complexity explains why IL-31 inhibition alone may not completely resolve the inflammatory component of atopic dermatitis and indicates potential additional therapeutic targets for comprehensive management of the disease.

How does the intradermal administration of IL-31 differ from intravenous administration in canine pruritus models?

The intradermal and intravenous IL-31 administration models offer different insights into pruritus mechanisms:

• Route of administration: Intravenous models deliver IL-31 systemically, while intradermal models deliver IL-31 directly to cutaneous tissues (typically the lateral thoracic region) .

• Physiological relevance: Intradermal models more closely mimic the "natural" itch sensation in atopic dermatitis, which originates from pruriceptive primary afferent neurons in the skin. Intravenous models bypass this initial cutaneous activation pathway .

• Temporal response patterns: Intradermal IL-31 administration produces a significant delayed pruritic response (150-300 minutes post-injection) but notably fails to induce acute itch within the first 30 minutes. This contrasts with the more immediate response typically seen with intravenous administration .

• Evaluation of therapeutics: Both models have been used to assess anti-pruritic medications, but the intradermal model may better represent the efficacy of treatments targeting cutaneous pathways. In studies using oclacitinib (JAK inhibitor), the medication significantly reduced pruritic behaviors in the intradermal IL-31 model .

• Experimental considerations: For intradermal studies, the skin areas are typically clipped ≥72 hours before injection to avoid potential localized skin irritation that might confound behavioral observations .

These differences highlight the importance of model selection depending on the specific research question, with intradermal models offering potential advantages for studies focused on cutaneous pruritus pathways.

What methodologies are used to quantify pruritic behaviors in canine IL-31 models?

Quantification of pruritic behaviors in canine IL-31 models typically employs these methodological approaches:

• Video monitoring: Dogs are video-recorded for extended periods (commonly 2-5 hours) following IL-31 administration . This provides objective documentation of behaviors for later analysis.

• Blinded observation: Two or more blinded investigators review the video recordings independently to minimize bias in behavior assessment .

• Behavioral classification: Pruritic behaviors are classified and quantified, including scratching, biting, licking, and rubbing at specific body regions .

• Temporal analysis: Behaviors are often analyzed in distinct time blocks (e.g., 0-30 minutes, 30-60 minutes) to characterize the temporal pattern of the pruritic response .

• Comparison to control: Pruritic behaviors in IL-31-treated dogs are compared with those receiving vehicle (e.g., phosphate-buffered saline) to establish the specific effect of IL-31 .

These methods allow for robust quantification of pruritic responses, enabling reliable comparison between different treatment conditions and accurate assessment of anti-pruritic interventions.

How can researchers verify the structural and functional properties of recombinant canine IL-31?

Verifying the structural and functional properties of recombinant canine IL-31 involves multiple complementary approaches:

• Sequence verification:

  • Mass spectrometry (tryptic digest and mapping) to confirm the amino acid sequence

  • N-terminal sequencing to verify the correct protein processing

• Structural analysis:

  • Secondary structure analysis reveals that canine IL-31 is primarily composed of alpha-helices

  • Comparison of amino acid sequences across species (canine, feline, human) to identify conserved structural elements

• Functional verification:

  • IL-31-induced STAT3 phosphorylation assay in canine DH82 cells to confirm biological activity

  • Receptor binding assays to measure affinity for IL-31RA and OSMRβ

  • In vivo pruritic response assessment following administration to laboratory animals

• Quantitative assessment:

  • Development of quantitative immunoassay techniques to measure serum levels of canine IL-31, with detection limits of approximately 13 pg/mL

These verification steps ensure that the recombinant protein maintains both the structural characteristics and biological functions of naturally occurring canine IL-31, which is essential for valid experimental outcomes.

What experimental controls should be included when designing studies using recombinant canine IL-31?

When designing studies using recombinant canine IL-31, researchers should include the following experimental controls:

• Vehicle control: Administration of the carrier solution (typically phosphate-buffered saline) without IL-31 to account for potential effects of the delivery medium or injection process .

• Baseline measurements: Collection of pre-treatment data from the same animals to establish normal behavior patterns or physiological parameters .

• Cross-over design: When feasible, using animals as their own controls by administering different treatments with appropriate wash-out periods (typically 4 weeks for IL-31 studies) .

• Positive control: Including a group treated with a known pruritogenic substance or a positive disease model when evaluating anti-pruritic interventions.

• Blinded assessment: Ensuring that investigators assessing outcomes are blinded to treatment allocation to minimize bias .

• Environmental controls: Standardizing housing conditions, handling procedures, and observation times to minimize external variables that might influence pruritic behaviors.

• Biological validation: Confirming the activity of the recombinant IL-31 batch using in vitro assays (e.g., STAT3 phosphorylation) before in vivo application .

These controls enhance the scientific rigor of IL-31 research and increase the reliability and reproducibility of experimental findings.

What are the future research directions for canine IL-31 studies?

Based on current findings, several promising research directions emerge for canine IL-31 studies:

• Combination therapy approaches: Given that IL-31 inhibition does not prevent expression of other proinflammatory mediators in atopic dermatitis, investigating combination therapies targeting multiple cytokine pathways simultaneously may yield more comprehensive treatment options .

• Receptor-specific interventions: The unique binding pattern of canine IL-31 to OSMRβ with higher affinity than to IL-31RA suggests potential for receptor-specific therapeutic approaches . Further characterization of these interactions could lead to novel targeted therapies.

• Biomarker development: Expanding on the finding that IL-31 is detectable in 57% of dogs with atopic dermatitis but not in healthy dogs, developing improved biomarkers for diagnosis and treatment monitoring represents an important research avenue .

• Neuronal mechanisms: Further investigation of how IL-31 directly induces itch sensation via its heterodimeric receptor complex on free nerve endings and/or through promoting other cell types to release additional pruritogens would enhance our understanding of pruritus pathways .

• Comparative species studies: The distinct binding patterns of IL-31 across species (human, canine, feline) warrant further comparative studies to elucidate the evolutionary and functional significance of these differences .