IL36RN Mouse

What is the IL36RN mouse model and how is it generated?

The IL36RN mouse model (Il36rn−/− mice) features knockout of the Il36rn gene, which encodes the interleukin-36 receptor antagonist (IL-36Ra). These mice are typically generated on a C57BL/6NCr1 background through targeted gene deletion approaches. The genotype is confirmed using allele-specific PCR techniques. These models serve as valuable tools for investigating the role of IL-36Ra deficiency in various inflammatory conditions and skin disorders .

What are the key phenotypic characteristics of IL36RN knockout mice?

IL36RN knockout mice (Il36rn−/− mice) exhibit enhanced inflammatory responses in various experimental settings. Key phenotypic characteristics include: enhanced contact hypersensitivity (CHS) responses, increased infiltration of inflammatory cells (neutrophils, CD4+ T cells, and CD8+ T cells), enhanced neutrophil extracellular trap (NET) formation, and elevated expression of pro-inflammatory cytokines and chemokines including IL-1β, CXCL1, CXCL2, and IL-36γ . Additionally, these mice show delayed wound healing and more severe psoriasis-like lesions in response to imiquimod treatment .

What is the role of IL-36Ra in normal immune function and homeostasis?

IL-36Ra functions as an anti-inflammatory mediator that regulates IL-36 signaling. Unlike IL-36α, IL-36β, and IL-36γ which act as receptor agonists with pro-inflammatory functions, IL-36Ra serves to tightly regulate the IL-36 pathway. In normal conditions, IL-36Ra helps maintain immune homeostasis by preventing excessive inflammatory responses. Loss of IL-36Ra function results in dysregulated immune responses characterized by enhanced neutrophil recruitment, excessive NET formation, and overproduction of inflammatory cytokines and chemokines .

How should researchers design a contact hypersensitivity (CHS) model using IL36RN knockout mice?

To establish a CHS model using IL36RN knockout mice, researchers should follow these methodological steps:

• Use gender-matched female mice aged 8-12 weeks, with both Il36rn−/− and wild-type controls.

• On day 0, sensitize mice by applying 25 μL of 0.5% DNFB in acetone/olive oil (4:1) on the shaved dorsal surface.

• On day 5, challenge mice by topically applying 15 μL of 0.2% DNFB in acetone/olive oil (4:1) on each side of both ears.

• Measure ear swelling using dial thickness gauges before the challenge and at 24 and 48 hours post-challenge. Perform measurements in triplicate and use the average value for analysis.

• Collect ear tissue samples on day 7 for RT-PCR and histopathological analysis.

This model effectively demonstrates enhanced CHS responses in Il36rn−/− mice compared to wild-type controls .

What techniques can be used to assess neutrophil extracellular trap (NET) formation in IL36RN mice?

NETs can be assessed in IL36RN mice using the following techniques:

• Immunofluorescence staining of tissue sections (e.g., ear tissue during CHS response) to visualize NET formation.

• Quantification of NET area in tissue samples using appropriate imaging software.

• In vitro assessment of NET formation by isolating neutrophils and stimulating them under controlled conditions.

• Use of NET inhibitors like Cl-amidine (a pan-peptidyl arginine deiminase inhibitor) to confirm the specificity of observed NETs.

• Co-staining for NET markers such as citrullinated histone H3, neutrophil elastase, and DNA.

Using these approaches, researchers have demonstrated significantly increased NET formation in Il36rn−/− mice compared to wild-type mice during inflammatory responses .

How should researchers establish and evaluate a wound healing model in IL36RN deficient mice?

To establish and evaluate a wound healing model in IL36RN deficient mice:

• Create full-thickness excisional wounds on the back of Il36rn−/− and wild-type control mice.

• Monitor macroscopic wound sizes at regular intervals (e.g., daily) to assess healing progression.

• Collect tissue samples at defined timepoints for histological assessment of re-epithelialization and granulation tissue formation.

• Quantify numbers of infiltrated inflammatory cells using appropriate staining techniques.

• Measure gene expression of inflammatory cytokines using RT-PCR or similar techniques.

• For intervention studies, consider administering compounds such as TAK-242 (a TLR4 inhibitor) to evaluate potential therapeutic approaches.

Research has demonstrated that wound healing, re-epithelialization, and granulation tissue formation are typically delayed by approximately 3 days post-injury in Il36rn−/− mice compared to wild-type controls .

How does NET formation contribute to enhanced inflammatory responses in IL36RN deficient mice?

NET formation plays a critical role in the enhanced inflammatory responses observed in IL36RN deficient mice through several mechanisms:

• NETs act as a source of autoantigens and damage-associated molecular patterns (DAMPs) that perpetuate inflammation.

• NET formation correlates with increased infiltration of inflammatory cells, including neutrophils, CD4+ T cells, and CD8+ T cells.

• NETs contribute to the enhanced expression of pro-inflammatory cytokines and chemokines, including IL-1β, CXCL1, CXCL2, and IL-36γ.

• Inhibition of NET formation using Cl-amidine (a PAD inhibitor) attenuates the CHS response in both Il36rn−/− and wild-type mice, demonstrating the causative role of NETs in inflammation.

• NET inhibition reduces the number of infiltrating inflammatory cells and decreases cytokine and chemokine expression levels.

These findings suggest that NET formation is a key mechanism by which IL-36Ra deficiency leads to exacerbated inflammatory responses .

What is the relationship between TLR4 signaling and delayed wound healing in IL36RN deficient mice?

The relationship between TLR4 signaling and delayed wound healing in IL36RN deficient mice involves several interconnected pathways:

• IL-36Ra deficiency leads to increased neutrophil and macrophage infiltration during wound healing.

• Enhanced expression of pro-inflammatory cytokines such as IL-36γ, CXCL1, and TGF-β is observed in wounds of Il36rn−/− mice.

• TLR4 signaling appears to mediate these enhanced inflammatory responses, as administration of TAK-242 (a TLR4 inhibitor) normalizes wound healing in Il36rn−/− mice.

• TLR4 inhibition abrogates the initial delay in tissue repair observed in IL-36Ra-deficient mice.

• This suggests that TLR4-mediated infiltration of immune cells and cytokine production contributes significantly to the delayed wound healing phenotype.

These findings indicate that targeting TLR4-mediated pathways could be beneficial in regulating wound healing in IL-36Ra-deficient skin disorders .

How do cytokine and chemokine expression patterns differ in IL36RN knockout mice compared to wild-type during inflammatory responses?

During inflammatory responses, IL36RN knockout mice exhibit distinct cytokine and chemokine expression patterns compared to wild-type mice:

• Significantly higher mRNA expression levels of IL-1β, IL-17A, TNF-α, CXCL1, CXCL2, and IL-36γ in inflamed tissues.

• Decreased mRNA levels of IL-36α compared to wild-type mice.

• No significant differences in mRNA expression of IFN-γ, IL-4, IL-6, IL-10, or IL-36β between Il36rn−/− and wild-type mice.

• Treatment with NET inhibitors like Cl-amidine significantly reduces the expression of most pro-inflammatory cytokines and chemokines in both Il36rn−/− and wild-type mice.

• The altered cytokine profile contributes to enhanced recruitment of inflammatory cells and exacerbated tissue inflammation.

These expression patterns help explain the mechanisms by which IL-36Ra deficiency leads to enhanced inflammatory responses and could identify potential therapeutic targets .

How effective is Cl-amidine treatment in modulating inflammatory responses in IL36RN deficient mice?

Cl-amidine treatment has demonstrated significant efficacy in modulating inflammatory responses in IL36RN deficient mice:

• Cl-amidine treatment attenuated the enhanced CHS response in Il36rn−/− mice, reducing ear swelling by approximately 28.49% at 24 hours and 23.32% at 48 hours post-challenge.

• The treatment significantly decreased inflammatory cell infiltration, including neutrophils, macrophages, CD4+ T cells, and CD8+ T cells in ear tissues.

• NET formation was substantially reduced in response to Cl-amidine treatment in both Il36rn−/− and wild-type mice.

• Cl-amidine treatment significantly decreased the elevated mRNA expression of multiple inflammatory cytokines and chemokines, including IL-1β, IFN-γ, IL-4, IL-6, IL-10, IL-17A, TNF-α, CXCL1, CXCL2, and IL-36γ.

• In vitro experiments confirmed that Cl-amidine reduces NET formation without exhibiting neutropenic effects.

These findings suggest that targeting NET formation with PAD inhibitors like Cl-amidine could be a viable therapeutic approach for conditions associated with IL-36Ra deficiency .

What is the role of TAK-242 in normalizing wound healing in IL36RN deficient mice?

TAK-242, a toll-like receptor 4 (TLR4) inhibitor, plays a crucial role in normalizing wound healing in IL36RN deficient mice:

• Administration of TAK-242 causes normalization of wound healing in Il36rn−/− mice, effectively abrogating the initial delay in tissue repair.

• TAK-242 likely reduces the enhanced inflammatory cell infiltration (neutrophils and macrophages) observed in wounds of Il36rn−/− mice.

• The TLR4 inhibitor appears to modulate the abnormal expression of pro-inflammatory cytokines such as IL-36γ, CXCL1, and TGF-β in IL-36Ra-deficient conditions.

• TAK-242 intervention suggests that TLR4-mediated pathways are critical in the delayed wound healing phenotype observed in IL-36Ra deficiency.

• These findings indicate that targeting TLR4-mediated inflammation could be beneficial for regulating wound healing in IL-36Ra-deficient skin disorders.

The efficacy of TAK-242 highlights the importance of TLR4 signaling in the pathophysiology of delayed wound healing associated with IL-36Ra deficiency .

How should researchers interpret differences in inflammatory cell infiltration between IL36RN deficient and wild-type mice?

When interpreting differences in inflammatory cell infiltration between IL36RN deficient and wild-type mice, researchers should consider:

• Temporal dynamics: The pattern and timing of infiltration may differ between genotypes, with Il36rn−/− mice typically showing earlier and more pronounced infiltration.

• Cell type specificity: Evaluate whether all inflammatory cell types are equally affected or if certain populations (e.g., neutrophils) show more dramatic differences.

• Correlation with functional outcomes: Relate cell infiltration data to functional outcomes such as tissue swelling, damage, or delayed healing.

• Mechanistic implications: Consider whether differences in cell infiltration are primary effects of IL-36Ra deficiency or secondary to altered cytokine/chemokine production.

• Statistical significance: Ensure that quantification of cell numbers is performed on multiple samples with appropriate statistical analysis.

• Technical considerations: Account for potential differences in tissue processing, staining efficiency, or counting methodology.

Research has shown that Il36rn−/− mice exhibit significantly higher numbers of MPO-positive neutrophils, F4/80-positive macrophages, CD4+ T cells, and CD8+ T cells compared to wild-type mice during inflammatory responses .

What controls and experimental conditions are crucial for reliable results in IL36RN mouse studies?

For reliable results in IL36RN mouse studies, the following controls and experimental conditions are crucial:

• Proper genotype controls: Always include wild-type mice of the same background strain (typically C57BL/6NCr1) as controls.

• Gender and age matching: Use gender-matched mice (either all male or all female) within the same age range (typically 8-12 weeks) to minimize variation.

• Vehicle controls: For intervention studies (e.g., with Cl-amidine or TAK-242), include proper vehicle control groups.

• Time course analysis: Collect data at multiple timepoints to capture the dynamic nature of inflammatory responses.

• Multiple readouts: Combine macroscopic assessments (e.g., ear thickness) with molecular and cellular analyses (histology, gene expression).

• Blinded analysis: Perform assessments in a blinded manner to avoid bias.

• Sample size determination: Use appropriate power calculations to determine required sample sizes (typically six mice per group in published studies).

• Housing conditions: Maintain mice in specific pathogen-free barrier facilities to avoid confounding infections.

• Experimental repetition: Verify key findings with independent experimental replicates.

Following these guidelines will help ensure that observed differences between Il36rn−/− and wild-type mice are reliable and reproducible .

How do findings from IL36RN mouse models relate to human IL36RN-associated disorders?

Findings from IL36RN mouse models have significant relevance to human IL36RN-associated disorders:

• Loss-of-function mutations in human IL36RN are implicated in generalized pustular psoriasis and other inflammatory skin conditions, paralleling the enhanced inflammatory phenotypes observed in Il36rn−/− mice.

• The enhanced NET formation observed in Il36rn−/− mice provides mechanistic insights into neutrophil-driven inflammation in human IL36RN-deficient conditions.

• Delayed wound healing in Il36rn−/− mice may explain compromised tissue repair in patients with IL36RN mutations.

• The efficacy of TLR4 inhibition (TAK-242) and NET inhibition (Cl-amidine) in mouse models suggests potential therapeutic approaches for human IL36RN-associated disorders.

• Cytokine and chemokine expression patterns in Il36rn−/− mice may help identify biomarkers and therapeutic targets for human conditions.

These translational connections highlight the value of IL36RN mouse models for understanding human disease pathophysiology and developing targeted interventions .

What are the implications of the IL36RN mouse model findings for developing treatments for psoriasis and other inflammatory skin conditions?

The findings from IL36RN mouse models have several important implications for developing treatments for psoriasis and other inflammatory skin conditions:

• NET-targeting therapies: The demonstration that NET inhibition with Cl-amidine ameliorates inflammatory responses suggests that targeting NETs could be a viable therapeutic approach.

• TLR4 inhibition: The efficacy of TAK-242 in normalizing wound healing indicates that TLR4 antagonists might be beneficial for treating IL36RN-associated disorders.

• Cytokine/chemokine targeting: The identification of key cytokines and chemokines that are dysregulated in Il36rn−/− mice (IL-1β, IL-17A, CXCL1, CXCL2, IL-36γ) provides potential targets for therapeutic intervention.

• Combination therapies: The complex inflammatory cascade in IL36RN deficiency suggests that combination therapies targeting multiple pathways might be most effective.

• Personalized approaches: The specific inflammatory signature of IL36RN deficiency indicates that patients with IL36RN mutations might benefit from tailored therapeutic strategies distinct from those used for other forms of psoriasis.

These implications highlight the potential for translating findings from IL36RN mouse models into novel therapeutic approaches for human inflammatory skin conditions .