IL36A Mouse

What is IL-36α and how does it function within the IL-1 cytokine family in mice?

IL-36α belongs to the IL-1 superfamily and is part of the IL-36 subfamily, which includes three agonists (IL-36α, IL-36β, IL-36γ) and one antagonist (IL-36Ra). All IL-36 isoforms bind to the IL-36 receptor (IL-36R) . When IL-36α binds to IL-36R, it recruits the IL-1 receptor accessory protein (IL-1RAcP) and activates downstream signaling pathways mediated by nuclear transcription factor kappa B and mitogen-activated protein kinase . This activates inflammatory cascades resulting in pro-inflammatory cytokine and chemokine production .

IL-36α functions independently of other IL-1 family members, as demonstrated by studies showing that intratracheal instillation of IL-36α can induce neutrophil influx in both wild-type C57BL/6 mice and IL-1αβ(-/-) mice . This functional independence makes IL-36α an important target for specific therapeutic interventions in inflammatory diseases.

Which cell types express IL-36α and IL-36R in mouse models?

In mouse models, IL-36α is expressed by a diverse range of cell types:

• Monocytes and B cells

• T cells (notably, IL-36α is the only novel IL-1 family member expressed on T-cells)

• Neutrophils (identified as a source in lung inflammation models)

• Epithelial cells in skin and lung tissues

The IL-36 receptor (IL-36R) expression has been documented in:

• Bone marrow-derived dendritic cells

• CD4+ T cells

• Monocytes and myeloid dendritic cells (mDCs)

• Monocyte-derived dendritic cells (MDDCs)

• Keratinocytes

This expression pattern explains the wide-ranging effects of IL-36α across multiple immune and non-immune cell types in inflammatory responses.

How does the regulation of IL-36α differ from other inflammatory cytokines in mice?

IL-36α regulation involves unique transcriptional control mechanisms that distinguish it from other inflammatory cytokines. One notable aspect is the regulation by C/EBPβ (CCAAT/enhancer-binding protein β), which binds specifically to a half-CRE- C/EBP motif in the IL-36α promoter to induce expression upon lipopolysaccharide (LPS) stimulation .

Interestingly, C/EBPβ binding to this regulatory element is insensitive to CpG methylation, allowing robust expression regardless of the methylation status of the promoter . This differs from many other cytokines whose expression is highly regulated by epigenetic modifications.

Additionally, IL-36α can be regulated by other inflammatory cytokines, creating feedback loops. IL-1α can induce IL-36α expression, and in return, IL-36α may regulate IL-1α in mouse keratinocytes . Similarly, IL-22, IL-17A, and TNF-α can induce the production of IL-36 subfamily members in human systems, suggesting similar regulatory mechanisms may exist in mice .

What are effective mouse models for studying IL-36α-mediated inflammation?

Several experimental mouse models have proven effective for studying IL-36α-mediated inflammation:

How should researchers quantify IL-36α-induced neutrophilic inflammation in mouse lungs?

Quantifying IL-36α-induced neutrophilic inflammation in mouse lungs requires a multi-parameter approach:

• Bronchoalveolar lavage (BAL) analysis:

  • Collect BAL fluid and perform total and differential cell counts

  • Quantify absolute neutrophil numbers and percentage of total cells

  • Measure neutrophil elastase or myeloperoxidase activity in BAL fluid

• Lung tissue analysis:

  • Perform histological assessment with H&E staining and immunohistochemistry

  • Score inflammatory infiltrates on a standardized scale

  • Use immunofluorescence to co-localize neutrophils with other cell types

• Gene expression analysis:

  • Quantify mRNA expression of neutrophil chemokines CXCL1 and CXCL2

  • Measure neutrophil-associated genes (e.g., Ly6G, elastase)

  • Assess IL-36α and IL-36R expression levels to evaluate potential feedback loops

• Protein measurements:

  • Perform ELISA or multiplex cytokine assays to measure chemokines in BAL fluid

  • Use Western blotting to assess activation of signaling pathways in lung tissue

  • Quantify neutrophil-derived proteins such as calprotectin

• Flow cytometry:

  • Analyze single-cell suspensions from digested lungs

  • Identify neutrophils using markers such as CD11b and Ly6G

  • Assess activation status with markers like CD66

Research shows that IL-36α significantly increases neutrophil numbers and CXCL1/CXCL2 expression in mouse lungs, making these key parameters to monitor .

What methodological considerations should be taken when using IL-36α or IL-36R knockout mice?

When using IL-36α or IL-36R knockout mice, researchers should consider several methodological aspects:

• Knockout verification:

  • Confirm gene deletion by genotyping

  • Verify protein absence using Western blot or ELISA

  • Check for compensatory upregulation of related cytokines (IL-36β, IL-36γ)

• Strain background effects:

  • Use mice backcrossed to a consistent genetic background (typically C57BL/6)

  • Include appropriate wild-type littermate controls

  • Consider potential strain-dependent variations in inflammatory responses

• Functional redundancy:

  • IL-36R knockout eliminates signaling from all IL-36 family members

  • IL-36α knockout may show partial phenotypes due to compensation by IL-36β/γ

  • Consider using combined knockouts or neutralizing antibodies to address redundancy

• Developmental effects:

  • Assess whether developmental abnormalities exist in knockout mice

  • Consider using conditional knockout models to avoid developmental adaptations

  • Compare results with pharmacological inhibition approaches

• Baseline characterization:

  • Evaluate steady-state immune parameters before experimental manipulation

  • Check for pre-existing differences in cell populations

  • Document any spontaneous phenotypes in unchallenged knockout mice

Research has shown that IL-36α knockout mice display reduced neutrophil recruitment to the epidermis and dermis, along with downregulated CXCL1 generation in inflammatory skin conditions, demonstrating the value of these models for understanding IL-36α function .

How does IL-36α signaling drive neutrophilic inflammation in mouse models?

IL-36α drives neutrophilic inflammation through multiple interconnected mechanisms in mouse models:

• Direct chemokine induction: IL-36α stimulation increases expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs, creating a chemotactic gradient for neutrophil recruitment .

• Activation of CD11c+ cells: IL-36α stimulates dendritic cells and macrophages to produce neutrophil-specific chemokines and TNFα, amplifying the inflammatory response. Additionally, IL-36α enhances expression of the co-stimulatory molecule CD40 on these cells .

• Positive feedback loops: Intratracheal IL-36α enhances expression of its own receptor (IL-36R) in the lungs, creating a self-amplifying inflammatory circuit .

• Synergistic effects: IL-36α cooperates with GM-CSF and viral mimics like poly(I:C) to promote activation of neutrophils, macrophages, and fibroblasts, further enhancing inflammation .

• NF-κB activation: Stimulation with IL-36α activates NF-κB in mouse macrophage cell lines, triggering pro-inflammatory gene expression .

• Cross-talk with adaptive immunity: IL-36α enhances the ability of dendritic cells to induce CD4+ T cell proliferation, linking innate and adaptive immune responses .

This multi-faceted role positions IL-36α as a crucial upstream amplifier of neutrophilic inflammation, particularly in lung diseases characterized by neutrophil infiltration.

What is known about the transcriptional regulation of IL-36α in mouse models?

Transcriptional regulation of IL-36α in mouse models involves several key mechanisms:

• C/EBPβ-mediated regulation: The transcription factor C/EBPβ binds specifically to a half-CRE- C/EBP motif in the IL-36α promoter to induce expression following LPS stimulation . This binding occurs regardless of CpG methylation status, allowing robust expression in different cellular contexts.

• LPS-induced expression: Lipopolysaccharide is a potent inducer of IL-36α transcription in mouse macrophages, suggesting a role in bacterial infection responses .

• Cytokine networks: In mouse keratinocytes, IL-1α can induce IL-36α expression, creating interconnected cytokine networks. The levels of IL-36α from inflamed IL-1R1−/− skin were significantly lower than those of wild-type mice, suggesting IL-1 receptor signaling contributes to IL-36α regulation .

• Cell type-specific mechanisms: Studies show differential methylation of the IL-36α promoter in the RAW264.7 macrophage cell line compared to primary murine macrophages, yet similar IL-36α mRNA and protein expression levels were observed following stimulation .

• Viral and bacterial stimuli: Beyond LPS, other pathogen-associated molecular patterns likely regulate IL-36α expression, as evidenced by its role in models combining cigarette smoke exposure and H1N1 influenza virus infection .

Understanding these regulatory mechanisms provides insights into how IL-36α expression is controlled during inflammatory responses and potential points for therapeutic intervention.

How does IL-36α interact with other cytokines in inflammatory cascades?

IL-36α engages in complex interactions with other cytokines within inflammatory cascades:

• IL-1 family cross-regulation: IL-36α and IL-1α appear to regulate each other in a feedback loop. IL-1α can induce IL-36α expression, and conversely, IL-36α stimulation leads to increased IL-1α production in mouse keratinocytes .

• Synergy with GM-CSF: IL-36α cooperates with GM-CSF to promote activation of neutrophils, macrophages, and fibroblasts, amplifying inflammatory responses in the lung .

• Enhancement of viral responses: IL-36α collaborates with the viral mimic poly(I:C) to potentiate inflammatory responses, suggesting an important role during viral infections .

• Independence from IL-1α/β: Studies in IL-1αβ(-/-) mice demonstrated that IL-36α can induce neutrophil influx and chemokine expression independently of IL-1α and IL-1β, highlighting its non-redundant role in inflammation .

• Th17 pathway connections: IL-36α can induce expression of IL-17A signaling-related genes, suggesting cross-talk with the Th17 inflammatory axis which is crucial in several autoimmune conditions .

• Pro-inflammatory cytokine induction: In mouse monocytes, IL-36 stimulation significantly upregulates expression of IL-1α, IL-1β, and IL-6, creating a broader cytokine response .

These interactions position IL-36α as an important node in inflammatory networks, with the ability to amplify and sustain inflammatory responses through multiple cytokine pathways.

How does IL-36α contribute to psoriasis-like inflammation in mouse models?

IL-36α plays several critical roles in psoriasis-like inflammation in mouse models:

• Disease exacerbation: In the imiquimod-induced psoriasis mouse model, IL-36α injection contributes to the development of severe skin lesions, demonstrating its pathogenic role .

• Neutrophil recruitment: IL-36α knockout (-/-) mice show reduced neutrophil infiltration in the epidermis and dermis following inflammatory stimuli, indicating its importance in the neutrophilic component of psoriasiform inflammation .

• Chemokine regulation: IL-36α deficiency results in downregulated CXCL1 generation, a key neutrophil-attracting chemokine in psoriatic lesions .

• Therapeutic target validation: Psoriatic mice treated with IL-36R-blocking antibodies show improved psoriatic dermatitis, providing evidence for IL-36 pathway inhibition as a therapeutic approach .

• Genetic associations: IL-36 signaling-related genes are enriched within psoriasis susceptibility loci, supporting a genetic basis for IL-36α involvement in psoriasis .

• Cytokine network amplification: IL-36α participates in inflammatory loops with other psoriasis-relevant cytokines. For example, IL-22, IL-17A, and TNF-α induce IL-36 production in keratinocytes, while IL-36α can promote IL-17A signaling pathways .

These findings from mouse models have helped establish IL-36α as a key mediator in psoriasis pathogenesis and have supported the development of targeted therapies against the IL-36 pathway, particularly for pustular forms of psoriasis.

What is the role of IL-36α in mouse models of lung inflammation?

IL-36α serves as a critical mediator in mouse models of lung inflammation:

• Key upstream amplifier: Research has identified IL-36α as a key upstream amplifier of neutrophilic lung inflammation, promoting activation of neutrophils, macrophages and fibroblasts .

• Direct neutrophil recruitment: Intratracheal instillation of recombinant IL-36α induces neutrophil influx in the lungs of both wild-type C57BL/6 mice and IL-1αβ(-/-) mice .

• Chemokine induction: IL-36α stimulation increases mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs, driving neutrophil infiltration .

• Synergistic pathways: IL-36α cooperates with GM-CSF and viral mimic poly(I:C) to amplify lung inflammation, suggesting important roles during respiratory infections .

• Cigarette smoke responses: IL-36 receptor deficient mice exposed to cigarette smoke show attenuated lung inflammation compared with wild-type controls, implicating IL-36α in smoking-related lung diseases .

• Viral exacerbations: IL-36 receptor deficient mice exposed to cigarette smoke and H1N1 influenza virus have attenuated lung inflammation, suggesting a role in viral exacerbations of chronic lung diseases .

• Self-amplification: Intratracheal IL-36α enhances mRNA expression of its own receptor (IL-36R) in the lungs, creating a positive feedback loop that magnifies inflammatory responses .

These findings provide a rationale for targeting IL-36α in various neutrophilic lung diseases, including chronic obstructive pulmonary disease (COPD), severe asthma, and respiratory infections.

How can IL-36α antagonism be achieved in mouse models and what therapeutic effects have been observed?

Several approaches to IL-36α antagonism have been implemented in mouse models, yielding significant therapeutic effects:

• IL-36R blocking antibodies: Treatment of psoriatic mice with IL-36R-blocking antibodies improves psoriatic dermatitis, demonstrating the potential of this approach .

• Genetic deletion models: IL-36α knockout mice show reduced neutrophil recruitment and chemokine production in inflammatory conditions, validating IL-36α as a therapeutic target .

• IL-36Ra administration: Recombinant IL-36 receptor antagonist (IL-36Ra) administration can inhibit IL-36 signaling. IL-36Ra binds to IL-36R but inhibits recruitment of IL-1RAcP, blocking downstream signaling pathways .

• Small molecule inhibitors: While still in development, targeting downstream signaling components such as MyD88 or IRAK proteins can inhibit IL-36α-mediated responses.

• Combined pathway inhibition: Simultaneous targeting of IL-36α and synergistic pathways (e.g., GM-CSF) has shown enhanced anti-inflammatory effects in lung inflammation models .

Therapeutic effects observed with these approaches include:

• Reduced neutrophilic infiltration in skin and lung tissues

• Decreased pro-inflammatory cytokine and chemokine production

• Attenuated tissue damage and remodeling

• Improved disease scores in psoriasis models

• Protection from cigarette smoke-induced lung inflammation

These findings from mouse models have translated to human studies, with monoclonal antibodies against IL-36R showing promise in clinical trials for pustular psoriasis patients .

What are the optimal methods for detecting and quantifying IL-36α in mouse samples?

Several complementary methods can be used for detecting and quantifying IL-36α in mouse samples:

For comprehensive analysis, researchers should use multiple complementary approaches. The Mouse IL-36 alpha/IL-1F6 DuoSet ELISA from R&D Systems provides a validated tool for quantitative measurements in mouse samples .

What are the best approaches for studying IL-36α signaling mechanisms in mouse cells?

Studying IL-36α signaling mechanisms in mouse cells requires several specialized approaches:

• Phosphorylation assays:

  • Western blotting for phosphorylated signaling proteins (p38 MAPK, JNK, ERK)

  • Phospho-flow cytometry for single-cell analysis of signaling activation

  • Phospho-proteomics to identify novel signaling targets

• Transcriptional regulation:

  • Chromatin immunoprecipitation (ChIP) to study transcription factor binding to IL-36α-regulated genes

  • Reporter assays with promoter constructs to identify regulatory elements

  • Analysis of histone modifications at IL-36α-responsive genes

• Receptor studies:

  • Binding assays with labeled recombinant IL-36α

  • Co-immunoprecipitation of IL-36R with IL-1RAcP following stimulation

  • CRISPR/Cas9 modification of receptor components

• Gene expression profiling:

  • RNA-sequencing of IL-36α-stimulated versus unstimulated cells

  • Pathway analysis to identify signaling networks

  • Comparison of wild-type versus IL-36R-deficient cells

• Genetic approaches:

  • Knockdown studies using siRNA targeting specific signaling components

  • Overexpression of dominant-negative mutants of signaling molecules

  • Pharmacological inhibitors of specific signaling pathways

• Cell-specific analyses:

  • Isolation of specific cell populations from IL-36α-treated mice

  • Single-cell RNA-seq to identify cell-specific responses

  • Conditional knockout of signaling components in specific cell types

Research has shown that IL-36α signaling activates NF-κB in mouse macrophage cell lines and induces p38-MAPK signaling-related genes, making these important pathways to investigate .

How should researchers design IL-36α dose-response studies in mouse models?

Designing effective IL-36α dose-response studies in mouse models requires careful methodological considerations:

• Dose range determination:

  • For intratracheal administration, test a range from 0.1-10 μg per mouse

  • Include at least four different doses to establish a complete dose-response curve

  • Use both sub-threshold and saturating doses to capture the full response range

• Time course considerations:

  • Perform both acute (hours) and extended (days) timepoints

  • Include measurements at 2h, 6h, 24h, and 48h post-administration

  • Consider multiple administrations for chronic models

• Readout selection:

  • Primary readouts: neutrophil counts, chemokine levels (CXCL1, CXCL2)

  • Secondary readouts: tissue histology, gene expression profiles

  • Functional measures: lung function tests, behavioral assessments

• Controls and validation:

  • Include inactive protein controls (heat-inactivated IL-36α)

  • Use IL-36R knockout mice to confirm specificity

  • Compare with positive controls (LPS, TNF-α) at established doses

• Route of administration considerations:

  • For lung studies: intratracheal, intranasal, or aerosolized delivery

  • For skin studies: intradermal or topical application

  • For systemic effects: intraperitoneal or intravenous administration

• Synergy assessment:

  • Combine IL-36α with other stimuli (GM-CSF, poly(I:C))

  • Test multiple combinations of concentrations

  • Calculate combination indices to quantify synergistic effects

• Statistical analysis:

  • Use appropriate statistical methods for dose-response curves (e.g., four-parameter logistic regression)

  • Calculate EC50 values to compare potency across different readouts

  • Consider area-under-curve analyses for time-dependent responses

Research has shown that intratracheal instillation of IL-36α induces dose-dependent neutrophil influx and chemokine expression in mouse lungs, providing a foundation for dose-response study designs .