IL36B Mouse

What is IL-36β and how does it function in the mouse immune system?

IL-36β is one of three agonistic cytokines within the IL-36 subfamily (alongside IL-36α and IL-36γ) of the larger IL-1 cytokine family. In mice, IL-36β functions as a pro-inflammatory mediator that signals through the IL-36 receptor (IL-36R) and requires IL-1RAcP as a co-receptor. Upon binding, this complex initiates downstream signaling primarily through activation of the NF-κB and MAPK pathways, resulting in the production of inflammatory mediators and chemokines that recruit immune cells to sites of inflammation . The mouse IL36B gene is located on chromosome 2 as part of a genomic cluster that includes other IL-1 family members .

How does mouse IL-36β differ structurally and functionally from other IL-36 family members?

While all three IL-36 agonists (α, β, and γ) signal through the same receptor complex, they exhibit differences in expression patterns and potency. In mouse models, IL-36β shares approximately 65-70% amino acid sequence identity with IL-36α and IL-36γ. All three require N-terminal processing for full biological activity, though the specific proteases responsible may differ. Expression analysis shows that IL-36β and IL-36γ are often co-expressed in response to inflammatory stimuli in epithelial cells, while their relative potency in activating downstream signaling pathways can vary by cell type and context .

What is known about the regulation of IL-36β expression in mice?

Expression of IL-36β mRNA is induced in mouse epithelial cells in response to pro-inflammatory stimuli including IL-1β, TNF, PMA, and bacterial components like flagellin. Notably, a synergistic effect occurs when TNF is combined with either PMA or flagellin, resulting in significantly enhanced IL-36β expression. Unlike some cytokines, IL-36β does not appear to induce its own expression in an autocrine manner in mouse models. At the transcriptional level, regulation involves NF-κB-dependent mechanisms, while post-translational regulation includes proteolytic processing of the inactive precursor to generate the fully active form .

What mouse models are most appropriate for studying IL-36β functions?

Several mouse models have proven valuable for studying IL-36β biology:

For acute inflammation models, cigarette smoke exposure with or without H1N1 influenza virus has shown significant differences between wild-type and IL-36R-deficient mice, making it a useful model to study IL-36 family members including IL-36β .

What are the optimal methods for detecting IL-36β expression in mouse tissues?

Detection of IL-36β in mouse tissues requires specialized approaches due to its relatively low expression levels under baseline conditions:

• mRNA detection: RT-qPCR remains the gold standard for quantifying IL-36β transcripts, with appropriate normalization to housekeeping genes. Primers should be designed to distinguish between IL-36β and other IL-36 family members due to sequence similarity .

• Protein detection: Western blotting using specific anti-IL-36β antibodies, often requiring immunoprecipitation to enrich for the protein. Importantly, researchers should consider the form being detected (full-length versus processed) as this affects biological activity .

• Immunohistochemistry/Immunofluorescence: For tissue localization, though signal amplification may be necessary. Controls using IL-36β knockout tissue are essential to confirm specificity .

• Reporter systems: When available, reporter mice expressing fluorescent proteins under the IL-36β promoter provide valuable tools for visualizing expression patterns .

For optimal results, stimulation with appropriate inducers (TNF plus PMA or bacterial components) is recommended when studying IL-36β expression in experimental settings .

How can researchers effectively generate and validate recombinant mouse IL-36β for experimental studies?

Generating biologically active recombinant mouse IL-36β requires specific considerations:

• Expression system selection: Bacterial systems (E. coli) can produce high yields but lack post-translational modifications. Mammalian expression systems may provide better folding but lower yields.

• N-terminal processing: Critical for activity, as full-length IL-36β has significantly lower biological activity. Express truncated forms beginning at key residues (equivalent to human n6 or n5 positions) or perform controlled proteolytic processing of full-length protein .

• Purification strategy: Affinity tags (His or GST) followed by size exclusion chromatography to ensure homogeneity.

• Activity validation: Functional testing using reporter cell lines expressing the mouse IL-36 receptor and an NF-κB reporter construct. The HT-29 cell line stably transfected with an NF-κB reporter gene has been used successfully for this purpose .

• Endotoxin removal: Critical for in vivo applications to prevent confounding inflammatory responses.

EC50 determination using dose-response curves is essential for comparing potency between different preparations and forms of IL-36β .

How do neutrophil-derived proteases regulate IL-36β activity in mouse models of inflammation?

Neutrophils play a dual role in IL-36β biology in mouse inflammation models - both as sources of IL-36 cytokines and as providers of proteases that can process and activate IL-36β. Research suggests that neutrophil serine proteases, including cathepsin G, elastase, and proteinase-3, can cleave the N-terminus of IL-36β, potentially at different sites than those identified through in vitro studies .

Methodologically, researchers investigating this interaction should:

• Use neutrophil depletion models (anti-Ly6G antibody treatment) to assess impact on IL-36β processing and activity

• Employ specific protease inhibitors in vivo and ex vivo to identify which neutrophil proteases are primarily responsible

• Generate site-directed mutagenesis variants of mouse IL-36β with altered predicted cleavage sites to determine processing specificity

• Use mass spectrometry to identify precise cleavage products in inflammatory exudates from mouse models

This research direction is particularly relevant for understanding the amplification of neutrophilic inflammation in conditions like COPD, where IL-36β processing may create positive feedback loops .

What are the molecular mechanisms by which IL-36β synergizes with other inflammatory mediators in mouse models?

IL-36β exhibits significant synergistic effects with other inflammatory mediators, particularly GM-CSF and viral mimics like poly(I:C), to amplify inflammatory responses in mouse models . The molecular basis for this synergy involves:

• Receptor cross-talk: IL-36R signaling may enhance expression or signaling capacity of receptors for other cytokines through shared adapter molecules like MyD88

• Convergent downstream pathways: Both IL-36β and GM-CSF activate overlapping but distinct aspects of the NF-κB and MAPK pathways, potentially leading to enhanced and sustained activation

• Cooperative transcription factor activation: Combined stimulation may recruit additional transcription factors or coactivators to inflammatory gene promoters

To investigate these mechanisms, researchers should employ:

• Phospho-proteomic analysis to identify shared and distinct signaling nodes

• ChIP-seq to map transcription factor binding patterns under single versus combined stimulation

• Proximity ligation assays to detect physical interactions between receptor components

• Single-cell RNA-seq to identify cell populations that are particularly responsive to the synergistic effects

Understanding this synergy is particularly important in mixed inflammatory environments such as viral-bacterial co-infections of the lung .

What is the temporal relationship between IL-36β expression and neutrophil recruitment in acute versus chronic mouse inflammation models?

The temporal dynamics between IL-36β expression and neutrophil recruitment represent a complex relationship with potential feedback loops. In acute inflammation models:

• Initial inflammatory triggers (pathogens, tissue damage) induce IL-36β expression primarily from epithelial cells

• IL-36β signaling promotes chemokine production (particularly CXCL1 and CXCL2 in mice)

• Neutrophils are recruited to the inflammatory site

• Recruited neutrophils can both produce additional IL-36β and process existing IL-36β to more active forms

• This creates a potential amplification circuit

In chronic models, such as repeated cigarette smoke exposure, this relationship becomes more complex, with:

• Sustained IL-36β expression from structural cells

• Persistent neutrophil recruitment and activation

• Tissue remodeling that may alter the cellular sources and responsiveness to IL-36β

Appropriate experimental approaches include:

• Time-course analyses with frequent sampling in both acute and chronic models

• Cell-specific conditional knockouts to determine the relative contribution of epithelial versus neutrophil-derived IL-36β

• Neutrophil transfer experiments using labeled cells to track their activation and cytokine production

How can researchers effectively distinguish between the biological activities of different IL-36 family members in mouse models?

Distinguishing the specific contributions of IL-36β from other family members presents a significant challenge due to shared receptors and similar biological activities. Advanced methodological approaches include:

• Selective neutralization: Using highly specific monoclonal antibodies against mouse IL-36β that don't cross-react with IL-36α or IL-36γ

• Cytokine replacement studies: Using IL-36β knockout mice reconstituted with recombinant IL-36β versus other family members

• Receptor mutants: Developing receptor variants with altered affinity for specific IL-36 family members

• Differential expression analysis: Comprehensive temporal profiling of all IL-36 family members during inflammatory responses to identify unique expression patterns

• Conditional deletion models: Cell-type specific deletion of IL-36β to determine tissue-specific roles

What are the most reliable protocols for studying IL-36β processing in mouse tissues and cell cultures?

Studying IL-36β processing in biological samples requires specialized techniques due to the low abundance of processed forms and technical challenges in their detection:

• Ex vivo processing assays: Incubate recombinant full-length mouse IL-36β with cellular fractions or purified enzymes, then analyze by:

  • Western blotting with antibodies specific to different regions

  • Mass spectrometry to identify precise cleavage sites

  • Functional assays using reporter cell lines to assess activity

• In vivo processing detection:

  • Immunoprecipitation with N-terminal versus C-terminal antibodies

  • Differential extraction protocols to enhance recovery of processed forms

  • Analysis of biological fluids (BAL, inflammatory exudates) by western blotting or mass spectrometry

• Live-cell imaging approaches:

  • Split fluorescent protein constructs that change localization or FRET properties upon processing

  • Activity-based probes that detect active proteases capable of processing IL-36β

A common methodological error is attempting to induce processing with inappropriate stimuli. Research indicates that classical inflammatory stimuli (TNF, IL-1) induce expression but not necessarily processing of IL-36β, while certain apoptotic stimuli (cycloheximide, staurosporine) may induce mobility changes suggesting processing .

How should researchers address the variability in IL-36β responses across different mouse strains?

Strain-dependent variability in IL-36β responses represents an important consideration in experimental design. Key methodological recommendations include:

• Strain selection considerations:

  • C57BL/6 mice: Most commonly used, with well-characterized IL-36 pathway components

  • BALB/c mice: May exhibit different T helper cell polarization affecting IL-36β responses

  • Background matching: Ensure knockout and control mice are on identical genetic backgrounds

• Experimental controls and reporting:

  • Always report complete strain information including substrain (e.g., C57BL/6J versus C57BL/6N)

  • Include wild-type littermate controls whenever possible

  • Consider using multiple strains for critical findings to establish generalizability

• Technical approaches to address variability:

  • Increased sample sizes based on power calculations from preliminary data

  • Mixed-effects statistical models that account for strain as a variable

  • Ex vivo validation using cells derived from different strains

• Mechanistic investigation of strain differences:

  • Sequence the IL-36β gene and its promoter regions across strains to identify polymorphisms

  • Compare IL-36 receptor expression levels and signaling responses

  • Evaluate strain-dependent differences in protease expression that might affect processing

Understanding strain variability not only improves experimental reproducibility but may also provide insights into genetic factors influencing IL-36β biology relevant to human disease heterogeneity.

What are the key differences between mouse and human IL-36β that researchers should consider when designing translational studies?

Understanding the species-specific differences in IL-36β biology is crucial for translational research. Key comparative aspects include:

For translational research, humanized mouse models expressing human IL-36 pathway components provide valuable tools to bridge these species differences. Additionally, parallel studies using both mouse and human primary cells are recommended to validate key findings across species .

How can mouse models of IL-36β biology inform therapeutic targeting strategies for human inflammatory diseases?

Mouse models have provided valuable insights for therapeutic targeting of the IL-36 pathway, with several key translational implications:

• Target identification and validation:

  • IL-36R antagonism: Mouse models of psoriasiform inflammation and neutrophilic lung inflammation support IL-36R as a therapeutic target

  • Processing inhibition: Understanding of mouse IL-36β processing suggests targeting specific proteases may prevent IL-36β activation

  • Signaling modulation: Mouse studies reveal key nodes in IL-36β signaling that might be targetable

• Therapeutic modality selection:

  • Biologics: Monoclonal antibodies against IL-36β or IL-36R validated in mice have translational potential

  • Small molecules: Inhibitors of processing enzymes or downstream signaling components

  • Cell-based therapies: Approaches targeting IL-36β-producing or responsive cells

• Biomarker development:

  • Mouse studies suggest processed forms of IL-36β or downstream products as potential biomarkers

  • Neutrophil activation markers correlate with IL-36 activity in mouse models

  • Combined biomarker panels may predict responsiveness to IL-36 pathway inhibition

• Potential clinical applications:

  • Pustular psoriasis: Strong genetic evidence links IL-36 pathway to human disease

  • COPD/neutrophilic asthma: Mouse models suggest IL-36β as an amplifier of neutrophilic inflammation

  • Inflammatory bowel disease: Emerging evidence for IL-36β in intestinal inflammation

The role of IL-36β as an upstream amplifier of neutrophilic inflammation in mouse models provides particular rationale for targeting this pathway in human neutrophil-mediated diseases .

What are the appropriate considerations when attempting to replicate human IL-36β-associated disease phenotypes in mouse models?

Effective disease modeling requires careful consideration of species differences and methodological approaches:

• Anatomical and physiological considerations:

  • Skin differences: Mouse skin is thinner with different hair follicle density affecting psoriasiform models

  • Lung architecture: Fewer airway generations in mice affecting respiratory disease models

  • Immune system: Different baseline neutrophil percentages and inflammatory mediator profiles

• Disease induction strategies:

  • Genetic approaches: Overexpression of processed IL-36β or deletion of IL-36Ra

  • Environmental triggers: Cigarette smoke, TLR ligands, or combined stimuli that better recapitulate complex human exposures

  • Humanized models: Reconstitution with human immune cells or expression of human IL-36 pathway components

• Assessment parameters:

  • Comprehensive phenotyping: Beyond gross pathology to molecular and cellular parameters

  • Temporal dynamics: Acute versus chronic phase analysis

  • Therapeutic response: Testing standard-of-care human treatments as benchmarks

• Limitations and interpretations:

  • Acknowledge differences in disease time course between mice and humans

  • Consider that successful treatment in mice may overpredict human efficacy

  • Validate key findings across multiple model systems when possible

Combined environmental exposures (such as cigarette smoke plus viral infection) often better recapitulate the complexity of human disease than single-trigger models, particularly for IL-36β-mediated conditions with multiple inflammatory inputs .

What emerging technologies are most promising for advancing our understanding of IL-36β biology in mouse models?

Several cutting-edge technologies offer significant potential for advancing IL-36β research:

• Single-cell multi-omics:

  • scRNA-seq to identify specific cellular sources and responders to IL-36β

  • Single-cell proteomics to detect low-abundance processed forms of IL-36β

  • Spatial transcriptomics to map IL-36β expression in tissue microenvironments

• CRISPR-based technologies:

  • Base editing for precise modification of processing sites or regulatory elements

  • CRISPRi/CRISPRa for temporal control of IL-36β expression

  • CRISPR screening to identify novel regulators of IL-36β processing or signaling

• Advanced imaging approaches:

  • Intravital microscopy with fluorescent reporter systems to track IL-36β expression in live animals

  • Multiplexed imaging mass cytometry to simultaneously visualize multiple IL-36 pathway components

  • Label-free imaging techniques to assess tissue structural changes in response to IL-36β

• Organoid and microphysiological systems:

  • Mouse-derived epithelial organoids for studying IL-36β in controlled tissue-like environments

  • Organ-on-chip systems incorporating multiple cell types for complex interaction studies

  • Co-culture systems with defined cellular components to dissect intercellular communication

These technologies promise to provide unprecedented resolution in understanding the spatiotemporal dynamics of IL-36β expression, processing, and signaling in complex inflammatory environments .

How might the study of IL-36β in mouse models contribute to our understanding of inflammatory disease heterogeneity?

The IL-36 pathway represents an important axis for understanding inflammatory disease heterogeneity. Mouse models can contribute in several ways:

• Identification of inflammatory endotypes:

  • IL-36β-high versus IL-36β-low inflammatory responses may define distinct disease subsets

  • Different processing mechanisms may contribute to disease variation

  • Distinct cellular sources of IL-36β might characterize different disease phenotypes

• Genetic and environmental interaction studies:

  • Models examining how IL-36β interacts with other genetic risk factors

  • Investigation of how environmental exposures modulate IL-36β responses

  • Development of combinatorial models that better reflect human disease complexity

• Response prediction:

  • Identification of biomarkers that predict responsiveness to IL-36 pathway inhibition

  • Characterization of compensatory mechanisms in IL-36β-deficient conditions

  • Understanding of resistance mechanisms to IL-36 pathway targeting

• Precision medicine approaches:

  • Definition of IL-36β-dependent versus IL-36β-independent disease variants

  • Stratification markers for patient selection in clinical trials

  • Combination therapy approaches based on mechanistic understanding

Mouse models incorporating genetic diversity (such as the Collaborative Cross or Diversity Outbred mice) may be particularly valuable for capturing the heterogeneity relevant to human disease .