IL 33 Mouse

What is IL-33 and how does it function in mice?

IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor. In mouse models, IL-33 drives cytokine production in multiple immune cell types, including mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells) .

The protein functions as a nuclear alarmin that alerts the innate immune system after tissue injury or infection, particularly in epithelial barrier tissues . Unlike many cytokines, endogenous IL-33 protein is constitutively expressed and stored in the nucleus of producing cells, with release occurring primarily during cell damage or stress .

Where is IL-33 primarily expressed in mouse tissues?

Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues. Using both the Il-33–LacZ gene trap reporter strain (Il-33 Gt/Gt) and immunostaining with anti-IL-33 antibodies, researchers have demonstrated that IL-33 is constitutively expressed in:

• Stratified squamous epithelia (vagina and skin)

• Cuboidal epithelium (lung, stomach, and salivary gland)

• Lymphoid organs

• Brain tissues

• Embryonic tissues

Notably, unlike in humans, constitutive expression of IL-33 is not detected in mouse blood vessels, highlighting important species-specific differences .

What mouse models are available for studying IL-33 function?

Several mouse models have been developed for IL-33 research:

• IL-33 Reporter Mice: The Il-33–LacZ gene trap reporter strain (Il-33 Gt/Gt) allows visualization of IL-33 promoter activity through β-galactosidase expression .

• IL-33 Knockout Mice: The Il-33 (Gt/Gt) mouse is also IL-33-deficient, making it useful as both a reporter and a functional knockout model .

• OVA-IL-33 Airway Inflammation Model: This model uses ovalbumin-sensitized C57BL/6 mice exposed to IL-33 before each OVA challenge to study exacerbation of allergic airway responses .

• LPS-Induced Endotoxin Shock Model: Used to study IL-33 expression during systemic inflammation .

• Papain-Induced Allergic Airway Inflammation Model: Used to study IL-33 expression during type 2 immune responses in the lung .

Each model provides unique insights into IL-33 biology depending on the research question being addressed.

How can I reliably detect IL-33 expression in mouse tissue samples?

Multiple complementary approaches are recommended for comprehensive IL-33 detection:

For mRNA detection:

• RT-qPCR using specifically designed primers:

  • IL-33 sense primer: 5'-TGAGACTCCGTTCTGGCCTC-3'

  • IL-33 anti-sense primer: 5'-CTCTTCATGCTTGGTACCCGAT-3'

For distinguishing between IL-33 mRNA species:

• Use specific forward primers corresponding to different 5'UTRs:

  • mRNA species "A": 5'-GGGGCTCACTGCAGGAAAGTA-3' (AK075849.1)

  • mRNA species "B": 5'-CAGCTGCAGAAGGGAGAAAT-3' (AK163464.1)

For protein detection:

• Immunostaining with anti-IL-33 antibodies (goat polyclonal antibody recommended)

• Flow cytometry with biotin-conjugated secondary antibodies and fluorophore-conjugated streptavidin

• Always include IL-33-deficient tissues (Il-33 Gt/Gt mice) as negative controls to validate antibody specificity

What are the optimal conditions for working with recombinant mouse IL-33 protein?

When working with recombinant mouse IL-33 protein:

Reconstitution and Storage:

• Reconstitute lyophilized protein at 100 μg/mL in sterile PBS

• Store reconstituted protein at -20°C to -80°C

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

Formulation Considerations:

• Standard formulation contains BSA as a carrier protein to enhance stability

• Carrier-free versions are available when BSA might interfere with experiments

Effective Concentration Range:

• Biological activity is observed at 0.0125-0.05 ng/mL in many experimental systems

Protein Characteristics:

• E. coli-derived mouse IL-33 corresponds to Ser109-Ile266 of the full-length protein

How does IL-33 contribute to airway inflammation in mouse models?

IL-33 plays a critical role in exacerbating antigen-driven airway responses in mouse models of allergic asthma. When administered to sensitized mice before antigen challenge, IL-33:

• Synergizes with allergen exposure: IL-33 acts synergistically with ovalbumin in sensitized mice to aggravate airway inflammation, hyperresponsiveness, and remodeling .

• Enhances mast cell activity: IL-33 exposure elevates levels of local and systemic mast cell protease mMCP-1 .

• Promotes antigen-specific IgE production: IL-33 administration to sensitized mice increases allergen-specific IgE levels .

• Amplifies Th2 cytokine responses: IL-33 increases IL-4, IL-5, and IL-13 levels in allergen-challenged mice .

• Expands ILC2 populations: IL-33 exposure increases innate lymphoid cell type 2 (ILC2) numbers in the lung, suggesting these cells mediate part of IL-33's effects in airway inflammation .

These findings suggest that IL-33 may be a critical factor in asthma exacerbations and could represent a therapeutic target for intervention.

What is the significance of IL-33 nuclear localization in mouse tissues?

The consistent nuclear localization of IL-33 in mouse tissues has important functional implications:

• Alarmin function: Nuclear localization supports IL-33's role as a nuclear alarmin, which is released during cell damage or stress to alert the immune system .

• Transcriptional regulation: Nuclear IL-33 may directly regulate gene transcription, suggesting dual functionality as both a cytokine and a nuclear factor .

• Release mechanism: Unlike canonically secreted cytokines, IL-33 lacks a signal sequence and is primarily released during cellular damage or necrosis .

• Species-specific patterns: The exclusively nuclear localization in mouse tissues differs from human IL-33, which can sometimes be detected in the cytoplasm .

Researchers should note that in all examined mouse tissues, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization, highlighting its primary role as a nuclear factor under homeostatic conditions .

How do the transcriptional regulation patterns of IL-33 differ in response to various stimuli?

IL-33 expression in murine macrophages and fibroblasts is regulated by multiple pathways:

• TLR Ligand Responses:

  • Different Toll-like receptor ligands can induce distinct patterns of IL-33 mRNA expression

  • LPS (TLR4 ligand) stimulation significantly upregulates IL-33 transcription in macrophages

• Promoter Utilization:

  • Two distinct mRNA species (A and B) are transcribed from different promoters

  • Stimuli may preferentially activate one promoter over another depending on cell type and context

• Tissue-Specific Regulation:

  • Constitutive expression in epithelial barriers versus inducible expression in immune cells

  • Strong expression of the Il-33–LacZ reporter observed in inflamed tissues:

    • Liver during LPS-induced endotoxin shock

    • Lung alveoli during papain-induced allergic airway inflammation

This complex regulation suggests context-specific roles for IL-33 in different physiological and pathological settings.

What controls should be included when studying IL-33-mediated effects in mouse models?

Proper experimental controls are critical for IL-33 research:

Essential Controls for IL-33 Studies:

• Genetic Controls:

  • Include Il-33 (Gt/Gt) IL-33-deficient mice as negative controls for antibody specificity

  • Use ST2-deficient mice to confirm receptor dependency of observed effects

• Reagent Controls:

  • Heat-denatured IL-33 to control for potential contaminants in recombinant protein preparations

  • Vehicle controls matching the reconstitution buffer of recombinant IL-33

• Dose-Response Analysis:

  • Include multiple concentrations of IL-33 (typically 0.0125-0.05 ng/mL for in vitro studies)

  • Time-course experiments to distinguish between acute and sustained effects

• Pathway Inhibitors:

  • Include ST2 blocking antibodies to confirm receptor specificity

  • Use appropriate inhibitors of downstream signaling pathways to delineate mechanism

• Cell-Specific Controls:

  • In cell depletion studies, include isotype control antibodies

  • For cell transfer experiments, use cells from IL-33-unresponsive donors as controls

How can I distinguish between direct and indirect effects of IL-33 in inflammatory models?

Distinguishing direct vs. indirect IL-33 effects requires systematic approach:

• Cell-Specific Receptor Expression Analysis:

  • Flow cytometric assessment of ST2 expression on potential target cells

  • Sorting of ST2+ and ST2- populations for ex vivo functional assays

• In Vitro vs. In Vivo Comparison:

  • Compare effects of IL-33 on isolated cell populations in vitro

  • Validate findings in vivo with cell-specific ST2 knockout models

• Kinetic Analysis:

  • Monitor early (0-6h) vs. late (24-72h) responses

  • Early responses often represent direct effects, while late responses may include indirect mechanisms

• Cytokine Neutralization:

  • Use neutralizing antibodies against secondary mediators to block indirect effects

  • Compare outcomes with and without neutralization to quantify direct vs. indirect contributions

• Bone Marrow Chimeras:

  • Generate mice with ST2 deficiency restricted to either hematopoietic or non-hematopoietic compartments

  • Assess which cell compartment mediates IL-33 responses

What factors might affect IL-33 detection in mouse tissues?

Several technical factors can influence IL-33 detection:

• Antibody Selection:

  • Antibody specificity varies significantly between vendors

  • Always validate antibodies using IL-33-deficient tissues as negative controls

• Sample Preparation:

  • Nuclear localization requires effective nuclear permeabilization for immunostaining

  • Cell lysis conditions must preserve nuclear proteins for western blotting

• mRNA vs. Protein Discrepancies:

  • IL-33 protein levels may not correlate with mRNA expression due to post-transcriptional regulation

  • Assess both mRNA and protein when possible

• Tissue-Specific Expression Patterns:

  • Expression varies substantially between tissues

  • Stratified squamous epithelia and cuboidal epithelium show highest expression levels

• Inflammation-Induced Changes:

  • Inflammatory stimuli can dramatically alter IL-33 expression

  • Include time-course analysis in inflammation models to capture dynamic changes

How should IL-33-induced ILC2 responses be measured in mouse models?

ILC2 responses to IL-33 can be comprehensively assessed through:

• Flow Cytometric Identification:

  • ILC2s are typically identified as lineage-negative (Lin-) cells that express:

    • KLRG1, ST2, ICOS, CD25, CD127

  • Include intracellular staining for GATA3 and IL-13 to confirm ILC2 identity

• Functional Assessments:

  • Intracellular cytokine staining for IL-5, IL-13 following PMA/ionomycin stimulation

  • Ex vivo culture of sorted ILC2s with IL-33 to assess cytokine production capacity

• Tissue Localization:

  • Immunofluorescence microscopy to visualize ILC2 distribution in tissues

  • Special focus on lung alveolar regions during airway inflammation models

• Quantitative Analysis:

  • Absolute numbers rather than percentages should be reported

  • Normalize to tissue weight or total cell count for accurate comparisons

• In Vivo Depletion Studies:

  • Use anti-CD90.2 or genetic approaches to deplete ILC2s

  • Assess IL-33-mediated effects before and after depletion to determine ILC2 contribution