IL33 Mouse, His

What is IL-33 and what are its primary functions in mice?

IL-33 is an epithelial-derived cytokine that belongs to the IL-1 family. It acts as an alarmin that is released upon tissue damage, stress, or infection to alert the immune system. In mice, IL-33 has been shown to modulate various immune responses, particularly Th2-type responses, but can also stimulate Th1-type immunity. It binds to its specific receptor ST2, which is expressed on most immune cell populations. IL-33 plays roles in multiple biological processes including inflammatory responses, tissue homeostasis, and neuronal function .

How does IL-33 expression differ across mouse tissues and developmental stages?

IL-33 is predominantly expressed in epithelial barrier tissues, endothelial cells, and tissue-resident immune cells in mice. During development, IL-33 expression patterns change dynamically, with particularly important roles in brain development and maturation. Altered IL-33 expression during development can lead to behavioral changes in adult mice, as evidenced by studies with Il33−/− mice that exhibit reduced anxiety-like behaviors and deficits in social novelty recognition .

What mouse models are commonly used to study IL-33 function?

Several mouse models are used to investigate IL-33 function:

• IL-33 knockout mice (Il33−/−): These mice have complete absence of IL-33 expression and show altered behaviors, including reduced anxiety and deficits in social novelty recognition .

• ST2 (IL-33 receptor) knockout mice: These models help distinguish receptor-dependent effects.

• D-galactose (D-gal)-induced aging models: Used to study IL-33's effects on age-related conditions, these mice exhibit features of accelerated aging and respond to IL-33 treatment with improved cognitive function and bone health .

• Conditional knockout models: Allow tissue-specific or inducible deletion of IL-33 to examine context-dependent functions.

How should researchers design experiments to discern the direct versus indirect effects of IL-33 in mouse models?

To distinguish direct from indirect effects of IL-33, researchers should:

• Use cell-specific conditional knockouts to determine which cell types mediate IL-33 responses

• Implement time-course experiments with acute and chronic IL-33 administration

• Compare local versus systemic IL-33 delivery methods

• Employ bone marrow chimeras to distinguish stromal versus hematopoietic IL-33 effects

• Use ex vivo cell culture systems alongside in vivo models to validate direct cellular effects

For example, in studies examining IL-33's effects on age-related bone loss, researchers used both in vitro osteoblast cultures and in vivo D-galactose-induced aging models to determine that IL-33 directly affects osteoblast function while also modulating T cell populations that indirectly impact bone homeostasis .

What are the critical considerations when generating recombinant IL-33 for mouse studies?

When generating recombinant IL-33 for mouse studies, researchers should consider:

• Expression construct design: The mature peptide sequence (S109 to I266) of murine IL-33 should be used, as full-length IL-33 is a nuclear protein that may not be properly released. Consider adding a signal sequence (e.g., human CD8α signal sequence) to ensure proper secretion .

• Protein purification strategy: His-tagged constructs are common for purification, but researchers should be aware that tags might affect protein function.

• Protein validation: Functional assays should confirm that recombinant IL-33 binds ST2 receptor and activates appropriate signaling pathways.

• Quality control: Endotoxin testing is essential as contamination can confound immune response studies.

• Storage conditions: Proper aliquoting and storage at -80°C is recommended to maintain protein activity.

• Dosing determination: Pilot dose-response studies should establish effective concentrations for specific experimental endpoints.

What methodological approaches provide the most reliable assessment of IL-33 levels in mouse tissues and fluids?

For reliable assessment of IL-33 levels in mouse specimens:

• ELISA: Use validated antibody pairs with appropriate standards. For example, purified anti-mouse IL-33 antibody (Goat Polyclonal IgG, Poly5165, Biolegend) as the capturing antibody and Biotin anti-mouse IL-33 antibody for detection, with recombinant mouse IL-33 as standard .

• Immunohistochemistry: Optimize fixation protocols as IL-33 is predominantly nuclear in non-stressed cells.

• Flow cytometry: For intracellular IL-33 detection in specific cell populations.

• qPCR: For mRNA expression analysis, though post-transcriptional regulation means protein levels may not correlate with mRNA.

• Western blotting: To distinguish between full-length and cleaved forms of IL-33.

• Multiple method validation: Combining techniques provides more robust data than relying on a single approach.

How does IL-33 modulate T cell populations in mouse models, and what are the implications for disease intervention?

IL-33 significantly affects T cell populations with important disease implications:

• Th17/Treg balance: IL-33 reduces the proliferation of proinflammatory Th17 cells while enhancing regulatory T cell (Treg) numbers. In D-galactose-induced aging mice, IL-33 treatment decreased IL-17A+ cells, suppressed ROR-γt and STAT-3 expression (Th17 transcription factors), and increased Foxp3 expression (Treg transcription factor) .

• Transcriptional regulation: IL-33 downregulates mRNA expression of ROR-γt and STAT-3 while enhancing Foxp3 expression, shifting the T cell balance toward an anti-inflammatory state .

• Cytokine modulation: IL-33 treatment reduces proinflammatory cytokines like TNF-α in aged mice .

• Disease implications: These immunomodulatory effects suggest therapeutic potential for:

  • Age-related bone loss (osteoporosis): Through suppression of inflammatory Th17 cells and enhancement of bone-protective Tregs

  • Neurodegenerative conditions: By reducing inflammatory processes in the brain

  • Allergic diseases: Context-dependent effects where IL-33 blockade may be beneficial

What explains the contradictory pro- and anti-tumorigenic effects of IL-33 in different mouse cancer models?

The dual role of IL-33 in cancer is context-dependent and can be explained by:

• Tumor microenvironment composition: The dominant immune cell populations present determine whether IL-33 promotes anti-tumor immunity or tumor-supporting inflammation.

• Specific immune cell activation: IL-33 can stimulate various anti-tumor immune effectors:

  • NK cells: IL-33 enhances NK cell activation markers (NKG2D, CD69), cytotoxic mediators, and tumor-killing capacity .

  • CD8+ T cells: IL-33 can promote CD8+ T cell functions in some contexts.

  • Eosinophils: IL-33 activates eosinophils to exert tumor cytotoxic functions through contact-dependent degranulation .

• Timing of IL-33 exposure: Early exposure may promote anti-tumor immunity, while chronic exposure might favor immunosuppressive mechanisms.

• Cancer type: Different tumors have varying sensitivities to immune effector mechanisms modulated by IL-33.

• Expression pattern: Tumoral expression of IL-33 may inhibit tumor growth by modifying the tumor microenvironment differently than systemic IL-33 .

Researchers should carefully control for these variables when designing experiments to study IL-33 in cancer models.

How does IL-33 signaling interact with helminth infection models in mice?

IL-33 plays a critical role in anti-helminth immunity:

• Susceptibility: Mice deficient in IL-33 or its receptor ST2 show increased susceptibility to various helminth infections .

• Immune evasion: Some helminths actively suppress the IL-33 pathway to avoid ejection by the host immune system. For instance, certain mouse-infective helminths secrete the Alarmin Release Inhibitor HpARI2, which suppresses IL-33 signaling .

• Molecular mechanisms: HpARI2 binds to mouse IL-33 with high affinity (KD ~48 pM) through its CCP domains, preventing IL-33 from interacting with its receptor ST2 .

• Structural basis: The binding interface between HpARI2 and IL-33 has been characterized, showing that HpARI2 contains three CCP-like domains that contact IL-33 primarily through the second and third domains. This interaction prevents the formation of a signaling complex between IL-33 and ST2 .

• Allergic connections: The suppression of IL-33 by helminths not only helps parasites evade immunity but may also reduce allergic responses, potentially explaining the observed reduction in asthma prevalence in helminth-endemic regions .

What neurobiological mechanisms explain the behavioral changes observed in IL-33 deficient mice?

IL-33 deficiency leads to notable behavioral alterations through several neurobiological mechanisms:

• Altered neuronal activity: IL-33−/− mice show changed patterns of c-Fos immunoreactivity (a marker of neuronal activation) in brain regions critical for anxiety-related behaviors, including the medial prefrontal cortex (mPFC), amygdala, and piriform cortex (PCX) .

• Neurodevelopmental effects: IL-33 appears to play a role in brain development and maturation, with its absence potentially altering neuronal connectivity patterns during critical developmental periods .

• Social cognition circuits: While IL-33−/− mice maintain intact sociability, they exhibit deficits in social novelty recognition in the three-chamber social interaction test, suggesting specific effects on neural circuits involved in social memory rather than general social motivation .

• Anxiety-related circuitry: IL-33 deficient mice show reduced anxiety-like behaviors in the elevated plus maze (EPM) and open field test (OFT), indicating altered function in anxiety-processing neural pathways .

• Neurotransmitter systems: Changes in excitatory/inhibitory balance or monoaminergic signaling may underlie the behavioral phenotypes, though these mechanisms require further investigation.

How should researchers design cognitive and behavioral tests to assess IL-33's effects on mouse brain function?

For robust assessment of IL-33's effects on cognition and behavior:

• Comprehensive test battery: Include tests for multiple domains:

  • Memory assessment: Novel object recognition test (measures recognition memory)

  • Spatial learning and memory: Morris water maze (evaluates spatial learning and memory retention)

  • Anxiety-like behaviors: Elevated plus maze and open field test

  • Social behaviors: Three-chamber social interaction test (distinguishes sociability from social novelty recognition)

  • Motor function: Analysis of locomotor activity

• Controlled testing conditions:

  • Use age and sex-matched controls

  • Conduct tests at consistent times of day

  • Control environmental variables (lighting, noise, handling)

  • Include appropriate habituation periods

• Complementary molecular analyses:

  • Assess c-Fos immunoreactivity to map activated brain regions following behavioral tests

  • Examine neuronal markers in regions showing altered activity

  • Investigate changes in neurotransmitter systems

• Quantitative assessment methods:

  • For Morris water maze: Track escape latency time across sessions, path length, and time spent in target quadrant

  • For novel object recognition: Calculate discrimination index and total investigation time

How does IL-33 treatment mitigate age-related pathologies in mouse models, and what are the translational implications?

IL-33 treatment shows remarkable efficacy in addressing multiple age-related pathologies:

• Oxidative stress reduction: IL-33 significantly decreases oxidative stress markers in aging mice:

  • Increases glutathione (GSH) content in cortex and hippocampus

  • Reduces malondialdehyde (MDA) levels and nitrite content

  • Enhances antioxidant defense systems in the brain

• Cognitive function improvement:

  • Enhances recognition memory (novel object recognition test)

  • Improves spatial learning and memory retention (Morris water maze)

  • Reverses D-galactose-induced memory deficits

• Amyloid pathology modulation:

  • Downregulates BACE1 protein levels in cortex and hippocampus

  • BACE1 inhibition reduces Aβ burden, a key pathological feature in Alzheimer's disease

• Tau pathology reduction:

  • Diminishes tau phosphorylation at Ser 396 in cortex and hippocampus

  • Abnormal tau phosphorylation is associated with neuronal death and dementia

• Immune system modulation:

  • Restores Th17/Treg balance

  • Reduces proinflammatory cytokines like TNF-α

Translational implications include potential therapeutic applications for age-related conditions like osteoporosis and dementia, though further research is needed to determine optimal dosing, administration routes, and safety profiles for human applications.

What methodological approaches best capture IL-33's multisystem effects in age-related pathologies?

To comprehensively evaluate IL-33's effects across multiple physiological systems:

• Integrated experimental design:

  • Use age-appropriate models (naturally aged mice or accelerated aging models like D-galactose-induced aging)

  • Include both male and female mice to capture sex differences

  • Employ longitudinal studies to track progression of intervention effects

• Multi-tissue analysis approach:

  • Brain: Assess cognitive function, oxidative stress markers, and protein aggregation

  • Bone: Evaluate bone density, microarchitecture, and cellular composition

  • Immune system: Analyze T cell populations and inflammatory markers

  • Metabolism: Monitor metabolic parameters and body composition

• Molecular and cellular methodologies:

  • Flow cytometry to analyze immune cell populations (Th17, Treg)

  • Biochemical assays for oxidative stress markers (MDA, nitrite, GSH)

  • Immunoblotting for protein expression (BACE1, phosphorylated tau)

  • RT-PCR for transcription factor analysis (ROR-γt, STAT-3, Foxp3)

• Functional assessments:

  • Cognitive tests (novel object recognition, Morris water maze)

  • Bone strength and microarchitecture (micro-CT)

  • Physiological parameters (metabolism, activity levels)

• Tissue-specific intervention comparisons:

  • Compare systemic versus tissue-targeted IL-33 delivery

  • Use tissue-specific IL-33 receptor knockouts to determine primary sites of action

What are the critical quality control steps when preparing and administering IL-33 in mouse experiments?

Ensuring reliable and reproducible IL-33 administration requires:

• Protein preparation:

  • Use the mature form of mouse IL-33 (S109 to I266) rather than full-length protein

  • Consider adding a signal sequence for secreted expression systems

  • Validate protein structure and function through binding assays

• Purity assessment:

  • Perform SDS-PAGE and Western blotting to confirm size and identity

  • Test for endotoxin contamination, which can confound immune response studies

  • Confirm protein concentration using validated methods (BCA assay, spectrophotometry)

• Activity validation:

  • Conduct cell-based assays to confirm biological activity before in vivo use

  • Test receptor binding using surface plasmon resonance (SPR) or similar techniques

• Administration considerations:

  • Optimize dose through pilot dose-response studies

  • Select appropriate administration route based on experimental goals

  • Maintain consistent timing of administration relative to outcome measurements

  • Use vehicle controls with identical composition minus IL-33

• Storage and handling:

  • Aliquot protein to avoid freeze-thaw cycles

  • Store at recommended temperatures (typically -80°C)

  • Follow validated reconstitution protocols

How can researchers effectively measure IL-33 signaling activity in different mouse tissues?

Multiple complementary approaches provide comprehensive assessment of IL-33 signaling:

• Receptor expression analysis:

  • Immunohistochemistry or flow cytometry to quantify ST2 receptor expression

  • RT-PCR for ST2 mRNA levels in different tissues

  • Single-cell RNA sequencing to identify ST2-expressing cell populations

• Signaling pathway activation:

  • Western blotting for phosphorylated signaling proteins (NF-κB, p38 MAPK, JNK)

  • Immunoprecipitation to detect protein-protein interactions in the signaling cascade

  • Transcriptional reporter assays in cell culture models

• Downstream gene expression:

  • RT-PCR arrays for IL-33-responsive genes

  • RNA-seq to comprehensively profile transcriptional changes

  • ChIP-seq to identify transcription factor binding sites in regulated genes

• Functional readouts:

  • Cell-type specific activation markers

  • Cytokine production profiles (ELISA, multiplex assays)

  • Cell population changes (flow cytometry)

• In vivo reporter systems:

  • Transgenic reporter mice with fluorescent proteins driven by IL-33-responsive elements

  • Bioluminescence imaging for real-time monitoring of signaling activity

How should researchers interpret contradictory findings regarding IL-33's effects across different mouse disease models?

When encountering contradictory findings about IL-33:

• Context-dependent factors to consider:

  • Disease model specifics (acute vs. chronic, infection vs. sterile inflammation)

  • Genetic background of mice (C57BL/6 vs. BALB/c responses can differ significantly)

  • Sex and age of mice (IL-33 effects may vary with age and between sexes)

  • Dose and timing of IL-33 administration or manipulation

• Methodological evaluation:

  • Different forms of IL-33 used (full-length vs. mature form)

  • Administration routes (local vs. systemic)

  • Assessment timepoints (early vs. late responses)

  • Knockout strategies (global vs. conditional knockouts)

• Mechanistic resolution approaches:

  • Cell-specific conditional knockouts to identify key responding cell types

  • Temporal manipulation using inducible systems

  • Dose-response studies across model systems

  • Comprehensive immune profiling to identify divergent downstream mechanisms

• Translational relevance assessment:

  • Compare findings to human data when available

  • Evaluate whether contradictions reflect genuine biological complexity or methodological issues

  • Consider evolutionary differences in IL-33 signaling between species

What are the most effective experimental approaches to study IL-33's role in neurodegenerative diseases using mouse models?

For studying IL-33 in neurodegeneration:

• Model selection considerations:

  • Use well-established models relevant to specific diseases (e.g., 5xFAD for Alzheimer's, MPTP for Parkinson's)

  • Consider naturally aged mice alongside genetic models

  • D-galactose-induced aging models are useful for preliminary studies

• Intervention design:

  • Compare preventive versus therapeutic IL-33 administration

  • Test central (intracerebroventricular) versus peripheral delivery

  • Include dose-response studies to establish optimal concentrations

• Comprehensive outcome assessment:

  • Cognitive function: Novel object recognition, Morris water maze, Y-maze

  • Neuropathology: Amyloid plaque and tau pathology quantification

  • Neuroinflammation: Microglial and astrocyte activation markers

  • Oxidative stress: MDA, nitrite, and GSH measurements in brain regions

  • Neuronal function: Electrophysiology, synaptic protein expression

• Molecular mechanism investigation:

  • Assess BACE1 protein levels, which IL-33 can downregulate

  • Measure tau phosphorylation at Ser 396, which IL-33 can diminish

  • Analyze effects on antioxidant defense systems in brain

• Cell-specific manipulations:

  • Target IL-33 or ST2 in specific cell populations (neurons, microglia, astrocytes)

  • Use cell-specific Cre lines with floxed IL-33 or ST2 alleles

  • Employ bone marrow chimeras to distinguish central vs. peripheral effects

What emerging technologies will advance our understanding of IL-33 biology in mouse models?

Cutting-edge approaches poised to transform IL-33 research include:

• Single-cell technologies:

  • Single-cell RNA sequencing to identify specific cell populations responding to IL-33

  • Single-cell proteomics to profile signaling pathways at cellular resolution

  • Spatial transcriptomics to map IL-33 and ST2 expression patterns in tissues

• Advanced imaging:

  • Intravital microscopy to visualize IL-33 responses in living tissues

  • PET imaging with radiolabeled IL-33 to track tissue distribution

  • Optogenetic control of IL-33 expression for precise temporal manipulation

• Genome editing advances:

  • CRISPR-Cas9 screens to identify novel components of IL-33 signaling

  • Base editing to introduce specific mutations in IL-33 or ST2

  • Tissue-specific and inducible CRISPR systems for precise manipulation

• Structural biology approaches:

  • Cryo-electron microscopy of IL-33/ST2 complexes in different states

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics

  • Structural studies of IL-33 antagonist interactions to guide therapeutic design

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Machine learning analysis of complex IL-33-dependent phenotypes

  • Network modeling of IL-33 signaling across tissues and disease states

How can mouse models of IL-33 function inform human therapeutic development?

Translating findings from mouse models to human applications requires:

• Comparative biology considerations:

  • Analyze structural and functional differences between mouse and human IL-33/ST2

  • Compare expression patterns across species in relevant tissues

  • Evaluate conservation of downstream signaling pathways

• Humanized mouse models:

  • Mice expressing human IL-33 or ST2 to test species-specific interactions

  • Humanized immune system mice to study human immune cell responses to IL-33

  • Patient-derived xenograft models to test IL-33 therapies in human disease contexts

• Therapeutic development approaches:

  • Testing recombinant IL-33 for conditions where enhancement is beneficial (neurodegeneration, osteoporosis)

  • Developing IL-33 antagonists or ST2-Fc fusion proteins for allergic or inflammatory conditions

  • Exploring cell-specific delivery systems to target particular tissues

• Biomarker identification:

  • Correlate mouse responses to IL-33 with potential human biomarkers

  • Develop companion diagnostics to identify patients likely to respond to IL-33-targeted therapies

  • Establish imaging or blood-based markers that predict treatment efficacy

• Safety assessment strategies:

  • Comprehensive toxicology studies addressing IL-33's pleiotropic effects

  • Long-term studies to identify delayed effects on multiple organ systems

  • Dose-finding studies to establish therapeutic windows