IL1B Mouse, His Active

What is mouse IL-1 beta and how does it compare to human IL1B?

Mouse IL-1 beta (IL-1F2) is a pro-inflammatory cytokine encoded by the Il1b gene that plays a crucial role in immune response and inflammation signaling. The mature mouse IL-1 beta protein spans from Val118 to Ser269 in the precursor protein (accession # NP_032387) .

When comparing mouse IL-1 beta to human IL1B, there are notable structural similarities but important species-specific differences. In humanized mouse models like B-hIL1B mice, the mouse Il1b gene is replaced with the human IL1B gene. ELISA analysis demonstrates that:

• Wild-type mice express only mouse IL1B protein

• Homozygous B-hIL1B mice (H/H) express only human IL1B protein

• Neither mouse expresses both species' proteins simultaneously

Despite replacing the mouse gene with the human version, leukocyte subpopulations in homozygous B-hIL1B mice remain similar to those in C57BL/6 wild-type mice, indicating that the substitution does not significantly alter immune cell development or distribution in the spleen .

How is active mouse IL-1 beta produced for research applications?

Active mouse IL-1 beta for research is typically produced as a recombinant protein in E. coli expression systems. The active form corresponds to the mature protein (Val118-Ser269), which requires processing from the pro-form. The production process generally involves:

• Bacterial expression of the protein, often with a histidine tag for purification

• Induction of protein expression in the bacterial culture

• Cell lysis and initial clarification of lysate

• Affinity chromatography using the His-tag for capture

• Secondary purification steps (ion exchange, size exclusion)

• Verification of biological activity through cell-based assays

The activity of recombinant mouse IL-1 beta can be verified using cell proliferation assays with the D10.G4.1 mouse helper T cell line, where the protein stimulates proliferation in a dose-dependent manner. This activity can be neutralized by specific antibodies, with a typical neutralization dose (ND50) of ≤0.25 μg/mL for high-quality antibodies when neutralizing 50 pg/mL of recombinant mouse IL-1 beta .

What are the main experimental applications for mouse IL-1 beta antibodies?

Mouse IL-1 beta antibodies are versatile tools with several key applications in research:

• Immunohistochemistry (IHC): Detection of IL-1 beta in tissue sections, as demonstrated in mouse thymus samples using antigen affinity-purified polyclonal antibodies (15 μg/mL) with HRP-DAB staining systems .

• Western Blotting: Identification of IL-1 beta in cell lysates and supernatants. Both pro-IL-1 beta and mature IL-1 beta can be detected. In RAW 264.7 mouse macrophage cells treated with LPS, IL-1 beta appears as a band at approximately 40 kDa under reducing conditions .

• Simple Western™ Analysis: Automated capillary-based immunoassay for IL-1 beta detection, particularly useful for quantitative analysis in complex samples .

• Neutralization Assays: Blocking IL-1 beta activity in functional assays, such as neutralizing IL-1 beta-induced proliferation in T cell lines .

• In vivo Applications: Neutralizing antibodies can be used to block IL-1 beta function in animal models, as demonstrated in studies examining MDSC (myeloid-derived suppressor cell) accumulation in lungs .

How does the NLRP3 inflammasome regulate IL-1 beta production in mouse models?

The NLRP3 inflammasome plays a critical role in regulating IL-1 beta production through a two-step process:

• Priming step: Activation of pattern recognition receptors (like TLR4 by LPS) induces NF-κB-dependent transcription of NLRP3 and pro-IL-1 beta.

• Activation step: Assembly of the NLRP3 inflammasome complex (NLRP3, ASC, pro-caspase-1), leading to caspase-1 activation, which cleaves pro-IL-1 beta to mature IL-1 beta.

Research using mouse macrophages shows that compounds like quercetin can inhibit auto-reactive NLRP3 inflammasome activation. In Nlrp3A350V/A350V bone marrow-derived macrophages (BMDMs), quercetin treatment significantly reduced IL-1 beta secretion in a dose-dependent manner after LPS priming . Western blot analysis of these samples showed decreased levels of ASC oligomerization, reduced caspase-1 activation, and lower IL-1 beta secretion in the supernatants, while maintaining normal levels of pro-IL-1 beta in the cell lysates .

The inflammasome activation pathway is also crucial in infection models. In Mycobacterium bovis infections, endoplasmic reticulum stress (ERS) mediates inflammasome activation, with increased levels of Bip and phosphorylated IRE1α correlating with enhanced IL-1 beta production. This activation can be inhibited by the ERS inhibitor 4-phenyl butyric acid (4-PBA), demonstrating the link between cellular stress and inflammasome function .

What post-translational modifications regulate mouse IL-1 beta processing and secretion?

Mouse IL-1 beta processing and secretion are regulated by several post-translational modifications, with ubiquitination playing a particularly important role:

• K63-linked polyubiquitination: This modification of pro-IL-1 beta promotes its interaction with the inflammasome complex and subsequent processing. K63-linked ubiquitin chains (rather than K48-linked chains) are specifically involved in this process .

• Deubiquitination by POH1: POH1 (also known as PSMD14) is a deubiquitinating enzyme that negatively regulates IL-1 beta processing by removing K63-linked ubiquitin chains from pro-IL-1 beta. Experimental evidence shows:

  • POH1 physically interacts with pro-IL-1 beta

  • POH1 overexpression reduces IL-1 beta secretion

  • The catalytically inactive POH1-H113Q mutant fails to inhibit IL-1 beta processing

  • Lysine 133 (K133) in mouse pro-IL-1 beta is a critical site for this regulation

• Caspase-1-mediated cleavage: The final step in IL-1 beta maturation, which is enhanced by proper ubiquitination and inhibited by deubiquitination.

This regulatory system creates a balance where:

• TLR3/4 activation upregulates POH1

• POH1 deubiquitinates pro-IL-1 beta

• Deubiquitination restricts IL-1 beta cleavage and secretion

• This serves as a negative feedback loop to prevent excessive inflammation

How do humanized IL1B mouse models differ from standard mouse models in cancer research?

Humanized IL1B mouse models (B-hIL1B mice) provide significant advantages over standard mouse models in cancer research:

• Species-specific targeting: B-hIL1B mice express human IL1B instead of mouse Il1b, allowing for direct testing of human-specific anti-IL1B antibodies. This is critical for translational research as many therapeutic antibodies are highly species-specific .

• Preserved immune architecture: Despite the replacement of mouse Il1b with human IL1B, these mice maintain normal proportions of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in the spleen. This demonstrates that the humanization does not disrupt normal immune development .

• Functional validation in tumor models: B-hIL1B mice show that anti-human IL1B antibodies can effectively inhibit MC38 colon cancer tumor growth without significant body weight changes, validating the model for preclinical evaluation of human IL1B-targeting therapies .

A comparative experiment demonstrated:

• MC38 murine colon cancer cells were implanted subcutaneously in homozygous B-hIL1B mice

• Treatment with anti-human IL1B antibody significantly controlled tumor growth

• This effect would not be possible to demonstrate in wild-type mice, as standard mouse models would require anti-mouse IL-1 beta antibodies

This makes B-hIL1B mice particularly valuable for preclinical testing of human-specific IL1B-targeting therapies in an in vivo context with an intact immune system.

What are the optimal protocols for detecting mouse IL-1 beta in different experimental systems?

Optimal protocols for mouse IL-1 beta detection vary by experimental system:

For Immunohistochemistry in Tissue Sections:

• Fix tissue appropriately (perfusion fixed frozen sections show good results)

• Section tissue at appropriate thickness

• Block endogenous peroxidase and non-specific binding

• Incubate with primary anti-IL-1 beta antibody (15 μg/mL) overnight at 4°C

• Apply appropriate detection system (e.g., Anti-Goat HRP-DAB)

• Counterstain with hematoxylin

• Mount and visualize

For Western Blotting of Cell Lysates and Supernatants:

• Stimulate cells appropriately (e.g., LPS 500 ng/ml for 90 min for macrophages)

• Collect cell supernatants for secreted (mature) IL-1 beta

• Prepare cell lysates for intracellular (pro) IL-1 beta

• Separate proteins by SDS-PAGE under reducing conditions

• Transfer to appropriate membrane

• Block and incubate with anti-IL-1 beta antibody

• Apply secondary antibody and develop

For Simple Western™ Analysis:

• Prepare lysates at concentration of 0.5 mg/mL

• Load samples in Simple Western system

• Use 2.5 μg/mL of anti-IL-1 beta antibody

• Follow with 1:50 dilution of HRP-conjugated secondary antibody

• Use 12-230 kDa separation system for optimal resolution

For ELISA of Secreted IL-1 beta:

• Stimulate cells (e.g., LPS or bacterial infection)

• Collect supernatants at appropriate timepoints

• Use species-specific IL-1 beta ELISA kit

• Important: Ensure species specificity when working with humanized models, as cross-reactivity between human and mouse IL-1 beta is minimal

How can researchers induce and measure inflammasome-dependent IL-1 beta production in mouse models?

Inducing and measuring inflammasome-dependent IL-1 beta production in mouse models involves several key steps:

For in vitro induction in bone marrow-derived macrophages (BMDMs):

• LPS Priming: Stimulate cells with LPS (typically 200-500 ng/mL) for 90 minutes to 4 hours to induce pro-IL-1 beta expression .

• Inflammasome Activation: Several methods can be used:

  • ATP (1 mM) as a second signal after LPS priming

  • Bacterial infection (e.g., M. bovis at MOI 10)

  • Pathogen-associated molecular patterns

  • Endoplasmic reticulum stress inducers

• Measurement Approaches:

  • ELISA: Quantify secreted IL-1 beta in supernatants

  • Western Blot: Analyze both pro-IL-1 beta (in lysates) and mature IL-1 beta (in supernatants)

  • Caspase-1 Activity: Using fluorescent caspase-1 substrates (FLICA) and flow cytometry

  • ASC Oligomerization: Visualize by western blot after chemical crosslinking

For in vivo induction and measurement:

• Infection Model: Infect mice with pathogens (e.g., M. bovis) or inject inflammatory stimuli

• Sample Collection: Collect serum for circulating IL-1 beta and harvest tissues (e.g., lung) for local IL-1 beta production

• Analysis Methods:

  • Serum ELISA: For systemic IL-1 beta levels

  • Tissue Immunoblotting: For NLRP3, AIM2, pro-IL-1 beta, and IL-1 beta levels

  • Histopathology: To correlate IL-1 beta levels with tissue damage

Experimental data shows that in M. bovis infection models, IL-1 beta production is dependent on endoplasmic reticulum stress, as demonstrated by the reduction in IL-1 beta levels when mice are treated with 4-PBA (18.6 mg/mouse/day). This treatment also reduced pathological lesions in mouse lungs after 3 and 6 weeks of infection .

What are the critical controls needed when working with IL-1 beta neutralizing antibodies?

When working with IL-1 beta neutralizing antibodies, several critical controls are essential to ensure valid and interpretable results:

1. Isotype Control Antibodies:

• Use matched isotype control antibodies (same species, isotype, and concentration) to distinguish specific IL-1 beta neutralization from Fc-mediated or non-specific effects

• Example: When testing anti-IL-1 beta effects on MDSC accumulation in lungs, control IgG treatment should be included in both wild-type and experimental groups

2. Dose-Response Controls:

• Include a range of antibody concentrations to establish dose-dependent neutralization

• Determine the neutralization dose (ND50) in relevant bioassays

• Typical ND50 for high-quality antibodies is ≤0.25 μg/mL when neutralizing 50 pg/mL of recombinant mouse IL-1 beta

3. Species Specificity Controls:

• When working with humanized models, verify that anti-human IL-1 beta antibodies do not cross-react with mouse IL-1 beta and vice versa

• Use ELISAs with species-specific detection antibodies to confirm target engagement

4. Positive Controls for Biological Activity:

• Include known IL-1 beta-dependent readouts

• For inflammasome activation, LPS+ATP treatment serves as a positive control

• For TNF-α and IL-6 production, LPS alone serves as a positive control

5. Validation of Target Engagement:

• Confirm reduced IL-1 beta signaling using downstream readouts (e.g., phosphorylation of signaling proteins)

• Measure IL-1 beta levels in treated vs. untreated samples to confirm antibody-mediated clearance or neutralization

The effectiveness of IL-1 beta neutralization can be assessed by examining biological outcomes. For example, in a study of DJ-1 knockout mice, anti-IL-1 beta neutralizing antibody treatment significantly reduced the number of MDSCs (Gr-1+/CD11b+ cells) accumulated in lungs compared to control IgG treatment, demonstrating successful neutralization of IL-1 beta biological activity in vivo .

How should researchers interpret contradictory results between different IL-1 beta detection methods?

When facing contradictory results between different IL-1 beta detection methods, researchers should consider several factors:

Form-specific detection considerations:

• Pro-IL-1 beta vs. Mature IL-1 beta: Different detection methods may preferentially detect either the pro-form (31-35 kDa) or mature form (17 kDa). Western blotting can distinguish between these forms, while some ELISAs may detect both or be specific to one form .

• Intracellular vs. Secreted IL-1 beta: Pro-IL-1 beta is primarily intracellular, while mature IL-1 beta is secreted. Contradictions may arise when comparing cell lysates (western blot) with supernatants (ELISA) . For example, in M. bovis-infected macrophages, immunoblot analysis of supernatants shows mature IL-1 beta bands while cell lysates show pro-IL-1 beta bands .

Method-specific considerations:

• Antibody Epitope Accessibility: Epitopes may be differentially accessible in native vs. denatured proteins. If an antibody recognizes a conformational epitope, it may work in ELISA but not in western blotting.

• Sensitivity Differences: Simple Western™ and ELISA systems typically offer higher sensitivity than traditional western blotting. For RAW 264.7 cells, Simple Western™ can detect IL-1 beta from samples loaded at 0.5 mg/mL after LPS stimulation .

• Post-translational Modifications: Ubiquitination and other modifications affect IL-1 beta detection. K63-linked polyubiquitination of pro-IL-1 beta can alter its apparent molecular weight and may affect antibody recognition .

Resolution strategies:

• Use Multiple Detection Methods: Confirm key findings with at least two independent techniques.

• Include Positive Controls: Use recombinant proteins and stimulation controls (like LPS+ATP for inflammasome activation) .

• Consider Modification Status: If studying ubiquitinated IL-1 beta, immunoprecipitation followed by ubiquitin-specific western blotting may resolve contradictions seen in direct IL-1 beta detection .

• Species-Specific Reagents: When working with humanized mouse models, ensure detection reagents are species-appropriate to avoid false negatives .

What are common artifacts in IL-1 beta detection assays and how can they be mitigated?

Common artifacts in IL-1 beta detection assays and their mitigation strategies include:

1. Non-specific antibody binding:

• Artifact: False positive signals or background noise

• Mitigation: Use antigen affinity-purified antibodies, include proper blocking steps, and validate specificity with knockout controls or competing peptides

• Example: For immunohistochemistry, using antigen affinity-purified polyclonal antibodies at optimized concentrations (e.g., 15 μg/mL) reduces background

2. Inflammasome activation during sample preparation:

• Artifact: Artificially elevated mature IL-1 beta due to cell damage during processing

• Mitigation: Maintain cold temperatures during processing, add protease inhibitors, and use gentle lysis methods

• Example: When comparing control vs. experimental conditions, consistent handling is critical as caspase-1 can be activated by mechanical stress

3. Cross-species reactivity issues:

• Artifact: False positives or negatives in humanized mouse models

• Mitigation: Use species-specific detection reagents and validate with appropriate controls

• Example: In B-hIL1B mice, human IL1B-specific ELISA kits detect the protein only in homozygous B-hIL1B mice (H/H) and not in wild-type mice, while mouse Il1b is detected only in wild-type mice

4. Confounding by other cytokines:

• Artifact: Misattribution of biological effects to IL-1 beta

• Mitigation: Measure multiple cytokines simultaneously and use specific neutralizing antibodies

• Example: In M. bovis infection studies, researchers measured IL-1 beta alongside TNF-α and IL-6 to distinguish inflammasome-specific effects from general inflammatory responses

5. Ubiquitination artifacts in molecular weight determination:

• Artifact: Altered molecular weight due to ubiquitination causing misinterpretation

• Mitigation: Use deubiquitinating enzymes as controls and perform immunoprecipitation followed by ubiquitin-specific western blotting

• Example: K63-linked polyubiquitination of pro-IL-1 beta can be distinguished from K48-linked ubiquitination using linkage-specific antibodies

How does strain background affect IL-1 beta expression and function in mouse models?

Strain background can significantly influence IL-1 beta expression and function in mouse models, impacting experimental outcomes and interpretation:

1. Baseline expression differences:
The baseline expression and inducibility of IL-1 beta can vary between mouse strains. C57BL/6 mice are commonly used as a reference strain for IL-1 beta studies, including as the background for B-hIL1B humanized mice . When comparing data across studies, it's essential to consider strain background as a potential source of variation.

2. Inflammasome activation thresholds:
Different mouse strains may have varying thresholds for inflammasome activation. This can affect:

• Sensitivity to pathogen-associated molecular patterns

• Requirement for secondary signals

• Magnitude of IL-1 beta production in response to stimuli

3. Humanized model considerations:
When creating humanized IL1B models, the strain background remains important:

• B-hIL1B mice developed on C57BL/6 background show species-specific expression patterns

• Human IL1B is expressed in homozygous B-hIL1B mice (H/H) but not in wild-type mice

• Mouse Il1b is detectable in wild-type mice but not in the humanized model

4. Immune cell population effects:
Analysis of spleen leukocyte subpopulations in B-hIL1B mice compared to C57BL/6 mice shows:

• Similar percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages

• This indicates that replacing mouse Il1b with human IL1B does not significantly alter immune cell development or distribution in this strain background

5. Cancer model responses:
In tumor models using MC38 murine colon cancer cells, B-hIL1B mice (on C57BL/6 background) respond effectively to anti-human IL1B antibody treatment, with:

• Significant inhibition of tumor growth

• No notable adverse effects on body weight

• Demonstration of the model's utility for preclinical evaluation of human IL1B-targeting therapies

How do findings from mouse IL-1 beta studies translate to human clinical applications?

Translating findings from mouse IL-1 beta studies to human clinical applications requires careful consideration of several factors:

1. Species-specific differences in IL-1 beta biology:
Despite structural similarities, mouse and human IL-1 beta have distinct regulation and signaling characteristics. This is evidenced by:

• Species-specific detection in ELISA assays with minimal cross-reactivity

• Differential responses to inhibitors and activators

• Evolutionary divergence in regulatory mechanisms

2. Value of humanized mouse models:
Humanized IL1B mouse models offer significant advantages for translational research:

• B-hIL1B mice express human IL1B instead of mouse Il1b

• These mice maintain normal immune cell development and distribution

• They enable direct testing of human-specific therapeutic antibodies

• Anti-human IL1B antibodies effectively inhibit tumor growth in B-hIL1B mice implanted with MC38 colon cancer cells

3. Conservation of core mechanisms:
Despite differences, several fundamental mechanisms are conserved between species:

• The two-signal requirement for inflammasome activation

• Post-translational regulation through ubiquitination

• Role of POH1 in deubiquitinating pro-IL-1 beta at conserved lysine residues

• Endoplasmic reticulum stress-mediated inflammasome activation

4. Therapeutic target validation:
Mouse models provide valuable insights for human therapeutic development:

• Neutralizing IL-1 beta in mouse models can reduce myeloid-derived suppressor cell (MDSC) accumulation in lungs

• Anti-IL-1 beta therapy shows efficacy in mouse cancer models

• Quercetin inhibition of NLRP3 inflammasome in mouse models suggests potential human applications

When translating findings from mouse to human applications, researchers should:

• Confirm key mechanisms in human cells/tissues

• Use humanized mouse models for testing human-specific therapies

• Consider species differences in drug metabolism and pharmacokinetics

• Validate biomarkers that translate between species

What are the main challenges when developing IL-1 beta-targeting therapeutics using mouse models?

Developing IL-1 beta-targeting therapeutics using mouse models presents several significant challenges:

1. Species specificity of therapeutic agents:

• Challenge: Many antibodies and inhibitors show strict species specificity, with limited cross-reactivity between mouse and human IL-1 beta

• Solution: Utilize humanized IL1B mouse models like B-hIL1B mice that express human IL1B instead of mouse Il1b, allowing direct testing of human-specific therapeutics

• Evidence: B-hIL1B mice respond to anti-human IL1B antibody treatment in MC38 tumor models, while such antibodies would be ineffective in wild-type mice

2. Differences in IL-1 beta processing and regulation:

• Challenge: Post-translational modifications and processing pathways may differ between species

• Solution: Focus on conserved regulatory mechanisms, such as the role of K63-linked polyubiquitination and POH1-mediated deubiquitination

• Evidence: Studies have identified specific lysine residues (e.g., K133 in mouse pro-IL-1 beta) as critical for ubiquitination-dependent regulation

3. Complex inflammatory microenvironments:

• Challenge: The inflammatory context in mouse models may not fully recapitulate human disease conditions

• Solution: Use multiple mouse models and validate findings across different inflammatory contexts

• Evidence: Studies show IL-1 beta processing is affected by endoplasmic reticulum stress during M. bovis infection, suggesting context-dependent regulation

4. Compensatory mechanisms:

• Challenge: Targeting IL-1 beta alone may trigger compensatory inflammatory pathways

• Solution: Assess effects on multiple cytokines (e.g., TNF-α, IL-6) when targeting IL-1 beta

• Evidence: In M. bovis infection models, researchers measured multiple cytokines to distinguish inflammasome-specific effects from general inflammatory responses

5. Translation of dosing and pharmacokinetics:

• Challenge: Mouse metabolism and body size necessitate adjustment of dosing for human applications

• Solution: Establish clear pharmacokinetic/pharmacodynamic relationships and biomarkers of target engagement

• Evidence: Neutralization dose assessments (ND50 ≤0.25 μg/mL) provide baseline parameters for translating antibody efficacy

For successful development of IL-1 beta-targeting therapeutics, researchers should:

• Select appropriate mouse models based on the specific therapeutic being tested

• Validate target engagement using multiple approaches

• Assess effects on both IL-1 beta levels and downstream biological outcomes

• Consider combination approaches that address potential compensatory mechanisms