Recombinant Mouse Interleukin-1 receptor-like 1 (Il1rl1)

What is Mouse Interleukin-1 receptor-like 1 (IL1RL1) and what are its key characteristics?

Mouse IL1RL1 is a member of the Toll-like receptor (TLR) superfamily that can affect Th2 responses by influencing Toll-like receptor pathway signaling . The gene is located on chromosome 2q12 and encodes the receptor for Interleukin-33 (IL-33) . IL1RL1 is expressed on multiple cell types including mast cells, T helper type 2 (Th2) cells, regulatory T cells, and macrophages .

Alternative splicing of the IL1RL1 gene leads to multiple isoforms with distinct functions:

• IL1RL1-a (soluble ST2, sST2): A soluble form that can be measured in serum

• IL1RL1-b (ST2L): A transmembrane receptor that mediates signaling

• Two less well-characterized isoforms: isoform 3 and IL1RL1-c (ST2V)

The IL1RL1 gene consists of a distal and proximal promoter and 13 exons, with the following structure:

![IL1RL1 Gene Structure]
The transmembrane form (IL1RL1-b) mediates IL-33 signaling, while the soluble form (IL1RL1-a) is thought to serve as a decoy receptor, sequestering IL-33 and blocking its function .

How does IL1RL1 signaling function in mouse models?

IL1RL1 signaling is initiated when IL-33 binds to a receptor complex consisting of IL1RL1-b and IL-1 receptor accessory protein (IL-1RAcP) present on Th2 cells, basophils, mast cells, and type 2 innate lymphoid cells . This binding triggers an inflammatory signaling cascade that results in:

• Activation of multiple adaptors and signaling proteins, such as Mal or MyD88

• Release of pro-inflammatory Th2 cytokines (IL-4, IL-5, IL-13)

• Inflammatory cell recruitment to affected tissues

• Promotion of airway inflammation, airway hyperresponsiveness, and mucus production

In contrast, the soluble IL1RL1-a isoform acts as a decoy receptor by binding IL-33 and preventing its interaction with membrane-bound IL1RL1-b, thereby inhibiting inflammatory signaling .

What is the biological activity of recombinant mouse IL1RL1 protein?

Recombinant mouse IL1RL1 protein typically refers to the soluble form (IL1RL1-a) that has been produced for research applications. According to product specifications, recombinant mouse IL1RL1 Fc chimera protein exhibits inhibitory activity against IL-1α-induced responses . Approximately 0.005-0.015 μg/mL of IL-1 sRI/Fc Chimera will inhibit 50% of the biological response due to 50 pg/mL of mouse IL-1α .

For research applications, recombinant IL1RL1 is available in both carrier-free formulations (without BSA) and with carrier protein added for enhanced stability. Carrier-free versions are recommended for applications where BSA might interfere with experimental outcomes .

How can recombinant mouse IL1RL1 be used in experimental research?

Recombinant mouse IL1RL1 can be utilized in several experimental applications:

• Binding studies: Used to characterize IL-33 binding properties and receptor dynamics

• Inhibition assays: As a competitive inhibitor of IL-33 signaling to assess the role of this pathway

• ELISA development: As capture antibodies or standards in immunoassays

• In vivo neutralization studies: To block IL-33 signaling in disease models

• Cell culture experiments: To modulate Th2 responses in primary cells or cell lines

For binding studies, researchers have used radiolabeled ligands like iodine-125-labeled recombinant human interleukin-1 alpha ([125I]IL-1α) to characterize IL-1 receptors in various tissues, a technique that could be adapted for IL1RL1 studies .

What methods are available for measuring IL1RL1 expression in mouse tissues?

Multiple techniques can be employed to measure IL1RL1 expression:

• ELISA: For quantifying soluble IL1RL1-a in serum, plasma, or tissue homogenates

• Western blotting: To detect protein expression in tissue lysates

• qRT-PCR: For measuring transcript levels of different IL1RL1 isoforms

• Immunohistochemistry/Immunofluorescence: For visualizing receptor expression in tissue sections

• Flow cytometry: To assess cell surface expression on immune cells

For accurate IL1RL1 isoform discrimination, researchers should design primers or antibodies specific to unique regions of each isoform. For example, to specifically measure IL1RL1-a versus IL1RL1-b transcript levels, primers spanning the transmembrane region (present only in IL1RL1-b) can be utilized .

What are the reconstitution and storage recommendations for recombinant mouse IL1RL1?

Based on product documentation, the following handling procedures are recommended:

Reconstitution:

• Lyophilized recombinant mouse IL1RL1 should be reconstituted at 100 μg/mL in sterile PBS

• Allow the reconstituted protein to sit for at least 15 minutes with gentle agitation before use

Storage:

• Ship at ambient temperature upon receipt

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

• After reconstitution, prepare aliquots to avoid repeated freeze-thaw cycles

• Reconstituted protein can typically be stored at -20°C for up to one month or at -80°C for longer periods

For carrier-free formulations, extra care should be taken as these preparations lack the stabilizing effects of carrier proteins like BSA .

How do genetic variants in IL1RL1 affect experimental outcomes in mouse models?

Genetic variations in IL1RL1 have been shown to significantly impact experimental outcomes in mouse models, particularly in inflammation and asthma research:

• SNP effects on protein expression: Studies have identified several SNPs (e.g., rs1921622, rs11685480, rs1420101) that affect IL1RL1 expression levels . These genetic variations can influence both baseline and inducible expression of IL1RL1 isoforms.

• Disease severity modulation: The rs1921622 polymorphism has been associated with disease severity in RSV bronchiolitis, demonstrating an effect at both allele and genotype levels (p = 0.011 and p = 0.040, respectively) .

• IL1RL1-a concentration differences: Ventilated infants with RSV showed >20-fold higher concentrations of soluble IL1RL1-a in nasopharyngeal aspirates compared to non-ventilated infants (median 9,357 [936–15,528] pg/ml vs. 405 [112–1,193] pg/ml; p<0.001) .

When designing experiments, researchers should consider:

• Genotyping experimental animals for relevant IL1RL1 SNPs

• Stratifying analysis based on genotype

• Measuring baseline IL1RL1 expression before intervention

• Including appropriate wild-type controls

What are the methodological considerations for generating and using IL1RL1 conditional knockout mice?

Conditional knockout models provide temporal and spatial control over IL1RL1 deletion, offering advantages over constitutive knockouts. Key methodological considerations include:

• Design strategy: IL1RL1 conditional knockout mice have been successfully generated using a floxed approach with loxP sites positioned to flank exons 3 and 4, targeting the signaling capacity of IL1RL1 .

• Verification steps:

  • Confirm normal IL1RL1 receptor expression in undeleted tissues (e.g., brain and spleen)

  • Verify normal IL1RL1-dependent biological responses (e.g., IL-1α-induced serum IL-6 elevation)

  • Test efficient excision when bred with Cre-expressing mice

• Control considerations:

  • Use littermate controls lacking Cre recombinase

  • Consider potential off-target effects of Cre expression

  • Include appropriate wild-type controls in all experiments

• Phenotyping approach:

  • Assess gross physical and behavioral phenotypes

  • Examine tissue-specific IL1RL1 expression patterns

  • Evaluate responses to inflammatory challenges

Researchers have observed that IL1RL1loxP/loxP mice breed normally, exhibit no gross physical or behavioral phenotypes, and display normal IL1RL1 receptor expression and function, making them valuable tools for cell-specific and temporally controlled studies .

How can soluble IL1RL1-a be measured in mouse biological samples?

Accurate quantification of soluble IL1RL1-a (sST2) in mouse biological samples requires attention to several methodological details:

• Sample collection and processing:

  • For serum: collect blood in serum separator tubes, allow to clot for 30 minutes, centrifuge at 1000×g for 10 minutes

  • For bronchoalveolar lavage fluid (BALF): standardize lavage volume (typically 0.8-1 mL PBS)

  • For tissue homogenates: consistent tissue:buffer ratios and homogenization protocols

  • Process samples quickly and keep cold to prevent degradation

• Detection methods:

  • ELISA: Commercial kits are available with typical detection ranges of 31.3-2000 pg/mL

  • Multiplex assays: Allow simultaneous measurement of IL1RL1-a alongside other inflammatory markers

  • Western blotting: For semi-quantitative analysis with antibody validation

• Calibration considerations:

  • Matrix matching: Prepare standards in the same matrix as samples when possible

  • Spike-and-recovery experiments to validate assay in specific sample types

  • Serial dilutions to ensure linearity within the detection range

• Data interpretation:

  • Compare levels between experimental groups rather than focusing on absolute values

  • Consider the ratio of soluble IL1RL1-a to IL-33 as a biomarker of pathway activation

  • Baseline levels vary by strain, age, sex, and housing conditions

Nasopharyngeal aspirates have shown dramatic differences in IL1RL1-a levels between ventilated and non-ventilated infants with RSV infection, suggesting this measurement could be valuable in respiratory infection mouse models .

What are the key differences between mouse and human IL1RL1 that impact translational research?

Several important differences between mouse and human IL1RL1 affect the translational relevance of mouse studies:

• Sequence homology: Mouse IL1RL1 shares 63% amino acid sequence homology with human IL1RL1 in their extracellular domains , creating potential differences in binding affinity and specificity.

• Expression patterns: While both species express IL1RL1 on similar cell types, the relative expression levels on specific immune cell populations may differ between mice and humans.

• Genetic associations: In humans, IL1RL1 genetic variants have been strongly associated with asthma in multiple large-scale genome-wide association studies (GWAS) . Mouse models may not fully recapitulate the complex genetic architecture of human asthma.

• IL-33 responsiveness: The downstream signaling cascade and cellular responses to IL-33 stimulation show similarities but also species-specific differences.

• Soluble isoform dynamics: The ratio of membrane-bound to soluble IL1RL1 and the regulation of these isoforms may differ between species.

When designing translational studies, researchers should:

• Consider using humanized mouse models for specific applications

• Validate key findings in human primary cells or tissues

• Be cautious when extrapolating dosing from mouse to human studies

• Acknowledge species differences when interpreting results

How is IL1RL1 involved in mouse models of asthma and airway inflammation?

IL1RL1 plays a critical role in asthma and airway inflammation models through several mechanisms:

• Type 2 inflammation regulation: IL1RL1 signaling stimulates Th2 cytokine responses (IL-4, IL-5, IL-13) that drive eosinophilic influx, airway inflammation, airway hyperresponsiveness, and mucus production .

• Genetic influences: SNPs in IL1RL1 (including rs1921622, rs11685480, and rs1420101) have been associated with asthma susceptibility and severity, with specific variants linked to the "type 2-high" asthma endotype .

• Balance of isoforms: The ratio between membrane-bound IL1RL1-b and soluble IL1RL1-a is crucial, as IL1RL1-a can sequester IL-33 and inhibit its pro-inflammatory function .

• Expression regulation: IL1RL1 expression is controlled by both genetic variants and epigenetic modifications, with blood DNA methylation levels potentially influencing IL1RL1-a protein levels .

In experimental asthma models, researchers have shown:

• IL1RL1 genetic variants specifically increase the risk of type 2-high disease

• These variants confer risk by reducing plasma and airway levels of soluble IL1RL1-a, an inhibitor of IL-33 signaling

• The IL1RL1-IL33 pathway is critical for allergen-induced airway hyperresponsiveness and inflammation

What is the role of IL1RL1 in neuroinflammation and brain injury models?

IL1RL1 and related IL-1 family receptors play significant roles in neuroinflammation and brain injury models:

• Expression patterns: IL-1 receptors show distinctive distribution in the mouse brain, with highest densities present in:

  • Molecular and granular layers of the dentate gyrus of the hippocampus

  • Choroid plexus

• Injury responses: After traumatic brain injury, IL-1 receptor signaling mediates:

  • Microglial/macrophage activation

  • Production of cyclooxygenase-2 (Cox-2)

  • Expression of vascular cell adhesion molecule-1

  • Diapedesis of peripheral macrophages into injured brain tissue

• Neuroprotective strategies: Blocking IL-1 signaling through IL-1R1 has shown neuroprotective effects in:

  • Experimental stroke models

  • Multiple sclerosis models

  • Traumatic brain injury

• Mechanism of neuroprotection: Cell preservation appears to be achieved by:

  • Abrogating microglial/macrophage activation

  • Disrupting the self-propagating cycle of inflammation

Studies using IL-1R1 null mice have demonstrated that while some molecular aspects of injury response remain normal (like TNF-α expression and nerve growth factor production), several inflammatory responses are significantly reduced, suggesting therapeutic potential for IL-1 pathway modulation in neurological conditions .

How does recombinant IL1RL1 administration affect disease progression in mouse models?

Administration of recombinant IL1RL1 (specifically the soluble IL1RL1-a form) has shown therapeutic potential in several disease models:

• Mechanism of action: Recombinant IL1RL1-a acts as a decoy receptor that binds IL-33, preventing its interaction with membrane-bound IL1RL1-b and inhibiting downstream inflammatory signaling .

• Dosing considerations: Studies indicate that approximately 0.005-0.015 μg/mL of IL-1 sRI/Fc Chimera will inhibit 50% of the biological response due to 50 pg/mL of recombinant mouse IL-1α . Similar dosing principles may apply when targeting IL-33/IL1RL1 interactions.

• Therapeutic applications:

  • Asthma models: Reduced eosinophilic airway inflammation and airway hyperresponsiveness

  • Viral infection models: Ameliorated inflammatory responses in RSV bronchiolitis

  • Autoimmune models: Decreased inflammatory cell infiltration and tissue damage

• Delivery approaches:

  • Systemic (intravenous/intraperitoneal) administration for widespread effects

  • Local (intranasal/intratracheal) delivery for respiratory conditions

  • Fusion proteins (e.g., IL1RL1-Fc chimeras) for extended half-life

Researchers should consider timing of administration (preventative vs. therapeutic), dose-response relationships, and potential immunogenicity of recombinant proteins when designing intervention studies.

What are the key quality control parameters for recombinant mouse IL1RL1?

When working with recombinant mouse IL1RL1, researchers should verify several quality control parameters:

• Purity assessment:

  • SDS-PAGE analysis showing a single band at the expected molecular weight

  • Confirmation of >95% purity by densitometry

  • Absence of degradation products

• Biological activity:

  • Functional testing of inhibitory activity against IL-1α or IL-33

  • Verification of dose-dependent effects

  • Comparison to established activity standards

• Endotoxin levels:

  • Confirmation of low endotoxin content (<1.0 EU/μg of protein)

  • Testing via Limulus Amebocyte Lysate (LAL) assay

• Protein concentration:

  • Accurate determination by validated methods (e.g., BCA assay)

  • Verification of concentration after reconstitution

• Post-translational modifications:

  • Confirmation of appropriate glycosylation patterns

  • Verification of correct disulfide bond formation

For carrier-free formulations, additional quality controls are necessary since the absence of carrier proteins like BSA can affect stability and activity .

How can researchers optimize experiments involving IL1RL1 genetic variants?

When studying IL1RL1 genetic variants, researchers should implement several optimization strategies:

• Genotyping approaches:

  • Multiple reliable methods exist for IL1RL1 SNP genotyping:

    • TaqMan genotyping assays for SNPs like rs1041973, rs1420101, rs10192157, rs1921622

    • LNA Primetime probes and primers for SNPs like rs11685480

    • Illumina Infinium Genome-wide genotyping arrays for broader coverage

• Experimental design considerations:

  • Include adequate sample sizes based on minor allele frequencies

  • Perform power calculations specific to the variants being studied

  • Consider linkage disequilibrium between IL1RL1 SNPs

• Functional validation strategies:

  • Measure IL1RL1 transcript levels in relevant tissues

  • Assess soluble IL1RL1-a protein concentrations in biological fluids

  • Evaluate IL-33 responsiveness in cells from subjects with different genotypes

• Data analysis approaches:

  • Test genetic associations at both allele and genotype levels

  • Consider multiple genetic models (dominant, recessive, additive)

  • Adjust for relevant covariates and multiple testing

Studies have successfully identified associations between IL1RL1 SNPs and disease severity in respiratory conditions, providing a framework for future genetic studies .

What are the most effective approaches for measuring the binding properties of recombinant IL1RL1?

Several sophisticated techniques can be employed to characterize the binding properties of recombinant IL1RL1:

• Radioligand binding assays:

  • Using iodine-125-labeled recombinant proteins (e.g., [125I]IL-1α)

  • Determining binding parameters: Kd (66 ± 10 pM for IL-1R in mouse kidney) and Bmax (1.04 ± 0.24 fmol/mg protein)

  • Performing competition studies with related ligands to establish specificity

• Surface Plasmon Resonance (SPR):

  • Real-time, label-free measurement of binding kinetics

  • Determination of association (kon) and dissociation (koff) rate constants

  • Calculation of equilibrium dissociation constant (KD)

• Biolayer Interferometry (BLI):

  • Alternative optical technique for measuring biomolecular interactions

  • Similar to SPR but with different detection principle

• Competitive binding assays:

  • Using known IL1RL1 ligands (e.g., IL-33) in competition with test compounds

  • Determining inhibitory binding affinity constants (Ki)

• Cellular binding/functional assays:

  • Flow cytometry-based binding assays using fluorescently labeled ligands

  • Reporter cell assays measuring downstream signaling activation

When conducting binding studies, researchers should:

• Use appropriate positive and negative controls

• Include competitive binding with structurally related molecules to establish specificity

• Perform assays at physiologically relevant temperatures

• Consider the effects of buffer composition on binding parameters