IL1RL1 Human, Sf9 Active

What is IL1RL1 and what are its primary structural features when expressed in Sf9 cells?

IL1RL1, also known as Interleukin-1 receptor-like 1 or Protein ST2, is a member of the IL-1 receptor family that functions as a receptor for interleukin-33. When produced in Sf9 Baculovirus cells, IL1RL1 is expressed as a single, glycosylated polypeptide chain containing amino acids 19-328 and is typically fused to an 8 amino acid His Tag at the C-terminus. The complete recombinant protein contains 318 amino acids with a theoretical molecular mass of 36.0kDa, though due to glycosylation it displays multiple bands between 40-57kDa when analyzed by SDS-PAGE under reducing conditions .

Why does IL1RL1 produced in Sf9 cells show multiple bands on SDS-PAGE despite being a single polypeptide?

The multiple bands observed between 40-57kDa on SDS-PAGE under reducing conditions are primarily attributed to post-translational modifications, specifically variable glycosylation patterns. Sf9 insect cells perform eukaryotic post-translational modifications including glycosylation, but with patterns that differ somewhat from mammalian cells. The heterogeneity in glycosylation results in proteins with slightly different molecular weights, appearing as multiple bands. This is a normal characteristic of glycosylated proteins expressed in insect cell systems and doesn't necessarily indicate protein degradation or contamination .

What expression system considerations are important when producing functional IL1RL1 in Sf9 cells?

For producing functional IL1RL1, the Sf9 Baculovirus expression system offers several advantages for researchers. This system supports proper folding and post-translational modifications of complex mammalian proteins. When designing expression constructs, researchers should consider: (1) Codon optimization for insect cell expression; (2) Inclusion of appropriate signal sequences for secretion; (3) Strategic placement of affinity tags (typically C-terminal His-tags) to avoid interference with protein folding or function; (4) Optimization of infection parameters including MOI (multiplicity of infection) and harvest timing to maximize yield of properly folded protein. The resulting recombinant IL1RL1 requires purification by proprietary chromatographic techniques to ensure high purity (>95%) suitable for functional studies .

What are the optimal storage conditions for maintaining IL1RL1 stability, and how can researchers evaluate protein integrity over time?

To maintain IL1RL1 stability, the purified protein should be stored in phosphate-buffered saline (pH 7.4) with 10% glycerol. For short-term storage (2-4 weeks), the protein may be kept at 4°C, but for longer periods, storage at -20°C is recommended. To enhance stability during long-term storage, the addition of carrier proteins (0.1% HSA or BSA) is advisable. Multiple freeze-thaw cycles should be avoided as they can lead to protein degradation and activity loss .

Researchers can evaluate protein integrity through several methods: (1) SDS-PAGE analysis to confirm molecular weight and assess degradation; (2) Western blotting with anti-IL1RL1 or anti-His antibodies; (3) Size-exclusion chromatography to detect aggregation; (4) Functional binding assays with IL-33 to confirm biological activity; and (5) Mass spectrometry for detailed structural analysis and identification of post-translational modifications.

How does IL1RL1 mediate downstream signaling upon IL-33 binding, and what experimental approaches can measure this activity?

IL1RL1 (particularly the transmembrane variant ST2L) mediates signaling upon IL-33 binding by recruiting a cascade of adapter proteins and kinases. Upon ligand binding, IL1RL1 recruits MYD88, IRAK1, IRAK4, and TRAF6, which leads to the phosphorylation of multiple MAP kinases including MAPK3/ERK1, MAPK1/ERK2, MAPK14, and MAPK8 . This signaling cascade ultimately leads to the activation of transcription factors that regulate inflammatory and immune responses.

Experimental approaches to measure IL1RL1 activity include:

• Phosphorylation assays to detect activated MAPKs using phospho-specific antibodies

• Reporter gene assays with promoters responsive to IL-33/IL1RL1 signaling

• Co-immunoprecipitation experiments to detect protein-protein interactions in the signaling complex

• Cytokine production measurement in cells expressing IL1RL1 after IL-33 stimulation

• Calcium flux assays to detect immediate signaling events

• Gene expression profiling to identify downstream transcriptional changes

What is the differential role of membrane-bound IL1RL1 (ST2L) versus soluble IL1RL1 (sST2) in inflammation and immune regulation?

Membrane-bound IL1RL1 (ST2L) and its soluble form (sST2) play distinct and sometimes opposing roles in inflammation and immune regulation:

ST2L (membrane-bound form):

• Functions as the receptor for IL-33, mediating its pro-inflammatory effects

• Expressed on various cell types including T helper cells, endothelial cells, epithelial cells, eosinophils, and mast cells

• Activates downstream signaling cascades leading to inflammatory responses

• Associated with Th2-related immune functions and allergic responses

sST2 (soluble form):

• Acts as a decoy receptor that binds free IL-33, preventing it from interacting with ST2L

• Serves as a negative regulator of Th2 cytokine production

• Elevated in various inflammatory conditions including asthma, sepsis, and myocardial infarction

• May have protective effects in certain inflammatory contexts

This dual system allows for fine-tuning of IL-33-mediated responses, with increased sST2 production potentially serving as a regulatory mechanism to control excessive inflammation .

How are IL1RL1 genetic variations associated with asthma and other respiratory conditions, and what functional mechanisms explain these associations?

Multiple genetic studies have identified IL1RL1 polymorphisms associated with asthma and respiratory conditions. In European birth cohorts, IL1RL1 rs102082293, rs10204137 (rs4988955), rs13424006, and rs13431828 (rs13048661) variations were associated with asthma development at school age . Additionally, the variant genotype of IL1RL1 rs13408661/13431828 was associated with current ICS (inhaled corticosteroid) use in former bronchiolitis patients at age 5-7 years and with persistent asthma at age 11-13 years .

These genetic associations may be explained by several functional mechanisms:

• Altered expression levels of IL1RL1, affecting the balance between membrane-bound and soluble forms

• Modified binding affinity to IL-33, changing downstream signaling intensity

• Differential regulation of Th2 inflammatory responses, which are central to allergic asthma pathogenesis

• Impact on epithelial barrier function and response to environmental triggers

Evidence suggests that IL1RL1 may be particularly relevant in Th2-like asthma phenotypes, as ST2L expression correlates with Th2 biomarkers in bronchial tissue samples . This supports the notion that targeting the IL-33/IL1RL1 pathway might have therapeutic benefits, especially in severe asthma with Th2 inflammation features .

What is the emerging role of IL1RL1 in cancer biology, particularly in lung cancer, and how might genetic polymorphisms influence cancer susceptibility?

Recent research has revealed important roles for IL1RL1 in cancer biology, particularly in lung cancer. Studies have shown that IL1RL1 expression is significantly downregulated in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues compared to normal lung tissues . Additionally, specific IL1RL1 polymorphisms, particularly rs12479210 and rs1420101, have been associated with increased lung cancer risk in the Chinese Han population .

The mechanistic involvement of IL1RL1 in cancer biology appears to function through several pathways:

Bioinformatics analyses have confirmed significant differences between six SNPs in IL1RL1 and the expression level of IL1RL1 in lung cancer tissues, suggesting that genetic variations may influence cancer risk through altered gene expression and protein function .

What are the optimal experimental conditions for studying IL1RL1 protein-protein interactions, and how can researchers assess binding affinity to IL-33?

For studying IL1RL1 protein-protein interactions and assessing binding affinity to IL-33, researchers should consider these methodological approaches:

Surface Plasmon Resonance (SPR):

• Immobilize purified IL1RL1 on a sensor chip using His-tag

• Flow IL-33 at different concentrations over the surface

• Analyze association and dissociation rates to determine Kd values

• Maintain physiological buffer conditions (PBS pH 7.4 with 0.05% surfactant)

Co-Immunoprecipitation (Co-IP):

• Express tagged versions of IL1RL1 and potential binding partners

• Use anti-tag antibodies to pull down protein complexes

• Analyze by Western blot to detect interactions

• Include appropriate controls to rule out non-specific binding

Fluorescence Resonance Energy Transfer (FRET):

• Generate fluorescently labeled IL1RL1 and IL-33

• Monitor energy transfer as an indication of binding

• Perform in solution or in cellular contexts for physiological relevance

Cellular Binding Assays:

• Use flow cytometry with fluorescently labeled IL-33

• Assess binding to cells expressing IL1RL1

• Develop competitive binding assays to determine specificity

For all these methods, it's crucial to use properly folded and glycosylated IL1RL1 from Sf9 cells to ensure physiologically relevant results, as glycosylation patterns may influence binding properties and protein-protein interactions .

How can researchers effectively distinguish between effects mediated by membrane-bound IL1RL1 versus soluble IL1RL1 in experimental systems?

Distinguishing between effects mediated by membrane-bound IL1RL1 (ST2L) versus soluble IL1RL1 (sST2) requires careful experimental design:

Selective Expression Systems:

• Generate expression constructs for ST2L (full-length) and sST2 (truncated form)

• Use cell lines that do not endogenously express IL1RL1

• Create stable cell lines expressing either form for comparative studies

Blocking Antibodies and Reagents:

• Utilize antibodies that specifically recognize the extracellular domain (both forms) versus transmembrane domain (ST2L only)

• Employ recombinant sST2 to competitively inhibit IL-33 binding to ST2L

• Design domain-specific peptides that selectively block protein-protein interactions

Genetic Approaches:

• Use siRNA/shRNA targeting exons specific to ST2L or sST2

• Employ CRISPR-Cas9 to selectively modify domains unique to each form

• Create conditional knockout models for temporal control of expression

Functional Readouts:

• Measure downstream signaling events (phosphorylation of MAPK3/ERK1, MAPK1/ERK2, etc.) that occur only with membrane-bound receptor activation

• Compare cytokine profiles induced by each form

• Analyze differential gene expression patterns specific to each form's activity

In vivo approaches:

• Administer recombinant sST2 to neutralize IL-33 in animal models

• Generate transgenic animals overexpressing either form selectively

• Use tissue-specific promoters to control expression in relevant cell types

These strategies enable researchers to parse the distinct biological roles of each form and understand their complementary functions in inflammatory and immune responses .

What are the most significant IL1RL1 SNPs associated with disease phenotypes, and what methodologies are recommended for their genotyping?

The search results identify several significant IL1RL1 SNPs associated with disease phenotypes:

Asthma-associated SNPs:

• rs102082293, rs10204137 (rs4988955), rs13424006, and rs13431828 (rs13048661) - associated with asthma at school age in European cohorts

• rs13408661/13431828 - associated with current ICS use in former bronchiolitis patients and persistent asthma

Bronchiolitis-associated SNPs:

• rs1921622 - associated with severe bronchiolitis compared with controls

Lung cancer-associated SNPs:

• rs12479210 and rs1420101 - associated with increased lung cancer risk in the Chinese Han population

Other allergic phenotype-associated SNPs:

• rs3771180, rs3771175, rs10208293, and rs10197862 - investigated in relation to lung cancer but associations not confirmed

• rs1420101 - associated with allergic phenotypes including asthma, eczema, and eosinophilia

For genotyping these SNPs, several methodological approaches are recommended:

• TaqMan SNP Genotyping Assays:

  • High-throughput method suitable for large sample sets

  • Provides accurate allelic discrimination

  • Requires specialized equipment but is widely available in research facilities

• Sequencing-based approaches:

  • Next-generation sequencing for comprehensive analysis of the IL1RL1 locus

  • Sanger sequencing for validation of specific variants

  • Allows detection of novel variants and haplotype determination

• Mass Spectrometry-based methods:

  • MALDI-TOF-based systems for multiplex SNP analysis

  • High accuracy and relatively high throughput

• PCR-RFLP (Restriction Fragment Length Polymorphism):

  • Cost-effective method for smaller studies

  • Allows visual confirmation of genotypes through gel electrophoresis

• Digital PCR:

  • Highly sensitive for detection of allele-specific expression

  • Useful for quantitative assessment of variant alleles

Researchers should select appropriate methods based on their study size, available resources, and required accuracy level.

How do IL1RL1 genotypes correlate with protein expression levels, and what implications does this have for interpreting functional studies?

The relationship between IL1RL1 genotypes and protein expression levels has important implications for interpreting functional studies:

IL1RL1 disease-associated SNPs correlate with IL1RL1 mRNA and serum protein levels of IL-1RL1a (sST2) . This genotype-phenotype correlation provides a mechanistic link between genetic variation and disease susceptibility. Research has shown that:

• Expression Quantitative Trait Loci (eQTL) effects:

  • Certain IL1RL1 SNPs function as eQTLs, directly influencing gene expression levels

  • Different alleles can lead to variable promoter activity or mRNA stability

  • This affects the balance between membrane-bound ST2L and soluble sST2 forms

• Tissue-specific expression patterns:

  • The impact of genotypes on expression may vary across different tissues

  • For example, expression differences between normal and lung cancer tissues show significant associations with specific SNPs

  • TCGA database analyses confirm differential IL1RL1 expression between normal tissues and lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)

• Biomarker potential:

  • IL-1RL1a serum levels can predict the development of eosinophilic asthma characterized by high FeNO in preschool wheezing children

  • IL-1RL1a levels increase during asthma exacerbations, suggesting utility as a marker of active inflammatory responses

These correlations have important implications for interpreting functional studies:

Understanding these genotype-expression relationships helps explain the mechanistic basis of disease associations and provides context for interpreting experimental results across different genetic backgrounds .

What approaches can be used to study IL1RL1 in complex diseases with heterogeneous phenotypes like asthma?

Studying IL1RL1 in complex diseases with heterogeneous phenotypes like asthma requires multi-faceted approaches:

Phenotype Stratification:

• Categorize patients based on clinical characteristics (age of onset, severity, treatment response)

• Use biomarkers to identify specific endotypes (e.g., Th2-high vs. Th2-low asthma)

• Apply cluster analysis to identify natural disease groupings based on objective parameters

Multi-omics Integration:

• Combine genomics (IL1RL1 SNPs), transcriptomics (gene expression), and proteomics (protein levels)

• Integrate with clinical data to correlate genetic variations with disease manifestations

• Use systems biology approaches to understand network effects beyond single-gene focus

Functional Validation in Model Systems:

• Develop mouse models with specific IL1RL1 variants to recapitulate human phenotypes

• Use primary human bronchial epithelial cells in air-liquid interface cultures to study IL1RL1 regulation

• Employ precision-cut lung slices to maintain tissue architecture while allowing experimental manipulation

Longitudinal Studies:

• Track IL1RL1 expression and sST2 levels over time in relation to disease progression

• Correlate with environmental exposures and clinical outcomes

• Identify temporal patterns that may indicate disease trajectory or treatment response

Clinical Trial Stratification:

• Use IL1RL1 genotypes and/or protein levels as stratification biomarkers in clinical trials

• Test for differential treatment response based on IL1RL1 status

• Develop targeted therapies against the IL-33/IL1RL1 pathway for specific patient subgroups

These comprehensive approaches allow researchers to address the heterogeneity inherent in complex diseases and identify the specific contexts in which IL1RL1 plays a critical role in pathogenesis .

How can researchers leverage IL1RL1 as a therapeutic target, and what experimental models best predict clinical efficacy?

Leveraging IL1RL1 as a therapeutic target requires understanding both its biology and appropriate experimental models for predicting clinical efficacy:

Therapeutic Targeting Strategies:

• Blocking antibodies: Develop antibodies against IL1RL1 to prevent IL-33 binding

• Soluble decoy receptors: Engineer optimized versions of sST2 with enhanced IL-33 binding

• Small molecule inhibitors: Design molecules that disrupt IL1RL1 signaling complexes

• Anti-sense oligonucleotides: Reduce IL1RL1 expression in specific tissues

• Gene editing approaches: Modify expression or function of IL1RL1 in relevant cell types

Experimental Models for Efficacy Prediction:

In vitro models:

• Primary human bronchial epithelial cells (HBECs) in air-liquid interface cultures to assess IL1RL1 regulation in response to therapeutic intervention

• Co-culture systems incorporating multiple cell types (epithelial cells, immune cells) to capture complex cellular interactions

• Precision-cut lung slices from human donors to maintain tissue architecture and evaluate responses in a more physiological context

Ex vivo models:

• Human lung explants to evaluate therapeutic effects on intact human tissue

• Bronchial biopsies from patients cultured with potential therapeutics

• Patient-derived organoids representing disease-specific phenotypes

In vivo models:

• Humanized mouse models expressing human IL1RL1 variants

• Mouse models with human-relevant IL1RL1 polymorphisms

• Challenge models that recapitulate specific disease triggers (allergen, viral, etc.)

Translational approaches:

• Early-phase clinical trials with extensive biomarker analysis

• Patient stratification based on IL1RL1 genotype or protein expression levels

• Proxy endpoints that correlate with clinical outcomes (e.g., changes in inflammatory biomarkers)

Predictive Biomarkers for Response:

• Baseline sST2 levels may predict response to anti-IL-33/IL1RL1 therapies

• IL1RL1 genotype could identify likely responders to pathway-targeted interventions

• Expression patterns of ST2L on specific immune cell populations might indicate therapeutic susceptibility

Evidence suggests that targeting this pathway may have particular therapeutic benefits in severe asthma, especially in phenotypes characterized by Th2-like inflammation . Both pathologic and genetic approaches support a role for IL1RL1 in severe and Th2-like asthma, making it a promising target for precision medicine approaches .

What emerging technologies might advance our understanding of IL1RL1 biology and its role in disease pathogenesis?

Several emerging technologies have potential to substantially advance our understanding of IL1RL1 biology:

Single-cell genomics and proteomics:

• Single-cell RNA sequencing to identify cell-specific expression patterns of IL1RL1 and its signaling partners

• Single-cell proteomics to detect protein-level variation not captured by transcriptomic approaches

• Spatial transcriptomics to map IL1RL1 expression within tissue microenvironments

CRISPR-based technologies:

• CRISPR activation/interference systems to modulate IL1RL1 expression with high specificity

• Base editing to introduce specific SNPs associated with disease risk

• Prime editing for precise genomic modifications of regulatory regions

• CRISPR screens to identify novel regulators of IL1RL1 expression and function

Advanced imaging technologies:

• Super-resolution microscopy to visualize IL1RL1 distribution and clustering at the cell membrane

• Live-cell imaging to track receptor dynamics in real-time

• Intravital microscopy to observe IL1RL1 function in intact tissues

Computational approaches:

• Machine learning algorithms to predict functional consequences of IL1RL1 genetic variants

• Network analysis to understand how IL1RL1 integrates with broader signaling systems

• Molecular dynamics simulations to model IL-33/IL1RL1 interactions at atomic resolution

Bioengineered model systems:

• Organ-on-chip platforms incorporating primary human cells expressing IL1RL1

• Microfluidic systems to analyze IL1RL1-mediated cell-cell communication

• 3D bioprinted tissues with controlled expression of IL1RL1 variants

These technologies will help address critical knowledge gaps, including: understanding cell-type specific roles of IL1RL1, elucidating the functional consequences of genetic variants, mapping temporal dynamics of IL1RL1 signaling, and identifying novel therapeutic strategies targeting this pathway in various disease contexts .

What are the current limitations in IL1RL1 research, and how might these challenges be addressed in future studies?

Current limitations in IL1RL1 research present several challenges that future studies must address:

Technical and Methodological Limitations:

• Protein heterogeneity: The multiple bands (40-57kDa) observed with recombinant IL1RL1 due to glycosylation patterns complicate structural and functional analyses

• Isoform-specific reagents: Limited availability of antibodies and tools that can specifically distinguish between membrane-bound ST2L and soluble sST2 forms

• Model systems: Incomplete representation of human disease complexity in current animal and cellular models

Knowledge Gaps:

• Regulatory mechanisms: Incomplete understanding of how IL1RL1 expression is regulated in different tissues and disease states

• Signaling complexity: Limited knowledge of how IL1RL1 signaling integrates with other pathways in different cellular contexts

• Genetic variation: Functional consequences of many IL1RL1 SNPs remain poorly characterized

• Tissue-specific roles: The function of IL1RL1 beyond immune and epithelial cells remains understudied

Future Approaches to Address These Challenges:

• Develop improved reagents and tools:

  • Generate isoform-specific antibodies and detection methods

  • Create reporter systems for tracking ST2L vs. sST2 expression

  • Design assays to measure specific post-translational modifications

• Enhance model systems:

  • Develop humanized mouse models with relevant IL1RL1 polymorphisms

  • Create patient-derived organoids that maintain genetic background and disease phenotypes

  • Establish co-culture systems that recapitulate complex cellular interactions

• Apply systems biology approaches:

  • Integrate multi-omics data to build comprehensive models of IL1RL1 function

  • Use network analysis to identify key regulators and interaction partners

  • Develop computational methods to predict functional consequences of genetic variants

• Expand clinical correlations:

  • Conduct larger, well-characterized cohort studies with comprehensive genotyping

  • Incorporate longitudinal sampling to capture dynamic changes in IL1RL1 expression

  • Correlate IL1RL1 genetics and expression with detailed clinical phenotyping

• Collaborative research initiatives:

  • Establish biobanks with well-characterized samples from diverse populations

  • Create shared resources for IL1RL1 reagents and model systems

  • Develop standardized protocols for measuring IL1RL1 expression and function

By addressing these limitations, future research can provide a more comprehensive understanding of IL1RL1 biology and accelerate the development of targeted therapies for conditions including asthma, bronchiolitis, and potentially cancer .

Frequently Asked Questions for Researchers on IL1RL1 Human, Sf9 Active

This comprehensive FAQ collection addresses key research considerations regarding IL1RL1 (Interleukin-1 receptor-like 1) human protein expressed in Sf9 systems. The questions are structured to support both fundamental understanding and advanced research applications, with emphasis on methodological approaches for academic researchers.

What is IL1RL1 and what are its primary structural features when expressed in Sf9 cells?

IL1RL1, also known as Interleukin-1 receptor-like 1 or Protein ST2, is a member of the IL-1 receptor family that functions as a receptor for interleukin-33. When produced in Sf9 Baculovirus cells, IL1RL1 is expressed as a single, glycosylated polypeptide chain spanning amino acids 19-328 and typically fused to an 8 amino acid His Tag at the C-terminus. The complete recombinant protein contains 318 amino acids with a theoretical molecular mass of 36.0kDa, though due to glycosylation it displays multiple bands between 40-57kDa on SDS-PAGE under reducing conditions .

Why does IL1RL1 produced in Sf9 cells show multiple bands on SDS-PAGE despite being a single polypeptide?

The multiple bands observed between 40-57kDa on SDS-PAGE under reducing conditions are primarily attributed to post-translational modifications, specifically variable glycosylation patterns. Sf9 insect cells perform eukaryotic post-translational modifications including glycosylation, but with patterns that differ from mammalian cells. The heterogeneity in glycosylation results in proteins with slightly different molecular weights, appearing as multiple bands. For research applications, this heterogeneity should be documented when characterizing the protein preparation, but doesn't necessarily indicate degradation or compromised functionality .

What expression system considerations are important when producing functional IL1RL1 in Sf9 cells?

For producing functional IL1RL1, the Sf9 Baculovirus expression system offers several advantages for researchers. This system requires careful optimization across multiple parameters:

• Expression construct design:

  • Codon optimization for insect cell expression

  • Inclusion of appropriate signal sequences for secretion

  • Strategic placement of affinity tags (typically C-terminal His-tags)

  • Selection of promoters for optimal expression timing

• Infection parameters:

  • MOI (multiplicity of infection) optimization

  • Time-course analysis to determine peak expression

  • Cell density at infection time

• Purification considerations:

  • Multiple chromatographic steps typically required

  • Careful buffer selection to maintain stability

  • Monitoring of glycosylation patterns during purification

The resulting recombinant IL1RL1 requires purification to ensure high purity (>95%) suitable for functional studies .

How can researchers evaluate the functional integrity of purified IL1RL1 protein?

Evaluating functional integrity of IL1RL1 requires multiple orthogonal approaches:

• Binding assays:

  • Surface plasmon resonance (SPR) with immobilized IL-33

  • ELISA-based binding assays

  • Cell-based binding assays using IL-33-responsive cells

• Structural analysis:

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to evaluate folding integrity

  • Thermal shift assays to determine stability

  • Mass spectrometry to confirm post-translational modifications

• Signaling activity:

  • Measuring downstream phosphorylation of MAPK3/ERK1, MAPK1/ERK2, MAPK14, and MAPK8 in appropriate cell lines

  • Analyzing recruitment of MYD88, IRAK1, IRAK4, and TRAF6 in cell-based systems

  • Reporter gene assays to monitor transcriptional activation

Researchers should maintain proper storage conditions in phosphate-buffered saline (pH 7.4) with 10% glycerol at -20°C for long-term storage with carrier proteins (0.1% HSA or BSA) while avoiding multiple freeze-thaw cycles .

How does IL1RL1 mediate downstream signaling upon IL-33 binding, and what experimental approaches can measure this activity?

IL1RL1 (particularly the transmembrane variant ST2L) initiates a complex signaling cascade upon IL-33 binding. This process involves:

• Receptor activation: Upon binding IL-33, IL1RL1 recruits MYD88, IRAK1, IRAK4, and TRAF6

• Kinase activation: This complex leads to phosphorylation of multiple MAP kinases including MAPK3/ERK1, MAPK1/ERK2, MAPK14, and MAPK8

• Transcriptional regulation: Activated kinases modulate transcription factors controlling inflammatory and immune responses

Experimental approaches to measure this activity include:

• Phospho-specific Western blotting to detect activated MAPK signaling

• Co-immunoprecipitation to analyze protein complex formation

• Luciferase reporter assays with IL-33-responsive promoters

• Quantitative RT-PCR to measure induction of downstream target genes

• Flow cytometry to assess phospho-protein levels at single-cell resolution

• Proximity ligation assays to detect protein-protein interactions in situ

These methods can be combined to provide a comprehensive understanding of IL1RL1 signaling dynamics in different cellular contexts .

What is the differential role of membrane-bound IL1RL1 (ST2L) versus soluble IL1RL1 (sST2) in inflammation and immune regulation?

Membrane-bound IL1RL1 (ST2L) and its soluble form (sST2) have distinct and sometimes opposing functions in inflammation and immune regulation:

ST2L (membrane-bound form):

• Functions as the receptor for IL-33, mediating its pro-inflammatory effects

• Expressed on various cell types including T helper cells, endothelial cells, epithelial cells, eosinophils, and mast cells

• Activates downstream signaling cascades leading to inflammatory responses

• Associated with Th2-related immune functions and allergic responses

sST2 (soluble form):

• Acts as a decoy receptor that binds free IL-33, preventing it from interacting with ST2L

• Serves as a negative regulator of Th2 cytokine production

• Elevated in various inflammatory conditions including asthma, sepsis, and myocardial infarction

• Functions as an immunomodulatory molecule with potential protective effects

This dual system allows for fine-tuning of IL-33-mediated responses, with increased sST2 production potentially serving as a regulatory mechanism to control excessive inflammation. Researchers investigating IL1RL1 should carefully distinguish between these forms when designing experiments and interpreting results .

How are IL1RL1 genetic variations associated with asthma and other respiratory conditions?

Multiple genetic studies have identified IL1RL1 polymorphisms associated with respiratory conditions:

Asthma associations:

• European birth cohorts identified rs102082293, rs10204137 (rs4988955), rs13424006, and rs13431828 (rs13048661) as significantly associated with asthma development at school age

• The variant genotype of IL1RL1 rs13408661/13431828 showed association with current ICS (inhaled corticosteroid) use in former bronchiolitis patients at age 5-7 years and with persistent asthma at age 11-13 years

• Evidence suggests IL1RL1 may be particularly relevant in Th2-like asthma phenotypes, as ST2L expression correlates with Th2 biomarkers in bronchial tissue samples

Bronchiolitis associations:

• The IL1RL1 rs1921622 SNP was associated with severe bronchiolitis compared with controls

• Severe bronchiolitis was associated with higher soluble IL1RL1-a concentrations in nasopharyngeal aspirates

Functionally, these genetic variations can impact:

Understanding these genetic associations helps identify at-risk populations and may guide development of targeted therapies for patients with specific IL1RL1 variants .

What is the emerging role of IL1RL1 in cancer biology, particularly in lung cancer?

Recent research has revealed important roles for IL1RL1 in cancer biology:

These findings suggest IL1RL1 may serve as both a biomarker and potential therapeutic target in certain cancers, particularly lung cancer, though mechanistic details require further investigation .

How can researchers effectively distinguish between effects mediated by membrane-bound IL1RL1 versus soluble IL1RL1 in experimental systems?

Distinguishing between effects mediated by membrane-bound IL1RL1 (ST2L) versus soluble IL1RL1 (sST2) requires sophisticated experimental design:

Selective Expression Systems:

• Generate expression constructs for ST2L (full-length) and sST2 (truncated form)

• Use cell lines that do not endogenously express IL1RL1

• Create stable cell lines expressing either form for comparative studies

Genetic Approaches:

• Use siRNA/shRNA targeting exons specific to ST2L or sST2

• Employ CRISPR-Cas9 to selectively modify domains unique to each form

• Design splice-switching oligonucleotides to modulate relative expression of isoforms

Blocking Reagents:

• Utilize antibodies that specifically recognize the extracellular domain (both forms) versus transmembrane domain (ST2L only)

• Employ recombinant sST2 to competitively inhibit IL-33 binding to ST2L

• Design domain-specific peptides that selectively block protein-protein interactions

Functional Readouts:

• Measure downstream signaling events (phosphorylation cascades) that occur only with membrane-bound receptor activation

• Compare cytokine profiles induced by each form

• Analyze differential gene expression patterns specific to each form's activity

Experimental Controls:

• Include appropriate negative controls lacking IL-33 stimulation

• Use IL-33 mutants with differential binding to ST2L vs. sST2

• Control for transfection/transduction efficiency in expression systems

These strategies enable researchers to parse the distinct biological roles of each form and understand their complementary functions in inflammatory and immune responses .

What methodological approaches should be used to study IL1RL1 polymorphisms in relation to disease phenotypes?

Studying IL1RL1 polymorphisms in relation to disease phenotypes requires comprehensive methodological approaches:

Study Design Considerations:

• Prospective cohort studies to assess polymorphism impact on disease development

• Case-control studies to evaluate associations with established disease

• Family-based designs to control for population stratification

• Longitudinal follow-up to capture age-dependent phenotypes

• Adequate sample size calculations based on expected effect sizes

Genotyping Methods:

• TaqMan SNP genotyping assays for targeted polymorphism analysis

• Next-generation sequencing for comprehensive variant detection

• Custom genotyping arrays for high-throughput analysis

• Digital PCR for highly sensitive allele discrimination

• Validation with secondary methods to confirm results

Phenotyping Approaches:

• Standardized clinical assessments (e.g., spirometry for asthma)

• Biomarker measurements (serum sST2 levels, IL-33 levels)

• Detailed clinical characterization to identify disease subtypes

• Objective measures of disease severity and progression

• Response to specific treatments or challenges

Statistical Analysis:

• Appropriate adjustment for multiple testing

• Haplotype analysis rather than single SNP associations

• Gene-environment interaction assessments

• Mediation analysis to determine mechanisms of effect

• Meta-analysis to combine results across studies

Functional Validation:

• Expression quantitative trait loci (eQTL) analysis

• In vitro reporter assays to assess variant impact on expression

• CRISPR-based editing to introduce specific variants

• Protein-level assessments (Western blotting, ELISA)

• Signaling studies to determine functional consequences

These methodological approaches have successfully identified significant associations, such as IL1RL1 rs13408661/13431828 with persistent asthma and rs12479210/rs1420101 with lung cancer risk .

How can researchers leverage multi-omics approaches to better understand IL1RL1 biology in complex diseases?

Multi-omics approaches provide powerful frameworks for understanding IL1RL1 in complex diseases:

Integrated Genomics:

• Whole genome/exome sequencing to identify rare variants beyond common SNPs

• Epigenomic profiling (methylation, chromatin accessibility) to understand regulatory mechanisms

• Targeted sequencing of the entire IL1RL1 locus including regulatory regions

• Analysis of structural variants and copy number variations affecting IL1RL1

Transcriptomics:

• RNA-seq to identify disease-specific expression patterns

• Single-cell RNA-seq to define cell-type specific IL1RL1 expression

• Alternative splicing analysis to quantify ST2L vs. sST2 isoform ratios

• Long-read sequencing to identify novel IL1RL1 isoforms

Proteomics:

• Mass spectrometry to analyze IL1RL1 post-translational modifications

• Targeted protein quantification of ST2L and sST2 in various tissues

• Interactome mapping to identify novel IL1RL1 binding partners

• Phosphoproteomics to characterize IL1RL1 signaling dynamics

Metabolomics:

• Identification of metabolic signatures associated with IL1RL1 activation

• Analysis of lipid mediators impacted by IL-33/IL1RL1 signaling

• Correlation of metabolic profiles with sST2 levels

Data Integration Approaches:

• Network analysis to position IL1RL1 within broader disease pathways

• Machine learning to identify patterns across multi-omic datasets

• Bayesian approaches to infer causal relationships

• Systems biology modeling of IL1RL1 signaling networks

This integrated approach can reveal mechanisms by which IL1RL1 variants influence disease risk and progression across conditions like asthma, bronchiolitis, and lung cancer, potentially identifying novel biomarkers and therapeutic targets .

What are the current challenges in translating IL1RL1 research findings into clinical applications?

Translating IL1RL1 research into clinical applications faces several significant challenges:

Biological Complexity:

• Dual roles of membrane-bound versus soluble forms with potentially opposing functions

• Tissue and cell-type specific expression patterns requiring targeted approaches

• Complex post-translational modifications affecting protein function

• Multiple genetic variants with different functional consequences

Technical Limitations:

• Lack of standardized assays for measuring ST2L versus sST2 in clinical samples

• Heterogeneity in glycosylation patterns affecting protein function and detection

• Challenges in developing specific antibodies distinguishing protein isoforms

• Difficulties in maintaining proper protein folding in recombinant systems

Clinical Translation Barriers:

• Phenotypic heterogeneity within disease categories (e.g., asthma subtypes)

• Population differences in genetic variant frequencies and functional impacts

• Need for large, well-characterized cohorts for validating genetic associations

• Determining optimal timing for therapeutic interventions targeting this pathway

Therapeutic Development Challenges:

• Specificity in targeting IL-33/IL1RL1 interaction without affecting other pathways

• Achieving tissue-specific delivery of therapeutics

• Identifying appropriate patient populations most likely to benefit

• Developing suitable biomarkers for monitoring treatment response

Regulatory and Practical Considerations:

• Standardizing IL1RL1/sST2 as a clinical biomarker across laboratories

• Cost-effectiveness of genotyping for clinical decision-making

• Integration with existing clinical algorithms and practice guidelines

• Demonstrating improved outcomes compared to standard treatments

Addressing these challenges requires coordinated efforts across basic science, translational research, and clinical studies to fully realize the potential of IL1RL1 as a therapeutic target and biomarker in conditions ranging from asthma and bronchiolitis to potentially lung cancer .

What novel experimental models might advance our understanding of IL1RL1 biology?

Several innovative experimental models show promise for advancing IL1RL1 research:

Advanced Cell Culture Systems:

• Air-liquid interface cultures of primary human bronchial epithelial cells for studying IL1RL1 regulation in a physiologically relevant context

• Co-culture systems incorporating multiple cell types (epithelial, immune, stromal) to recapitulate complex cellular interactions

• Microfluidic organ-on-chip platforms to study IL1RL1 function under physiological flow conditions

Organoid Technologies:

• Patient-derived lung organoids for studying IL1RL1 in disease-specific contexts

• Immune-competent organoid systems incorporating both structural and immune cells

• Brain organoids to study neuroinflammatory aspects of IL1RL1 signaling

Advanced Animal Models:

• Humanized mouse models expressing human IL1RL1 variants

• CRISPR-engineered mice harboring specific human IL1RL1 polymorphisms

• Conditional and inducible IL1RL1 knockout/knockin models for temporal control

• Tissue-specific IL1RL1 models to delineate organ-specific functions

Ex Vivo Human Systems:

• Precision-cut lung slices from human donors with defined IL1RL1 genotypes

• Explant cultures from surgical specimens for pharmacological studies

• Human lung-on-chip microfluidic devices incorporating IL1RL1 variants

Computational and In Silico Models:

• Machine learning approaches to predict functional consequences of IL1RL1 variants

• Molecular dynamics simulations of IL-33/IL1RL1 interactions

• Network models integrating IL1RL1 signaling with broader inflammatory pathways

• Virtual patient cohorts for in silico clinical trials of IL1RL1-targeted therapies

These advanced models can help address key knowledge gaps, including understanding cell type-specific functions, elucidating the consequences of genetic variations, and identifying optimal therapeutic targeting strategies .

How might targeting the IL-33/IL1RL1 pathway translate into novel therapeutic strategies?

The IL-33/IL1RL1 pathway presents several promising therapeutic targeting strategies:

Direct Pathway Inhibition:

• Anti-IL1RL1 monoclonal antibodies to block IL-33 binding

• Recombinant sST2 proteins as decoy receptors

• Small molecule inhibitors of IL1RL1 signaling

• IL-33 neutralizing antibodies or traps

• Peptide inhibitors targeting critical interaction domains

Genetic and RNA-based Approaches:

• Antisense oligonucleotides to modulate ST2L vs. sST2 splicing ratios

• siRNA/shRNA to selectively knock down IL1RL1 expression

• mRNA therapeutics to deliver modified sST2 as a decoy receptor

• CRISPR-based gene editing for patients with high-risk variants

Pathway-Specific Targeting:

• Inhibitors of downstream signaling components (IRAK1/4, TRAF6)

• Modulators of IL1RL1 expression (targeting transcriptional regulators)

• Agents affecting IL1RL1 trafficking and membrane localization

• Compounds influencing IL1RL1 post-translational modifications

Precision Medicine Applications:

• IL1RL1 genotype-guided therapy selection

• sST2 levels as biomarkers for patient stratification and response monitoring

• Combination therapies targeting IL1RL1 along with other pathways

• Tissue-specific delivery approaches for localized effects

Disease-Specific Approaches:

• For asthma: Inhaled formulations targeting bronchial epithelium

• For lung cancer: Combination with immune checkpoint inhibitors

• For bronchiolitis: Early intervention in genetically susceptible infants

• For allergic conditions: Desensitization protocols with IL1RL1 modulation

The strongest evidence currently supports targeting this pathway in Th2-driven inflammatory conditions like severe asthma . Both pathologic and genetic approaches confirm IL1RL1's role in these conditions, suggesting that these therapeutic strategies may benefit specific patient populations identified through biomarker and genetic screening .

What quality control measures are essential when working with recombinant IL1RL1 from Sf9 cells?

Rigorous quality control is critical when working with recombinant IL1RL1 from Sf9 cells:

Purity Assessment:

• SDS-PAGE analysis with Coomassie or silver staining (>95% purity standard)

• Size-exclusion chromatography to detect aggregates and multimers

• Western blot with specific antibodies to confirm identity

• Mass spectrometry for accurate molecular weight determination

• Endotoxin testing to ensure preparation is endotoxin-free (<1 EU/mg)

Structural Integrity:

• Circular dichroism spectroscopy to assess secondary structure

• Thermal shift assays to determine stability profiles

• Limited proteolysis to evaluate folding quality

• Dynamic light scattering to assess homogeneity and aggregation state

• N-terminal sequencing to confirm correct processing

Glycosylation Analysis:

• Lectin blotting to characterize glycan patterns

• PNGase F treatment to assess contribution of N-glycans

• Mass spectrometry to map glycosylation sites

• Comparison of glycoform patterns between batches for consistency

• Functional impact assessment of different glycoforms

Functional Validation:

• IL-33 binding assays with quantitative affinity measurements

• Cell-based activity assays measuring downstream signaling

• Comparative analysis with mammalian-expressed IL1RL1

• Lot-to-lot consistency testing with reference standards

• Stability studies under various storage conditions

Documentation and Reporting:

• Detailed batch records including expression conditions

• Certificate of analysis with all quality parameters

• Stability data under recommended storage conditions

• Validation of performance in standard assay systems

• Transparent reporting of heterogeneity in publications

These quality control measures ensure that experimental results using IL1RL1 are reliable and reproducible, which is essential for advancing our understanding of its biology and therapeutic potential .

What are the optimal experimental designs for studying IL1RL1 genetic variants in human populations?

Optimal experimental designs for studying IL1RL1 genetic variants require careful consideration of multiple factors:

Cohort Selection and Design:

• Prospective cohorts with longitudinal follow-up

  • Enables assessment of variant impact on disease development

  • Allows evaluation of age-dependent effects

  • Provides opportunity for repeated biomarker measurements

• Nested case-control studies within established cohorts

  • Efficient use of resources

  • Matching on important confounders

  • Availability of pre-disease samples

• Family-based designs

  • Controls for population stratification

  • Enables transmission disequilibrium testing

  • Allows study of rare variants in familial cases

Sample Size Considerations:

• Power calculations based on expected effect sizes from previous studies

• Adjustment for multiple testing when analyzing multiple variants

• Consideration of gene-environment interactions requiring larger samples

• Planning for replication cohorts to validate findings

Genotyping Strategy:

• Targeted approach focusing on known IL1RL1 SNPs:

  • rs13408661/13431828, rs1921622, rs12479210, rs1420101 (established associations)

  • Tag SNPs covering major haplotype blocks

  • Functional variants affecting expression or protein structure

• Comprehensive sequencing approach:

  • Capture of regulatory regions beyond coding sequences

  • Identification of rare variants not covered in genotyping arrays

  • Detection of structural variants affecting IL1RL1 locus

Phenotyping Depth:

• Detailed clinical characterization to identify disease subtypes

• Molecular phenotyping (IL1RL1 expression levels, ST2L vs. sST2 ratio)

• Biomarker panels to capture pathway activity

• Treatment response as a phenotypic outcome

• Environmental exposure assessment for interaction studies

Analysis Considerations:

• Haplotype analysis rather than single SNP approach

• Proper adjustment for ancestry and population stratification

• Assessment of gene-environment interactions

• Integrative analysis incorporating expression data (eQTL)

• Meta-analysis across multiple cohorts for robust findings