Recombinant Mouse Interleukin-36 alpha protein (Il36a) (Active)

What is the molecular structure of recombinant mouse IL-36 alpha protein?

Recombinant Mouse IL-36 alpha (also known as IL-1F6) is a full-length protein spanning amino acids 1-160, expressed in Escherichia coli expression systems. The protein has a specific amino acid sequence (M N K E K E L R A A S P S L R H V Q D L S S R V W I L Q N N I L T A V P R K E Q T V P V T I T L L P C Q Y L D T L E T N R G D P T Y M G V Q R P M S C L F C T K D G E Q P V L Q L G E G N I M E M Y N K K E P V K A S L F Y H K K S G T T S T F E S A A F P G W F I A V C S K G S C P L I L T Q E L G E I F I T D F E M I V V H) and exhibits ≥95% purity with endotoxin levels ≤0.1 EU/mg, making it suitable for various experimental applications including SDS-PAGE, HPLC, and mass spectrometry analysis .

How does IL-36 alpha compare structurally to other IL-36 family members?

IL-36 alpha is a member of the broader IL-1 cytokine family and specifically belongs to the IL-36 subfamily. Unlike IL-36 beta (IL-1F8), which is typically used in its truncated form (aa 31-183) , IL-36 alpha is available in forms spanning amino acids 1-160 or 8-160 . While all IL-36 family members share structural similarities and the capacity to signal through the IL-36 receptor (IL1RL2), they may exhibit differential potency and tissue-specific expression patterns, which researchers should consider when designing comparative studies of IL-36 family proteins.

What are the optimal storage and handling conditions for maintaining IL-36 alpha activity?

For maintaining optimal biological activity of recombinant mouse IL-36 alpha, researchers should store the lyophilized protein at -20°C to -80°C and reconstitute it immediately before use in appropriate buffers (typically PBS or similar physiological buffers). Once reconstituted, the protein should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly reduce biological activity. When handling the protein for experimental applications, it's advisable to maintain sterile conditions and use low protein-binding tubes to prevent loss of protein through adsorption to container surfaces.

What experimental systems are most effective for studying IL-36 alpha expression in vitro?

The murine RAW264.7 macrophage cell line and primary bone marrow-derived macrophages (BMDMs) are well-established systems for studying IL-36 alpha expression. These cells effectively respond to lipopolysaccharide (LPS) stimulation by inducing IL-36 alpha expression . For quantification, absolute mRNA copy numbers can be determined using quantitative RT-PCR, while protein levels can be assessed through Western blot analysis. When designing such experiments, researchers should consider inclusion of appropriate time points (typically examining expression at 8 hours post-stimulation) and controls to account for cell type-specific variations in baseline expression levels .

How can researchers detect and quantify IL-36 alpha protein in biological samples?

Detecting and quantifying IL-36 alpha in biological samples requires combining multiple techniques for comprehensive analysis:

• Western blotting: Effective for detecting pro-IL-36α in cell lysates from stimulated cells (such as RAW264.7 or BMDMs after LPS treatment)

• ELISA: Enables quantitative measurement of IL-36 alpha in serum samples or cell culture supernatants

• Mass spectrometry: Particularly useful for confirming protein identity and modifications

• Immunohistochemistry: For tissue localization studies

For accurate quantification, researchers should establish standard curves using purified recombinant IL-36 alpha protein with known concentrations and validate their detection methods with appropriate positive and negative controls.

What are the methodological considerations for IL-36 alpha promoter analysis studies?

When analyzing the IL-36 alpha promoter, researchers should consider:

• Methylation analysis: The half-CRE- C/EBP element in the IL-36 alpha promoter can be differentially methylated across cell types, although this methylation does not significantly affect transcription factor binding or gene expression

• Chromatin immunoprecipitation (ChIP): For studying C/EBPβ binding to the IL-36 alpha promoter following LPS stimulation

• Luciferase reporter assays: To evaluate promoter activity under different stimulation conditions and with various promoter mutations

• Electrophoretic mobility shift assays (EMSA): To assess protein-DNA interactions between transcription factors and the IL-36 alpha promoter elements

These approaches should be complemented with expression analysis to correlate promoter activity with actual IL-36 alpha production .

What are the primary signaling pathways activated by IL-36 alpha?

IL-36 alpha binds to and signals through the IL1RL2/IL-36R receptor, which leads to the activation of several downstream signaling pathways:

• NF-kappa-B signaling pathway: Critical for inflammatory gene expression

• MAPK signaling pathways: Including p38, ERK, and JNK pathways

These pathways collectively drive pro-inflammatory responses in target cells. IL-36 alpha signaling requires the co-receptor IL1RAP, which it shares with the IL-1 signaling system. This signaling cascade ultimately leads to the expression of various pro-inflammatory mediators, making IL-36 alpha an important orchestrator of inflammatory responses, particularly in epithelial barriers .

How does IL-36 alpha affect dendritic cell maturation and function?

IL-36 alpha plays a significant role in dendritic cell maturation and function through multiple mechanisms:

• Surface marker expression: IL-36 alpha stimulates the increased surface expression of CD80, CD86, and MHC class II molecules on dendritic cells, which are essential for antigen presentation and T cell activation

• Cytokine production: Induces production of pro-inflammatory cytokines, including IL-12, IL-1 beta, IL-6, TNF-alpha, and IL-23 in bone marrow-derived dendritic cells (BMDCs)

• T-cell regulation: Indirectly affects T-cell responses by prompting dendritic cells to create an environment conducive to T-cell proliferation and differentiation

• CD40 expression: May be involved in T-cell maturation by stimulating CD40 expression in splenic CD11c+ cells

These effects collectively position IL-36 alpha as an important regulator of the interface between innate and adaptive immunity through its influence on dendritic cell function .

What is the relationship between IL-36 alpha and metabolic regulation?

IL-36 alpha demonstrates significant involvement in metabolic regulation, particularly in the context of obesity and metabolic disorders:

• Serum levels in obesity: Elevated IL-36 cytokine expression is found in the serum of obese patients and, importantly, shows a negative correlation with blood glucose levels among individuals with type 2 diabetes

• Protection against metabolic disease: Studies using mice lacking IL-36Ra (the IL-36 receptor antagonist) found they develop less diet-induced weight gain, hyperglycemia, and insulin resistance, suggesting IL-36 signaling may have protective effects

• Microbiome modulation: IL-36 cytokines promote the growth of metabolically protective bacteria, particularly Akkermansia muciniphila, in the intestinal microbiome

• Mucus secretion: IL-36 cytokines increase colonic mucus secretion, which may contribute to maintaining gut barrier integrity

These findings indicate IL-36 alpha may have therapeutic potential in metabolic disorders through its effects on gut microbiota and metabolic function .

What are the optimal conditions for using recombinant IL-36 alpha in cell culture experiments?

For optimal results in cell culture experiments with recombinant IL-36 alpha:

• Concentration range: Use concentrations between 1-20 ng/mL, with typical effective doses (ED50) for inducing IL-6 secretion in responsive cell lines being 3-18 ng/mL

• Exposure time: For most cellular responses, 8-24 hours of exposure is sufficient, though this should be optimized for specific endpoints

• Cell types: Most responsive cell types include keratinocytes, dendritic cells, T cells, fibroblasts like NIH-3T3, and macrophages

• Medium supplements: Standard cell culture medium (DMEM or RPMI) supplemented with 10% FBS is typically sufficient, though serum should be reduced for specific signaling studies

• Controls: Include both negative controls (untreated cells) and positive controls (cells treated with known inducers of your endpoint of interest)

How can researchers effectively measure IL-36 alpha-induced cytokine production?

To accurately measure IL-36 alpha-induced cytokine production, researchers should implement a multi-faceted approach:

• ELISA: For quantitative measurement of secreted cytokines in cell culture supernatants

  • Recommended for IL-12, IL-1 beta, IL-6, TNF-alpha, and IL-23 quantification

• qRT-PCR: For measuring cytokine mRNA expression

  • Include time-course experiments to capture both early and late expression patterns

  • Use appropriate housekeeping genes for normalization

• Flow cytometry: For intracellular cytokine staining in specific cell populations

  • Particularly useful for heterogeneous cell populations

• Multiplex assays: For simultaneous detection of multiple cytokines

  • Provides comprehensive cytokine profile with minimal sample volume

• Western blotting: For detecting intracellular pro-forms of cytokines

When designing these experiments, include appropriate positive controls (like LPS stimulation) and titrate IL-36 alpha concentrations to establish dose-response relationships for each cytokine of interest .

What are the methodological considerations for studying IL-36 alpha effects on specific cell populations?

When investigating IL-36 alpha effects on specific cell populations, researchers should consider:

• Cell isolation techniques:

  • For BMDMs: Proper bone marrow isolation and differentiation protocols (typically 7 days with M-CSF)

  • For dendritic cells: CD11c+ selection from spleen or bone marrow culture with GM-CSF

• Purity assessment:

  • Flow cytometry verification of population purity before experimentation

  • Consideration of contaminating cell types that may influence results

• Receptor expression verification:

  • Confirm IL-36R expression on target cells before interpretation of results

  • Consider receptor expression changes during cell activation/differentiation

• Functional readouts:

  • For dendritic cells: Surface marker expression (CD80, CD86, MHC-II, CD40) by flow cytometry

  • For T cells: Proliferation assays and cytokine production (IFN-gamma, IL-4, IL-17)

  • For macrophages: Phagocytosis assays and polarization markers

• Co-culture systems:

  • For studying interactions between IL-36 alpha-treated cells and other immune cells

  • Careful consideration of cell ratios and contact-dependent vs. soluble factor effects

How is IL-36 alpha gene expression regulated at the transcriptional level?

IL-36 alpha gene (Il36a) expression is regulated through several transcriptional mechanisms:

• Transcription factor binding: C/EBPβ is a critical transcription factor that binds specifically to an essential half-CRE- C/EBP motif in the IL-36 alpha promoter to induce expression upon LPS stimulation

• CpG methylation: The half-CRE- C/EBP element in the IL-36 alpha promoter can be differentially methylated across cell types. Interestingly, unlike many genes, IL-36 alpha expression is not significantly affected by the methylation status of this site. C/EBPβ binding and subsequent gene activation occurs independently of the CpG methylation status of the half-CRE- C/EBP motif

• Promoter structure: The IL-36 alpha promoter contains multiple CpGs proximal to the transcriptional start site, which may contribute to its regulation in different contexts

• Cell type-specific regulation: IL-36 alpha is expressed at different levels across various cell types and tissues, suggesting complex regulatory mechanisms beyond the core promoter elements

Understanding these regulatory mechanisms provides insights into potential therapeutic targets for modulating IL-36 alpha expression in various disease contexts .

What post-translational modifications affect IL-36 alpha activity?

IL-36 alpha activity is significantly influenced by post-translational modifications:

• Proteolytic processing: Similar to other IL-1 family members, IL-36 alpha is produced as a precursor (pro-IL-36α) that requires N-terminal truncation to reach full biological activity. Different truncated forms may exhibit varying levels of biological activity

• Glycosylation: Unlike many cytokines, recombinant IL-36 alpha produced in E. coli lacks glycosylation, which should be considered when comparing activity to naturally produced cytokine

• Oxidation/reduction: The protein sequence contains cysteine residues that may form disulfide bonds or be subject to oxidation, potentially affecting protein structure and activity

• Protein-protein interactions: Interaction with binding partners or chaperones may affect IL-36 alpha stability and activity in different cellular contexts

Researchers investigating IL-36 alpha should consider these modifications when interpreting experimental results, especially when comparing recombinant proteins from different sources or naturally produced cytokine .

What environmental factors influence IL-36 alpha production and signaling?

Various environmental factors influence IL-36 alpha production and signaling:

• Pathogen-associated molecular patterns (PAMPs):

  • LPS is a potent inducer of IL-36 alpha expression in macrophages and dendritic cells through TLR4 signaling

  • Other TLR ligands may also induce IL-36 alpha in a cell type-specific manner

• Inflammatory cytokines:

  • TNF-α and IL-1β may synergize with or prime cells for IL-36 alpha production

  • Type I interferons may modulate IL-36 alpha expression in specific contexts

• Tissue microenvironment:

  • Oxygen tension and metabolic state of the tissue may affect IL-36 alpha expression

  • pH changes during inflammation may modify IL-36 alpha activity or receptor binding

• Gut microbiota:

  • IL-36 signaling promotes the growth of metabolically protective bacteria, particularly Akkermansia muciniphila

  • Conversely, the microbiome composition may influence IL-36 alpha expression in intestinal tissues

These environmental factors should be carefully controlled or accounted for in experimental designs investigating IL-36 alpha biology .

How does IL-36 alpha contribute to metabolic disorders and potential treatments?

IL-36 alpha demonstrates significant involvement in metabolic regulation and potential therapeutic applications:

• Obesity correlation: Elevated IL-36 cytokine expression is found in the serum of obese patients, but importantly, it negatively correlates with blood glucose levels among those with type 2 diabetes

• Protective metabolic effects: Mice lacking IL-36Ra (the IL-36 receptor antagonist) show reduced diet-induced weight gain, hyperglycemia, and insulin resistance, suggesting enhanced IL-36 signaling may have protective effects against metabolic disease

• Microbiome modulation: IL-36 cytokines promote the growth of metabolically protective bacteria, particularly Akkermansia muciniphila, in the intestinal microbiome

• Mucus production: IL-36 increases colonic mucus secretion, potentially improving gut barrier function and reducing metabolic endotoxemia

These findings suggest potential therapeutic strategies focused on enhancing IL-36 signaling or targeting downstream effects like microbiome modulation for treating metabolic disorders. Future research should explore the therapeutic window for IL-36 modulation and potential side effects related to its pro-inflammatory properties in other contexts .

What role does IL-36 alpha play in inflammatory diseases?

IL-36 alpha functions as a key mediator in various inflammatory conditions:

• Skin inflammation: IL-36 alpha acts on keratinocytes, dendritic cells, and indirectly on T-cells to drive tissue infiltration, cell maturation, and proliferation, suggesting involvement in inflammatory skin diseases

• Pulmonary inflammation: IL-36 alpha induces the expression of CXCL1 and CXCL2 in the lung, and stimulates TNF-alpha, IL-36c, IL-1A, IL-1B, CXCL1, and CXCL2 expression in alveolar macrophages, indicating potential roles in respiratory inflammatory conditions

• Inflammatory cascade regulation: IL-36 alpha induces production of pro-inflammatory cytokines, including IL-12, IL-1 beta, IL-6, TNF-alpha, and IL-23 in dendritic cells, potentially amplifying inflammatory responses

• NF-kappa B activation: IL-36 alpha induces NF-kappa B activation in macrophages, a central pathway in many inflammatory diseases

These mechanisms position IL-36 alpha as both a potential biomarker for inflammatory conditions and a therapeutic target, particularly in diseases affecting epithelial barriers where IL-36 signaling is prominent .

How can researchers design experimental models to evaluate IL-36 alpha as a therapeutic target?

Designing robust experimental models to evaluate IL-36 alpha as a therapeutic target requires multifaceted approaches:

• Genetic models:

  • IL-36R knockout mice to study complete pathway ablation

  • IL-36Ra knockout mice to study enhanced signaling

  • Conditional/tissue-specific knockouts to examine context-dependent effects

  • Humanized mouse models expressing human IL-36 pathway components

• Pharmacological interventions:

  • Recombinant IL-36 alpha administration at physiologically relevant doses

  • IL-36R antagonizing antibodies or small molecules

  • Targeted delivery systems for tissue-specific modulation

• Disease-specific models:

  • Diet-induced obesity models for metabolic effects

  • DSS-induced colitis for intestinal inflammation

  • IMQ-induced psoriasis for skin inflammation

  • LPS challenge for acute inflammatory responses

• Readouts and endpoints:

  • Molecular: Signaling pathway activation, transcriptional changes

  • Cellular: Immune cell infiltration and activation

  • Physiological: Weight, glucose tolerance, insulin sensitivity

  • Microbiome: 16S rRNA sequencing to assess microbiota changes

  • Tissue analysis: Histopathology, mucus production, barrier integrity

• Translational considerations:

  • Correlation studies between animal models and human patient samples

  • Ex vivo studies using human tissues

  • In vitro studies with human cells

These approaches should be integrated to develop a comprehensive understanding of IL-36 alpha's therapeutic potential across different disease contexts .