Recombinant Mouse Interleukin-36 gamma protein (Il36g) (Active)

What is IL-36 gamma and what are its key characteristics?

IL-36 gamma (also known as IL-1F9) is a 17-18 kDa cytokine belonging to the IL-1 family. The recombinant mouse IL-36 gamma protein typically contains amino acids 13-164 and is expressed in E. coli expression systems. It functions as an agonist of NF-kappa B activation through the orphan IL-1-receptor-related protein 2 (IL-1Rrp2). IL-36 gamma is part of the IL-36 signaling system present in epithelial barriers and participates in local inflammatory responses similar to the IL-1 system, with which it shares the coreceptor IL-1RAP. This protein is primarily involved in skin inflammatory responses, neutrophilic airway inflammation, and innate immune responses to fungal pathogens .

Which cell types primarily express IL-36 gamma?

IL-36 gamma is predominantly expressed by multiple cell types involved in first-line defense against pathogens. The highest levels of IL-36 gamma are produced by Langerhans cells, keratinocytes, and stomach chief cells and parietal cells. These cells contribute significantly to the first-line defense against pathogens in the skin, lungs and digestive tract. Additionally, expression can be induced in monocytes, bronchial epithelia, and other epithelial cells under specific stimulatory conditions . Within research contexts, understanding the cell-specific expression patterns is crucial for designing physiologically relevant experiments.

What induces IL-36 gamma expression in different cell types?

Several stimuli can induce IL-36 gamma expression:

• Lipopolysaccharide (LPS) treatment of monocytes

• IL-alpha/beta treatment of keratinocytes and bronchial epithelia

• IL-17 stimulation of keratinocytes and bronchial epithelia

• TNF-alpha treatment of keratinocytes and bronchial epithelia

• Pro-inflammatory cytokines including IL-1β, IL-18, and IFN-γ in dendritic cells and macrophages

Additionally, IL-36 cytokines can enhance their own expression, creating a positive feedback loop that amplifies inflammatory responses. This auto-induction has been observed in multiple cell types including dendritic cells, keratinocytes, and epithelial cells .

How does N-terminal processing affect IL-36 gamma activity?

N-terminal processing is critical for the full biological activity of IL-36 gamma. Unlike other IL-1 family members that possess caspase cleavage sites, IL-36 cytokines require post-translational processing at the N-terminal region to be fully active. Specifically, processing of IL-36γ proximal to S18 dramatically enhances its biological activity by 1,000-10,000 fold. This explains why early research showed variable results regarding the concentration of IL-36γ required to observe biological effects in vitro (ranging from 50 ng/ml to 500 ng/ml in different studies). Full-length recombinant IL-36 proteins typically appear less active than their endogenous counterparts due to this requirement for N-terminal trimming .

What are the key signaling pathways activated by IL-36 gamma and how can they be monitored experimentally?

IL-36 gamma primarily activates two major signaling pathways:

• NF-κB Signaling Pathway:

  • Activation occurs through binding to IL-1Rrp2 (IL-36R) which recruits IL-1RAcP

  • This forms a functional receptor complex that initiates signal transduction

  • Leads to nuclear translocation of NF-κB and subsequent gene transcription

• MAPK Signaling Pathway:

  • Includes p38, JNK, and ERK1/2 activation

  • Leads to activation of transcription factors like c-Jun

These pathways can be monitored experimentally through:

• Western blotting for phosphorylated forms of IκB, p38, JNK, and ERK1/2

• Nuclear translocation assays for NF-κB p65 subunit

• Reporter gene assays using NF-κB responsive elements

• RT-qPCR for downstream target genes

• Pharmacological inhibitors can be used to confirm pathway involvement (e.g., BAY 11-7082 for NF-κB, SB203580 for p38 MAPK) .

How can researchers distinguish between the biological activities of processed versus unprocessed IL-36 gamma?

Distinguishing between processed and unprocessed IL-36 gamma requires careful experimental design:

• Activity Comparison:

  • Use dose-response curves to compare potency (ED50 values)

  • Processed IL-36 gamma typically shows ED50 values in the low ng/mL range (3-18 ng/mL)

  • Unprocessed forms require 1000-10000 fold higher concentrations

• Biochemical Verification:

  • SDS-PAGE analysis under reducing conditions (processed IL-36 gamma appears at ~17 kDa)

  • N-terminal sequencing to confirm processing site (proximal to S18)

  • Mass spectrometry to determine exact molecular weight

• Functional Assays:

  • NF-κB activation assays in responsive cell lines (e.g., NIH-3T3)

  • IL-6 secretion assays (a common readout for IL-36 activity)

  • Cell-specific responses (e.g., DC maturation markers CD83 and CD86)

For controlled experiments, researchers should consider using commercially available truncated recombinant proteins (aa 13-164) which mimic the processed form, or enzymatically process full-length proteins in vitro prior to use .

What methodological approaches can be used to study IL-36 gamma's role in inflammatory diseases?

Several methodological approaches can be employed:

• In Vitro Models:

  • Primary cell cultures (keratinocytes, bronchial epithelial cells, dendritic cells)

  • Co-culture systems to study cell-cell interactions

  • Organotypic 3D cultures to mimic tissue architecture

  • Stimulation with disease-relevant triggers (e.g., TLR ligands, allergens)

• In Vivo Models:

  • Transgenic mouse models with IL-36 pathway alterations

  • Disease-specific models:

    • Imiquimod-induced psoriasis

    • Oxazolone-induced colitis

    • Asthma models

    • Viral infection models

• Mechanistic Studies:

  • Neutralizing antibodies against IL-36 gamma or its receptor

  • siRNA knockdown in relevant cell types

  • CRISPR/Cas9-mediated gene editing

  • Receptor antagonist (IL-36Ra) studies to block signaling

• Translational Approaches:

  • Analysis of patient samples for IL-36 gamma expression

  • Correlation with disease severity and biomarkers

  • Ex vivo studies using patient-derived cells .

How does IL-36 gamma interact with other cytokines in inflammatory networks?

IL-36 gamma operates within complex inflammatory networks:

• Synergistic Interactions:

  • IL-36 gamma synergizes with IL-1β, TNF-α, and IL-17A to induce robust inflammatory responses

  • Acts with IL-12 to drive Th1 differentiation and IFN-γ production

  • Potentiates responses to pathogen-associated molecular patterns (PAMPs)

• Amplification Loops:

  • IL-36 gamma induces production of IL-1β, TNF-α, and IL-6

  • These cytokines further induce IL-36 gamma expression

  • Creates a positive feedback loop that can amplify inflammation

• Cross-regulation:

  • IL-36Ra antagonizes IL-36 gamma effects by competing for receptor binding

  • IL-10 and TGF-β may suppress IL-36 gamma-induced responses

  • Type I interferons can modulate IL-36 pathway activation

• Cell-specific Effects:

  • In dendritic cells: induces IL-12, IL-1β, IL-6, TNF-α, and IL-23

  • In T cells: promotes Th1 and Th9 responses while inhibiting Treg cells

  • In epithelial cells: induces antimicrobial peptides and chemokines

This complex network can be studied using cytokine blocking antibodies, receptor antagonists, and analysis of downstream signaling pathway interactions .

What are the optimal storage and handling conditions for recombinant IL-36 gamma protein?

For optimal results with recombinant mouse IL-36 gamma:

• Storage Conditions:

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

  • After reconstitution, store at -80°C in single-use aliquots

  • Avoid repeated freeze-thaw cycles (no more than 3)

  • Short-term storage (1-2 weeks) at 4°C may be acceptable for reconstituted protein

• Reconstitution Guidelines:

  • Reconstitute in sterile water or PBS

  • Gentle swirling is recommended rather than vortexing

  • Allow protein to stand for 10-15 minutes at room temperature

  • Filter sterilization (0.22 μm) may be necessary for cell culture applications

• Stability Considerations:

  • Working stock concentrations typically 0.1-1 mg/mL

  • Addition of carrier protein (0.1% BSA) may improve stability

  • Monitor activity periodically if stored for extended periods

  • Document lot number and date of reconstitution .

What assays can be used to measure IL-36 gamma activity in experimental systems?

Several reliable assays can measure IL-36 gamma activity:

• Cell-based Bioassays:

  • NIH-3T3 mouse embryonic fibroblast IL-6 secretion assay (ED50: 3-18 ng/mL)

  • NF-κB reporter cell lines (e.g., HEK293 cells transfected with IL-36R and NF-κB reporter)

  • Dendritic cell maturation assay (measuring CD83, CD86 upregulation)

• Biochemical Assays:

  • SDS-PAGE with silver staining for protein integrity (17 kDa band)

  • ELISA for protein quantification

  • Western blot for detection of IL-36 gamma in complex samples

• Molecular Assays:

  • RT-qPCR for downstream gene expression (IL-6, IL-8, CCL20)

  • Signaling pathway activation (phospho-IκB, phospho-p38)

  • Chromatin immunoprecipitation for NF-κB binding to target promoters

• Functional Readouts:

  • Cytokine production (IL-6, IL-12p40, IL-12p70, CCL11, CCL4, TNF-α, G-CSF)

  • Cell proliferation assays

  • Migration and chemotaxis assays .

How can researchers optimize experimental design when studying IL-36 gamma in different model systems?

Optimization strategies for different model systems include:

• In Vitro Cell Culture Systems:

  • Cell type selection: Choose physiologically relevant cells (keratinocytes, bronchial epithelial cells, dendritic cells)

  • Concentration range: Titrate IL-36 gamma between 0.1-100 ng/mL for processed forms

  • Time course: Monitor responses at multiple time points (4, 8, 24, 48 hours)

  • Synergy testing: Combine with other stimuli (TLR ligands, cytokines) at sub-optimal doses

  • Controls: Include IL-36Ra as negative control and known IL-36 inducers as positive controls

• Ex Vivo Tissue Explants:

  • Tissue preparation: Maintain consistent size and viability

  • Culture conditions: Use specialized media with minimal serum

  • Delivery method: Consider slow-release delivery systems for sustained exposure

  • Analysis: Combine histology, gene expression, and secreted factors

• In Vivo Models:

  • Delivery route: Local vs. systemic administration affects outcomes

  • Dosing schedule: Single bolus vs. repeated administration

  • Genetic background: Consider strain-specific differences in inflammatory responses

  • Age and sex: Control for these variables as they affect inflammatory responses

  • Readouts: Combine systemic (serum cytokines) and local (tissue) measurements

• Translational Research:

  • Patient stratification: Based on IL-36 pathway activity

  • Sample collection: Standardize processing and storage

  • Paired analyses: Compare affected vs. unaffected tissue from same patient

  • Validation: Confirm findings across multiple patient cohorts .

What are the critical quality control parameters for recombinant IL-36 gamma protein in research applications?

Critical quality control parameters include:

• Purity Assessment:

  • SDS-PAGE analysis (>95% purity recommended)

  • Silver staining for visualization of contaminants

  • High-resolution techniques like capillary electrophoresis for detailed analysis

• Biological Activity:

  • Specific activity measurement (units/mg)

  • Dose-response curves in standard bioassays (e.g., NIH-3T3 IL-6 induction)

  • Comparison to reference standards or previous lots

• Endotoxin Testing:

  • LAL (Limulus Amebocyte Lysate) assay

  • Acceptable levels: ≤0.005 EU/μg protein

  • Critical for preventing false inflammatory responses

• Protein Characterization:

  • Mass spectrometry for molecular weight confirmation

  • N-terminal sequencing to verify processing site

  • Circular dichroism for secondary structure analysis

  • Size exclusion chromatography for aggregation assessment

• Stability Testing:

  • Accelerated and real-time stability studies

  • Activity retention over time under recommended storage conditions

  • Freeze-thaw stability assessment

• Lot-to-Lot Consistency:

  • Comparison of activity between different production lots

  • Standardized testing protocols

  • Certificate of Analysis documentation .

How can IL-36 gamma be applied in investigating specific disease mechanisms?

IL-36 gamma offers valuable insights into several disease mechanisms:

• Psoriasis Research:

  • IL-36 gamma is markedly elevated in psoriatic skin

  • Can be used to model keratinocyte activation and proliferation

  • Useful for studying cross-talk between keratinocytes and immune cells

  • Applications in testing anti-inflammatory compounds targeting the IL-36 pathway

• Inflammatory Bowel Disease (IBD):

  • IL-36 gamma promotes CD4+ T cell-dependent colitis

  • Can be used to study epithelial-immune cell interactions in intestinal inflammation

  • Useful for investigating the role of IL-36/IL-36R signaling in producing pro-inflammatory cytokines

  • Applications in developing biomarkers for intestinal inflammation

• Pulmonary Inflammation:

  • IL-36 gamma is elevated in asthma and during viral infections

  • Contributes to neutrophil influx in the lungs

  • Can be used to model inflammatory disorders of the lung

  • Applications in understanding airway epithelial responses to pathogens

• Cardiovascular Research:

  • IL-36 improves age-related coronary microcirculatory dysfunction

  • Can attenuate myocardial ischemia-reperfusion injury in mice

  • Applications in developing therapeutic approaches for cardiac protection

What are the current technical challenges in IL-36 gamma research and how can they be addressed?

Researchers face several technical challenges when working with IL-36 gamma:

• Protein Processing and Activation:

  • Challenge: Full-length recombinant proteins often show reduced activity

  • Solution: Use pre-processed recombinant proteins (aa 13-164) or develop controlled enzymatic processing protocols

  • Alternative: Generate expression constructs lacking N-terminal sequences

• Receptor Complexity:

  • Challenge: IL-36R forms complexes with IL-1RAcP and exhibits cell-type specific expression

  • Solution: Perform receptor expression profiling before experiments

  • Alternative: Use reporter cell lines with controlled receptor expression

• Downstream Signaling Redundancy:

  • Challenge: Overlapping signaling pathways with other IL-1 family members

  • Solution: Use combination approaches with selective inhibitors and genetic knockdowns

  • Alternative: Develop more specific readouts for IL-36-specific signaling

• Translating In Vitro Findings:

  • Challenge: Cell culture conditions don't replicate the complex tissue environment

  • Solution: Adopt 3D culture systems and co-culture approaches

  • Alternative: Validate findings in ex vivo tissue explants before animal studies

• Species Differences:

  • Challenge: Mouse and human IL-36 systems show biological differences

  • Solution: Perform comparative studies with both mouse and human proteins

  • Alternative: Use humanized mouse models for specific applications .

What are the emerging research directions for IL-36 gamma in immunology and inflammation?

Several promising research directions are emerging:

• IL-36 in Trained Immunity:

  • Investigation of IL-36 gamma's role in programming innate immune memory

  • Study of epigenetic changes induced by IL-36 signaling

  • Potential applications in vaccine adjuvant development

• Tissue-Specific Functions:

  • Exploration of IL-36 functions beyond skin and mucosa

  • Investigation of IL-36 in adipose tissue inflammation and metabolism

  • Study of neuron-glia interactions mediated by IL-36

• Precision Medicine Applications:

  • Development of IL-36 pathway biomarkers for patient stratification

  • Identification of genetic variants affecting IL-36 responsiveness

  • Design of targeted therapies for IL-36-driven inflammatory conditions

• Microbiome Interactions:

  • Study of how commensal and pathogenic microbes regulate IL-36 expression

  • Investigation of IL-36's role in maintaining barrier immunity

  • Exploration of microbiome manipulation as a strategy to modulate IL-36 pathways

• Novel Therapeutic Approaches:

  • Development of small molecule inhibitors targeting IL-36 processing

  • Design of bispecific antibodies targeting IL-36/IL-36R interactions

  • Engineering of cell-specific delivery systems for IL-36 modulators .

How does IL-36 gamma interact with different immune cell populations, and what methods best study these interactions?

IL-36 gamma interacts with diverse immune cell populations through direct and indirect mechanisms:

• Dendritic Cells (DCs):

  • Direct effects: Induces maturation (CD83, CD86 upregulation)

  • Cytokine production: IL-12, IL-1β, IL-6, TNF-α, IL-23

  • Methods: Flow cytometry for surface markers, cytokine ELISAs, transcriptional profiling

  • Applications: Studying vaccine adjuvant potential

• T Cells:

  • Direct effects: Enhances IFN-γ, IL-4, IL-17 production in CD4+ T cells

  • Differentiation: Promotes Th1 and Th9 responses, inhibits Treg functions

  • Methods: Intracellular cytokine staining, CFSE proliferation assays, ChIP for epigenetic changes

  • Applications: Understanding T cell polarization in inflammatory conditions

• Macrophages:

  • Direct effects: Induces pro-inflammatory cytokine production

  • Function: Promotes antimicrobial peptide production and inflammasome activation

  • Methods: RNA-seq for transcriptional profiles, phagocytosis assays, metabolic flux analysis

  • Applications: Studying host defense mechanisms

• Neutrophils:

  • Indirect recruitment: Via IL-36-induced chemokines

  • Function: Contributes to neutrophil influx in lungs during inflammation

  • Methods: Chemotaxis assays, intravital microscopy, neutrophil extracellular trap (NET) formation

  • Applications: Understanding neutrophilic inflammation in asthma and other conditions