Recombinant Human Interleukin-36 gamma protein (IL36G) (Active)

What is the structural and functional characterization of recombinant human IL-36γ?

Recombinant human IL-36γ (formerly known as IL-1F9) is a member of the IL-1 family of cytokines. It is synthesized as a 19 kDa, 169 amino acid protein without a signal sequence, prosegment, or N-linked glycosylation sites . The protein shares 30% amino acid identity with IL-1ra, and various degrees of identity with other IL-1 family members, including 23% with IL-1β, 33% with IL-36ra, 57% with IL-36α, 35% with IL-37, 45% with IL-36β, and 32% with IL-1F10 . Functionally, IL-36γ activates NF-κB and MAPK pathways through binding to its receptor complex consisting of IL-1Rrp2 and IL-1RAcP .

Which cell types express IL-36γ and what are its primary biological activities?

IL-36γ is expressed by multiple cell types including Langerhans cells, keratinocytes, monocytes, bronchial epithelium, and gastric cells (Chief cells and Parietal cells) . The protein is secreted via a nonclassical pathway that likely requires extracellular ATP . Primary biological activities include:

• Activation of NF-κB and MAPK signaling pathways

• Downregulation of betacellulin and upregulation of MMP-9 and MMP-10

• Activation of macrophages and fibroblasts

• Induction of multiple chemokines (CXCL1, 2, 3, 8 and CCL2, 3, 20)

• Promotion of type-1 immune responses in bacterial infections

• Enhancement of CD8+ T cell, NK cell, and γδ T cell activation

How should researchers store and reconstitute recombinant IL-36γ protein?

For optimal results when working with recombinant human IL-36γ:

• Store lyophilized protein at -20°C to -70°C for up to 12 months from date of receipt

• Once reconstituted, store at 2-8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution, store at -20°C to -70°C for up to 3 months under sterile conditions

• Avoid repeated freeze-thaw cycles by aliquoting the reconstituted protein

• Reconstitute in sterile PBS or specified buffer according to manufacturer's instructions

• Centrifuge the vial prior to opening to ensure complete recovery of the protein

What is the optimal concentration range for IL-36γ in cell-based assays?

The optimal concentration range of IL-36γ varies depending on the experimental system and readout:

• For induction of IL-8 in human epidermoid carcinoma cell line A431, the EC50 is approximately 1.5-9 ng/mL

• In studies measuring IL-36γ stimulation of CD8+ T cells, concentrations ranging from 10-100 ng/mL have shown dose-dependent effects

• When evaluating inflammatory responses in bronchial epithelial cells, concentrations of 10-100 ng/mL are typically used

• For 3D skin equivalent models, 300 ng/mL of IL-36γ has been effective in inducing inflammatory responses

It is recommended to perform dose-response experiments to determine the optimal concentration for your specific cell system and experimental endpoint.

How can researchers verify the biological activity of recombinant IL-36γ?

Several methods can be used to confirm the biological activity of recombinant IL-36γ:

• IL-8 induction assay: Measure IL-8 secretion from IL-36γ-responsive cells such as HaCaT keratinocytes or A431 cells. The activity can be confirmed by inhibition with IL-36Ra

• NF-κB activation assay: Use reporter cell lines expressing NF-κB response elements to measure activation following IL-36γ treatment

• TR-FRET binding assay: Utilize Time-Resolved Fluorescence Resonance Energy Transfer to verify binding of IL-36γ to the IL-36R/IL-1RAcP heterodimer

• MAPK phosphorylation: Measure phosphorylation of ERK1/2, JNK1/2, p38, and c-Jun following IL-36γ stimulation using Western blotting or phospho-specific flow cytometry

• Multiplex cytokine assays: Measure downstream cytokine and chemokine production (IL-6, IL-8, CXCL1) in responsive cell types

What controls should be included when using recombinant IL-36γ in experiments?

To ensure robust experimental design when working with IL-36γ:

• Negative controls: Untreated cells or cells treated with heat-inactivated IL-36γ

• Positive controls: Cells treated with well-characterized inflammatory cytokines like IL-1β or TNF-α

• Specificity controls: Treatment with IL-36Ra (receptor antagonist) to block IL-36γ activity

• Receptor validation: Knockdown or knockout of IL-36R in target cells to confirm receptor-specific effects

• Dose-response: Include multiple concentrations of IL-36γ to establish dose dependency

• Time course: Multiple time points to determine optimal kinetics of the response

• Carrier controls: If using carrier proteins like BSA, include carrier-only controls

How does processed/truncated IL-36γ differ from full-length protein in biological activity?

Full-length IL-36γ (Met1-Asp169) exhibits significantly lower biological activity compared to N-terminally truncated forms:

• Truncation of IL-36γ through proteolytic processing dramatically enhances its biological activity

• The most active form of human IL-36γ begins at Ser18, resulting in the protein Ser18-Asp169

• Various proteases from neutrophils, antigen-presenting cells, and epithelial cells can activate IL-36γ through processing

• Activity assays show that truncated forms induce significantly higher IL-8 production compared to full-length protein

• In experimental settings, researchers should consider using the truncated, more active form (Ser18-Asp169) for maximum biological effect

For comparative studies, the following table summarizes activity differences:

What experimental models are most suitable for studying IL-36γ functions in disease contexts?

Several experimental models have been validated for studying IL-36γ in different disease contexts:

In vitro models:

• Human keratinocyte cultures (HaCaT cells) for skin inflammation studies

• Bronchial epithelial cell cultures for respiratory research

• 3D skin equivalents combining keratinocytes and fibroblasts for more physiologically relevant skin models

• Primary immune cell cultures (T cells, NK cells, macrophages) for immunomodulatory studies

In vivo models:

• IL-36γ and IL-36R knockout mice for loss-of-function studies

• Bacterial pneumonia models (Streptococcus pneumoniae, Klebsiella pneumoniae) to study protective immune responses

• Cancer models using IL-36γ-overexpressing tumor cells (B16 melanoma, 4T1 breast cancer) to evaluate anti-tumor effects

• Microparticle-delivered IL-36γ for reconstitution studies in knockout mice

When selecting a model, consider the expression pattern of IL-36R in your model organism, as expression levels vary between tissues and species .

How can researchers effectively measure IL-36γ-induced signaling pathways?

To comprehensively evaluate IL-36γ-induced signaling:

• NF-κB pathway activation:

  • TransAM NF-κB p65 assay to measure nuclear translocation

  • IκBα degradation assessment by Western blot

  • NF-κB reporter assays using luciferase-based systems

• MAPK pathway activation:

  • Western blotting for phosphorylated ERK1/2, JNK1/2, p38, and c-Jun

  • Flow cytometry with phospho-specific antibodies for single-cell analysis

  • Kinase activity assays for downstream substrates

• Transcriptional profiling:

  • RNA-seq or microarray analysis to identify IL-36γ-regulated genes

  • Real-time PCR for targeted gene expression analysis (IL-8, CXCL1, IL-6)

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding sites

• Temporal considerations:

  • Early signaling events (5-30 minutes): MAPK phosphorylation

  • Intermediate events (30-120 minutes): NF-κB nuclear translocation

  • Late events (2-24 hours): Gene expression and protein secretion

• Pathway inhibitors for validation:

  • IKK inhibitors (e.g., BMS-345541) for NF-κB pathway

  • MAPK inhibitors (U0126 for ERK, SP600125 for JNK, SB203580 for p38)

  • MyD88 inhibitors to block upstream signaling

What are the common technical challenges when working with recombinant IL-36γ?

Researchers frequently encounter these challenges when working with IL-36γ:

• Low biological activity: Full-length IL-36γ requires processing for full activity. Solution: Use truncated forms (Ser18-Asp169) or pre-activate with specific proteases

• Species-specificity issues: Human IL-36γ shows only 53% amino acid identity with mouse IL-36γ . Solution: Use species-matched recombinant proteins for in vivo or ex vivo studies

• Receptor expression variability: IL-36R expression varies across cell types. Solution: Verify IL-36R expression in your experimental system before studying IL-36γ effects

• Protein aggregation: IL-36γ may form aggregates during storage or thawing. Solution: Filter reconstituted protein through a 0.2μm filter and avoid repeated freeze-thaw cycles

• Carrier protein interference: BSA in some preparations may interfere with certain assays. Solution: Use carrier-free formulations when possible

• Signal detection sensitivity: Low-level responses may be difficult to detect. Solution: Use sensitive detection methods like ELISA or multiplex assays, and include positive controls

How can researchers effectively compare results obtained with different sources or batches of recombinant IL-36γ?

To ensure reproducibility when working with different IL-36γ preparations:

• Standardize activity measurements:

  • Establish an in-house bioassay (e.g., IL-8 induction in HaCaT cells)

  • Calculate specific activity (units/mg) for each batch

  • Normalize dosing based on activity rather than protein concentration

• Check protein specifications:

  • Verify amino acid sequence (full-length vs. truncated forms)

  • Confirm purity (≥95% by SDS-PAGE is standard)

  • Validate by Western blot with specific antibodies

• Internal reference standards:

  • Maintain an internal reference preparation

  • Perform side-by-side comparisons with new batches

  • Generate relative potency calculations

• Documentation practices:

  • Record lot numbers, sources, and formulations

  • Document storage conditions and reconstitution methods

  • Note any carrier proteins or additives

• Cross-validation:

  • Test multiple biological readouts with each batch

  • Perform dose-response curves to identify potential shifts in potency

  • Consider multiple time points to account for kinetic differences

What techniques are recommended for detecting endogenous versus exogenous IL-36γ in experimental systems?

To distinguish between endogenous and exogenously added recombinant IL-36γ:

• Tagged recombinant proteins:

  • Use His-tagged, Flag-tagged, or Fc-fusion IL-36γ

  • Detect with tag-specific antibodies via Western blot or immunofluorescence

  • Separate from endogenous protein by size difference on Western blots

• Species-specific detection:

  • Use human IL-36γ in mouse systems with human-specific antibodies

  • Apply species-specific qPCR primers to differentiate transcript origin

• Truncation-specific antibodies:

  • Generate antibodies that specifically recognize the N-terminus of truncated forms

  • Use these for selective detection of processed IL-36γ

• Mass spectrometry-based approaches:

  • Use isotope-labeled recombinant protein (15N or 13C labeled)

  • Perform mass spectrometry to differentiate labeled vs. unlabeled peptides

  • Quantify relative abundance of endogenous vs. exogenous forms

• Timing considerations:

  • Measure baseline levels before adding recombinant protein

  • Track kinetics of IL-36γ degradation in your experimental system

  • Consider that exogenous IL-36γ may induce endogenous IL-36γ expression

How does IL-36γ interact with other inflammatory pathways in complex disease models?

Recent research reveals complex interactions between IL-36γ and other inflammatory mediators:

• IL-36γ and IL-1 family cross-regulation:

  • IL-36γ and IL-1β show reciprocal regulation in various tissues

  • IL-36Ra and IL-1Ra have opposing effects on infectious keratitis outcomes

  • IL-36γ can induce IL-1β expression in some contexts but suppress it in others

• Interface with type-1 immunity:

  • IL-36γ synergizes with IL-12 to enhance CD8+ T cell and NK cell activity

  • It promotes IFN-γ production from multiple lymphocyte populations

  • IL-36γ acts as a crucial proximal component in protective type-1-mediated lung immunity

• Role in tissue remodeling and fibrosis:

  • IL-36 signaling promotes secretion of profibrotic mediators

  • IL-36γ may serve as a bridge between inflammation and fibrosis in multiple organs

  • This pathway influences tissue repair mechanisms in inflammatory conditions

• Wnt signaling pathway interactions:

  • IL-36γ stimulates autophagy and induces WNT5A expression

  • It activates the COX-2/AKT/mTOR pathway via noncanonical Wnt signaling

  • Wnt signaling inhibitors can blunt IL-36γ-driven inflammatory responses

These complex interactions suggest that targeting IL-36γ may have context-dependent effects depending on the disease state and inflammatory milieu.

What are the therapeutic implications of using recombinant IL-36γ or IL-36γ antagonists?

The development of IL-36γ-targeting therapeutics presents various opportunities and considerations:

What advanced analytical methods are being developed to study IL-36γ binding and activation mechanisms?

Cutting-edge approaches for investigating IL-36γ molecular mechanisms include:

• TR-FRET displacement assays:

  • Novel Time-Resolved Fluorescence Resonance Energy Transfer methods measure IL-36γ binding to IL-36R/IL-1RAcP heterodimer

  • Allow high-throughput screening of potential antagonists

  • Enable quantitative measurement of binding kinetics and affinities

• 3D skin equivalent models:

  • Complex in vitro systems combining keratinocytes and fibroblasts

  • Enable assessment of IL-36γ effects in physiologically relevant contexts

  • Allow evaluation of both cytokine release and tissue morphology changes

• Protease activity profiling:

  • Identification of specific proteases that cleave and activate IL-36γ

  • Determination of precise cleavage sites and resulting activity modulation

  • Understanding the role of neutrophil-, APC-, and epithelial cell-derived enzymes in IL-36γ processing

• Structural biology approaches:

  • Crystallography studies of IL-36γ in complex with IL-36R

  • Molecular dynamics simulations to understand conformational changes

  • Structure-based drug design for developing targeted IL-36γ modulators

• Single-cell analysis:

  • Single-cell RNA sequencing to identify IL-36γ-responsive cell populations

  • CyTOF (mass cytometry) for high-dimensional analysis of signaling responses

  • Spatial transcriptomics to map IL-36γ expression and activity in tissue contexts

These advanced methods are expanding our understanding of IL-36γ biology and facilitating development of targeted therapeutics.