Recombinant Human Interleukin-36 alpha protein (IL36A) (Active)

What is the structural composition of recombinant human IL-36α protein?

Recombinant human IL-36α protein is typically produced as a truncated form (amino acids 6-158) that exhibits enhanced biological activity compared to the full-length protein. The most common commercially available recombinant forms are produced in E. coli expression systems. The protein shares 57-68% amino acid sequence identity with mouse, rabbit, equine, and bovine IL-36α homologs, and 27-57% amino acid sequence identity with other IL-1 family members .

The active form of IL-36α is not the full-length protein but rather a processed form. IL-36 cytokines are expressed as inactive precursors that require proteolytic processing to become fully active . This processing is crucial for the protein's ability to bind effectively to its receptor and initiate downstream signaling cascades.

What are the primary expression patterns and cellular sources of IL-36α?

IL-36α demonstrates tissue-specific expression patterns primarily in:

• Epithelial barriers (skin, bronchial epithelium, intestine)

• Lymphoid tissues

• Fetal brain

• Trachea

• Stomach

At the cellular level, IL-36α is expressed by:

• Monocytes

• B cells

• T cells

• Keratinocytes (particularly when activated)

Importantly, IL-36α expression can be induced by various stimuli. For instance, IL-17 and TNF can induce IL-36α expression in keratinocytes, with IL-22 synergizing this induction. Epidermal growth factor (EGF) also regulates IL-36α expression in the skin . In immune cells, Toll-like receptor (TLR) activation through various ligands can trigger IL-36α expression in a cell type-specific manner .

What is the receptor-mediated signaling pathway for IL-36α?

IL-36α signals through a receptor complex composed of:

• IL-36 receptor (IL-36R, also known as IL-1Rrp2 or IL-1RL2) - primarily expressed in epithelial cells and keratinocytes

• IL-1 receptor accessory protein (IL-1RAcP) - widely expressed co-receptor

Upon binding to this receptor complex, IL-36α activates:

• Nuclear factor kappa B (NF-κB) signaling

• Mitogen-activated protein kinase (MAPK) pathways

These signaling cascades ultimately lead to the induction of pro-inflammatory genes and cytokine production. The effective concentration (EC50) for IL-36α-induced responses varies by cell type and readout, typically ranging from 0.4-24 ng/mL .

How should researchers determine optimal concentrations for in vitro experiments with recombinant IL-36α?

When designing in vitro experiments with recombinant IL-36α, researchers should consider:

• Cell type-specific sensitivity: Different cell types respond to IL-36α at varying concentrations. For example:

  • Human epidermoid carcinoma cells (A-431) respond to IL-36α with IL-8 production at an ED50 of 0.4-2.4 ng/mL

  • Other cell systems may require concentrations of 4-24 ng/mL for observable effects

• Experimental readout: The concentration required may vary based on the specific endpoint being measured:

  • For NF-κB activation assays: Start with concentrations of 1-10 ng/mL

  • For cytokine induction studies: 5-50 ng/mL is typically effective

  • For cellular phenotypic changes: Higher concentrations (10-100 ng/mL) may be necessary

• Methodology for determining optimal concentration:

  • Perform dose-response curves using 2-5 fold serial dilutions

  • Include positive controls (such as IL-1β or TNFα) for comparison

  • Assess multiple time points (4, 8, 24 hours) as response kinetics may vary

  • Measure multiple outputs when possible (e.g., both mRNA using qRT-PCR and protein using ELISA)

What cellular and molecular assays are most appropriate for evaluating IL-36α activity?

Several complementary assays can effectively measure IL-36α activity:

Gene Expression Assays:

• Quantitative RT-PCR targeting downstream genes including IL-8, IL-6, CXCL1, CXCL2, CXCL10, S100A8/A9, and β-defensins

• RNA-seq for genome-wide assessment of transcriptional responses

Protein Production Assays:

• ELISA for quantification of induced cytokines/chemokines

• Multiplexed cytokine arrays for broader profiling

• Western blotting for signaling protein phosphorylation (p-p38, p-ERK, p-JNK, IκB degradation)

Functional Cellular Assays:

• Cell migration assays to assess chemotactic responses

• Reporter cell lines with NF-κB or AP-1 responsive elements

• Flow cytometry to assess surface marker changes

Ex Vivo Tissue Models:

• Human skin biopsies or reconstructed 3D skin models can be used to evaluate IL-36α effects in a complex tissue environment

• Gene expression analysis using skin biopsies can effectively demonstrate IL-36 pathway activation or inhibition

How can researchers effectively validate IL-36α specificity in their experiments?

To ensure experimental results are specifically attributable to IL-36α activity:

• Include appropriate controls:

  • Use heat-inactivated IL-36α protein

  • Include the IL-36 receptor antagonist (IL-36Ra) to block specific signaling

  • Use isotype-matched irrelevant recombinant proteins

• Receptor validation approaches:

  • Employ IL-36R knockdown/knockout models (siRNA, CRISPR-Cas9)

  • Use blocking antibodies against IL-36R

  • Test newly developed small molecule IL-36R antagonists like 36R-P192 or 36R-D481

• Genetic validation:

  • Compare responses in wild-type vs. IL-36R-deficient cells

  • Use cells from different sources with confirmed IL-36R expression levels

• Downstream signaling verification:

  • Monitor NF-κB and MAPK pathway activation

  • Compare signaling kinetics with other IL-1 family members

How does IL-36α contribute to inflammatory skin conditions, particularly psoriasis?

IL-36α plays a critical role in inflammatory skin pathologies:

• Expression pattern in psoriasis:

  • IL-36α is significantly upregulated in psoriatic lesions

  • Activated keratinocytes are a primary source of IL-36 cytokines in inflamed skin

  • Creates an amplification loop where IL-36α stimulates further cytokine production

• Mechanistic contributions:

  • Activates keratinocytes to produce additional pro-inflammatory mediators

  • Recruits and activates immune cells including neutrophils and T cells

  • Promotes tissue remodeling through induction of matrix metalloproteinases

  • Synergizes with other cytokines (IL-17, TNF) to enhance inflammatory responses

• Therapeutic implications:

  • Targeting the IL-36 pathway may provide benefit in psoriasis treatment

  • Small molecule IL-36R antagonists like 36R-P192 effectively suppress IL-36-induced gene expression in human skin biopsies

  • Combines with existing psoriasis treatments may offer synergistic benefits

What roles does IL-36α play in lung inflammation and respiratory diseases?

IL-36α is increasingly recognized as an important mediator in pulmonary inflammation:

• Expression in respiratory tissues:

  • Bronchial epithelial cells and pulmonary macrophages produce IL-36α in response to pathogen-associated molecular patterns

  • Toll-like receptor (TLR) stimulation induces IL-36α expression in lung cells

• Functional effects in the lung:

  • Intratracheal administration of IL-36α induces CXCL1 and CXCL2 expression

  • Promotes neutrophil recruitment to the lungs

  • Activates lung fibroblasts

  • May contribute to both protective anti-microbial responses and pathological inflammation

• Research implications:

  • IL-36α levels may serve as biomarkers for specific lung pathologies

  • Targeting IL-36 signaling could provide therapeutic benefit in certain pulmonary inflammatory conditions

  • Understanding the balance between IL-36α-mediated protection versus pathology is crucial for therapeutic development

What is the current understanding of IL-36α's role in kidney inflammation and injury?

IL-36α has emerged as a significant factor in renal pathology:

• Expression and regulation in kidney tissue:

  • IL-36R is expressed in kidney tissue, particularly in proximal tubules

  • IL-36α expression increases significantly following kidney injury

• Experimental evidence from kidney disease models:

  • In renal ischemia-reperfusion models, IL-36R depletion protects against kidney inflammation

  • IL-36α expression increases within 24 hours of unilateral ureter obstruction (UUO)

  • IL-36α levels correlate with kidney dysfunction in folic acid-induced acute kidney injury models

  • In chronic glomerulonephritis models, IL-36α levels correlate with tubular damage severity and renal interstitial fibrosis

• Cellular mechanisms:

  • In renal tubular epithelial cells, IL-36α treatment increases NF-κB activity and ERK phosphorylation

  • Contributes to inflammatory cytokine production in the kidney microenvironment

• Clinical implications:

  • IL-36α is consistently upregulated in various mouse models of kidney disease

  • May serve as a biomarker for earlier detection of kidney injuries

  • Represents a potential therapeutic target for kidney inflammatory conditions

How does the proteolytic processing of IL-36α affect its bioactivity, and what are the key methodological considerations?

The proteolytic processing of IL-36α is critical for its full biological activity:

• Processing requirements:

  • IL-36 cytokines are expressed as inactive precursors requiring proteolytic processing to attain full activity

  • Proper N-terminal processing significantly enhances receptor binding and biological activity

  • Commercial recombinant preparations typically use the truncated form (aa 6-158) to maximize activity

• Methodological implications for researchers:

  • When designing experiments, researchers should:

    • Verify the form of IL-36α being used (full-length vs. truncated)

    • Consider that full-length IL-36α may show substantially less activity

    • Account for endogenous processing that may occur in complex biological systems

    • Consider co-expression or treatment with relevant proteases if working with full-length protein

• Research directions:

  • Identification of specific proteases responsible for IL-36α processing in different tissues

  • Development of processing-resistant forms for mechanistic studies

  • Investigation of how processing is regulated in different disease states

What are the molecular mechanisms underlying the differential effects of IL-36α versus other IL-36 family members?

Despite signaling through the same receptor complex, IL-36α, IL-36β, and IL-36γ exhibit distinct biological effects:

• Sequence and structural differences:

  • IL-36α shares only partial sequence homology with IL-36β and IL-36γ

  • These differences likely affect receptor binding kinetics and downstream signaling intensity

• Expression pattern variations:

  • Cell type-specific expression of different IL-36 family members

  • Differential regulation of IL-36α vs other family members:

    • TLR stimulation induces different IL-36 family members depending on cell type and specific TLR ligand

    • IL-36α and IL-36γ, but not IL-36β, are induced in bronchial epithelial cells by various TLR ligands

• Functional distinctions:

  • While all IL-36 agonists activate NF-κB and MAPK pathways, they may do so with different kinetics or magnitude

  • Tissue-specific responses may vary due to differential receptor expression or co-factors

• Research design considerations:

  • Comparative studies should include multiple IL-36 family members

  • Dose-matching is essential when comparing effects between family members

  • Analysis of signaling kinetics rather than single time points may reveal differences

How can researchers effectively study IL-36α in the context of complex disease models?

Studying IL-36α in complex disease systems requires sophisticated approaches:

• Animal model selection and development:

  • Consider species-specific differences in IL-36 signaling

  • Humanized mouse models may be necessary for certain applications

  • Tissue-specific conditional knockout models provide advantages over global knockouts

• Combination treatments and pathway interactions:

  • IL-36α rarely acts alone in disease states

  • Consider combination treatments with other cytokines (IL-17, TNF, IL-22) that synergize with IL-36α

  • Study pathway crosstalk, particularly between IL-36 and other IL-1 family members

• Translational approaches:

  • Ex vivo culture of patient-derived samples with IL-36α

  • Correlation of IL-36α levels with disease parameters in patient cohorts

  • Evaluation of genetic variations in IL-36 pathway components

• Opposing roles of IL-36 pathway components:

  • The opposing effects of IL-1Ra and IL-36Ra must be considered in experimental design

  • In corneal inflammation models, IL-1Ra improves outcomes while IL-36Ra worsens them

  • Administration of IL-36γ stimulates expression of innate defense molecules but suppresses IL-1β expression

What is the current understanding of the role of IL-36α in cancer biology?

The role of IL-36α in cancer development represents an emerging research area:

• Expression patterns in cancer:

  • Decreased IL-36α expression correlates with poor prognosis in hepatocellular carcinoma

  • Expression patterns vary across cancer types, with some showing upregulation and others downregulation

• Potential mechanisms in tumor biology:

  • May influence tumor microenvironment through recruitment and activation of immune cells

  • Limited studies suggest potential anti-tumor effects

  • IL-36α may affect cancer cell proliferation, invasion, and response to therapy

  • Could have context-dependent pro- or anti-tumor effects depending on cancer type and stage

• Research opportunities:

  • Characterizing IL-36α expression across different cancer types and stages

  • Investigating the impact of IL-36α on tumor-infiltrating immune cells

  • Exploring potential applications in cancer immunotherapy

  • Studying the interaction between IL-36α and established cancer-associated inflammatory pathways

• Methodological approaches:

  • Cancer cell line studies with recombinant IL-36α treatment

  • IL-36α overexpression or knockout in tumor models

  • Analysis of patient tumor samples for IL-36 pathway component expression

  • Correlation of IL-36α levels with response to immunotherapy

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

Understanding the transcriptional regulation of IL-36α is crucial for experimental design and interpretation:

• Transcription factor involvement:

  • C/EBPβ binds specifically to an essential half-CRE- C/EBP motif in the IL-36α promoter

  • This binding is crucial for promoter activation in the cellular context

• Epigenetic regulation:

  • DNA methylation influences IL-36α expression

  • Different cell types show varying methylation levels at the half-CRE- C/EBP site

  • This may explain cell type-specific expression patterns

• Induction mechanisms:

  • Lipopolysaccharide (LPS) stimulation induces IL-36α expression in multiple cell types

  • In RAW264.7 cells and bone marrow-derived macrophages (BMDMs), LPS triggers IL-36α expression despite different methylation levels at regulatory sites

  • Multiple TLR ligands can induce IL-36α expression with cell type-specific patterns

• Research applications:

  • Promoter analysis can help identify key regulatory elements

  • Understanding transcriptional regulation provides insight into targeting IL-36α expression therapeutically

  • Cell-specific expression patterns should inform experimental design

What are the latest therapeutic approaches targeting the IL-36 pathway?

Recent advances in targeting the IL-36 pathway include:

• Small molecule inhibitors:

  • Development of low molecular weight (<1000 Da) IL-36R antagonists

  • Compound 36R-D481, identified through DNA encoded libraries (DEL) screening, effectively inhibits IL-36 signaling

  • X-ray crystallography shows these molecules bind to the IL-36R's D1 domain, potentially disrupting IL-36 cytokine binding

• Macrocyclic peptides:

  • 36R-P138, identified through mRNA-based display technique, blocks IL-36R signaling

  • Optimized analog 36R-P192 effectively suppresses expression of marker genes induced by IL-36 in human skin biopsies

• Receptor antagonist applications:

  • Recombinant IL-36Ra for therapeutic applications

  • Understanding the opposing effects of IL-1Ra and IL-36Ra in different disease models

• Combination approaches:

  • Targeting multiple inflammatory pathways simultaneously

  • Combining IL-36 pathway inhibition with established therapies