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

What is IL-36α and how does it relate to other IL-1 family cytokines?

IL-36α, also known as interleukin-1 family member 6 (IL1F6), is a member of the IL-1 cytokine family. Human IL-36α is synthesized as a 158 amino acid protein that contains no signal sequence, no prosegment, and no potential N-linked glycosylation sites. Within the IL-1 family, IL-36α shares 30% amino acid identity with IL-1ra, 27% with IL-1β, 31% with IL-36Ra/IL-1F5, 36% with IL-37/IL-1F7, 46% with IL-36β/IL-1F8, 57% with IL-36γ/IL-1F9, and 28% with IL-1F10 . Like other IL-1 family members, IL-36α displays a 12 β-strand, β-trefoil configuration, suggesting they evolved from a common ancestral gene that underwent multiple duplications .

What is the molecular structure of recombinant human IL-36α?

Recombinant human IL-36α presents as an 18 kDa monomer when found in cell lysate . The protein lacks a traditional signal sequence yet appears to be actively secreted . For research applications, various recombinant forms exist, including truncated versions (aa 6-158) that show improved bioactivity compared to the full-length protein . The recombinant protein can be produced with N-terminal tags (such as His or FLAG tags) that can later be cleaved to obtain the tag-free protein for experimental use .

Where is IL-36α naturally expressed in human tissues?

IL-36α is predominantly expressed in skin and lymphoid tissues, but is also found in fetal brain, trachea, stomach, and intestine . At the cellular level, IL-36α is expressed by monocytes, B cells, and T cells . Notably, IL-36α is the only novel IL-1 family member found to be expressed on T cells .

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

IL-36α expression is regulated through transcriptional activation by CCAAT enhancer binding protein β (C/EBPβ). C/EBPβ binds specifically to an essential half-cAMP response element (half-CRE)- C/EBP motif in the IL36A promoter to induce expression upon lipopolysaccharide (LPS) stimulation . Interestingly, this binding and subsequent promoter activation is insensitive to CpG methylation of the binding site, as demonstrated in studies comparing the methylation states in RAW264.7 macrophage cell lines and primary murine macrophages . This suggests a robust transcriptional regulation mechanism that functions independently of this epigenetic modification.

What factors induce IL-36α expression in experimental settings?

In experimental settings, IL-36α expression can be induced by:

• Lipopolysaccharide (LPS) stimulation in macrophages

• Inflammatory conditions, particularly in epithelial tissues

• Certain pathogenic stimuli, including bacterial and viral components

Quantitative RT-PCR studies have shown significant upregulation of IL-36α mRNA following LPS stimulation in both cell lines (RAW264.7) and primary cells (bone marrow-derived macrophages) .

What is the receptor for IL-36α and how does signaling occur?

The receptor for IL-36α is a heterodimeric complex consisting of:

• IL-36 receptor (IL-36R, also called IL-1Rrp2, IL1RL2, or IL-1R6) - mainly found in epithelia and keratinocytes

• IL-1 receptor accessory protein (IL-1RAP) - widely expressed and shared with IL-1 and IL-33 receptors

Upon binding to this receptor complex, IL-36α activates NF-κB and MAPK signaling pathways in an IL-36R-dependent manner . This activation leads to the induction of various inflammatory mediators and chemokines.

What are the primary pro-inflammatory effects of IL-36α in the lungs?

IL-36α functions as a key upstream amplifier of neutrophilic lung inflammation. Its primary effects include:

• Neutrophil recruitment: Intratracheal instillation of recombinant IL-36α induces significant neutrophil influx in the lungs of mice

• Chemokine production: IL-36α stimulation increases mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in lung tissue

• Pro-inflammatory cytokine induction: IL-36α increases expression of TNFα, IL-1α, and IL-1β in the lungs

• Receptor upregulation: IL-36α enhances the expression of its own receptor (IL-36R), potentially creating a positive feedback loop that amplifies inflammation

• Immune cell activation: IL-36α stimulates CD11c+ cells to produce neutrophil-specific chemokines and increases co-stimulatory molecule expression

These pro-inflammatory effects occur independently of IL-1α and IL-1β, as demonstrated in studies using IL-1αβ-/- mice .

How does IL-36α cooperate with other immune mediators to amplify inflammation?

IL-36α acts as a critical upstream amplifier of inflammation by cooperating with other immune mediators:

• IL-36α works synergistically with GM-CSF and viral mimics like poly(I:C) to promote activation of neutrophils, macrophages, and fibroblasts

• IL-36α induces expression of multiple cytokines (IL-6, IL-12 p40, CXCL1, CCL1, IL-12 p35, IL-1β, IL-19 p19, GM-CSF, CXCL10, TNFα, CCL3) and adhesion molecules (VCAM-1, ICAM-1) in mouse bone marrow-derived dendritic cells and CD4 T cells

• IL-36α activates NF-κB in macrophages, enhancing their inflammatory responses

This cooperative activity positions IL-36α as a key orchestrator of inflammatory responses, particularly in conditions involving neutrophilic inflammation.

What are the recommended methods for producing recombinant IL-36α for research applications?

The production of bioactive recombinant IL-36α typically involves:

• Cloning approach:

  • Engineering a fusion protein with N-terminal purification tags (His, FLAG)

  • Example primer design: Forward 5′-CGGAATTCCgattacaaggatgacgatgacaagAATAAGGAGAAAGAACTAAGAG-3′ and reverse 5′-AAGGAAAAAAGCGGCCGCTTAATGTACCACAATCATCTC-3′

  • Cloning into an appropriate expression vector (e.g., pETDuet-1)

• Expression system:

  • Bacterial expression in E. coli

  • Induction with IPTG (typically 1 mM) for 4 hours during log-phase

• Purification process:

  • Ni²⁺ column chromatography for His-tagged proteins

  • Tag cleavage using enterokinase

  • Removal of enterokinase and cleaved tags

  • Dialysis against PBS

  • LPS removal with polymyxin-B agarose beads

Quality control should include verification of bioactivity by testing the protein's ability to induce pro-inflammatory responses in appropriate cell types.

What cell types and assays are most suitable for studying IL-36α signaling in vitro?

Recommended experimental systems for studying IL-36α signaling include:

• Cell types:

  • Dendritic cells and macrophages (CD11c+ cells)

  • RAW264.7 macrophage cell line

  • Bone marrow-derived macrophages (BMDMs)

  • Primary CD4+ T cells

  • Epithelial cells and keratinocytes (express IL-36R)

  • Fibroblasts

• Signaling assays:

  • NF-κB activation assays (reporter systems, phosphorylation studies)

  • MAPK pathway analysis

  • Luciferase reporter assays for promoter studies

• Functional readouts:

  • Cytokine/chemokine production (ELISA, qPCR for CXCL1, CXCL2, TNFα, etc.)

  • Flow cytometry for co-stimulatory molecule expression (e.g., CD40)

  • T cell proliferation assays

  • Neutrophil migration assays

Each experimental system provides different insights into IL-36α biology, from receptor signaling to downstream functional effects.

What are effective in vivo models for studying IL-36α function?

Effective in vivo models for studying IL-36α function include:

• Intratracheal administration model:

  • Direct instillation of recombinant IL-36α into mouse lungs

  • Assessment of neutrophil influx by bronchoalveolar lavage (BAL)

  • Analysis of chemokine and cytokine expression in lung tissue

• Genetic models:

  • IL-36R knockout mice to assess receptor-dependent effects

  • IL-1αβ-/- mice to study IL-36α functions independent of classical IL-1 cytokines

• Disease models:

  • Cigarette smoke exposure models (with or without H1N1 influenza virus)

  • Bacterial and viral pneumonia models

  • Potential models for chronic kidney disease and rheumatoid arthritis

These models allow for studying the role of IL-36α in different physiological and pathological contexts.

What is the role of IL-36α in inflammatory lung diseases?

IL-36α plays a significant role in inflammatory lung diseases as:

• An upstream amplifier of neutrophilic inflammation, independent of other IL-1 family members

• A promoter of neutrophil recruitment through induction of CXCL1 and CXCL2

• A mediator of lung inflammation in response to cigarette smoke exposure and viral infection

• A potential therapeutic target for neutrophilic lung diseases, based on attenuated lung inflammation observed in IL-36R-deficient mice exposed to cigarette smoke or cigarette smoke plus H1N1 influenza virus

The central role of IL-36α in orchestrating neutrophilic inflammation suggests that targeting this pathway could benefit patients with conditions such as COPD, severe asthma, and viral pneumonia.

How is IL-36α involved in other inflammatory conditions beyond the lung?

IL-36α has been implicated in several inflammatory conditions:

• Rheumatoid arthritis: Elevated expression in synovium

• Chronic kidney disease: Increased expression

• Skin inflammation: Critical role in neutrophilic skin diseases like psoriasis

• Hepatocellular carcinoma: Decreased expression correlates with poor prognosis, suggesting a potential protective role

• Adipose tissue: Reduces adipocyte differentiation and induces inflammatory gene expression in mature adipocytes

These diverse disease associations highlight the pleiotropic effects of IL-36α across multiple tissue types and suggest potential therapeutic applications beyond lung diseases.

What insights do IL-36α studies provide for developing targeted therapies?

Research on IL-36α offers several insights for therapeutic development:

• Pathway specificity: IL-36α functions independently of IL-1α/β, suggesting that IL-36 pathway blockade might provide therapeutic benefits in cases where IL-1 blockade is ineffective

• Amplification mechanism: As an upstream amplifier of inflammation, targeting IL-36α could potentially interrupt inflammatory cascades at an early stage

• Functional redundancy: Understanding the functional overlap between IL-36α and other IL-1 family cytokines helps identify where selective targeting might be beneficial versus where broader inhibition is needed

• Cell-type specific effects: IL-36α's actions on specific cell populations (neutrophils, macrophages, fibroblasts) could allow for more targeted therapeutic approaches

These insights provide a foundation for developing novel therapeutics targeting the IL-36 pathway for inflammatory diseases, particularly those characterized by neutrophilic inflammation.

What are the critical quality control parameters for recombinant IL-36α?

When working with recombinant IL-36α, researchers should verify:

• Protein purity: >95% purity by SDS-PAGE is standard for research-grade recombinant proteins

• Endotoxin levels: LPS contamination should be minimal (<1 EU/μg protein), as it can confound inflammatory assays

• Biological activity: Confirm the protein induces expected responses in appropriate cell types (e.g., NF-κB activation, chemokine production)

• N-terminal processing: Truncated versions (aa 6-158) typically show higher bioactivity than full-length protein

• Storage stability: Monitor for activity loss during storage and avoid repeated freeze-thaw cycles

How can researchers differentiate IL-36α-specific effects from those of other IL-1 family members?

To distinguish IL-36α-specific effects:

• Use genetic approaches:

  • IL-36R knockout or knockdown models

  • IL-1αβ-/- mice or cells (to eliminate IL-1 effects)

• Employ selective blocking:

  • Anti-IL-36R antibodies

  • IL-36Ra (natural antagonist of IL-36 cytokines)

• Compare response profiles:

  • Parallel experiments with IL-1β, IL-36β, and IL-36γ

  • Analysis of downstream gene expression signatures

• Examine cell type specificity:

  • Focus on cells expressing high levels of IL-36R but low levels of other IL-1 receptors

These approaches help delineate the specific contributions of IL-36α to observed biological effects.

What methodological considerations are important when measuring IL-36α expression and activity?

Important methodological considerations include:

• RNA quantification:

  • For absolute quantification, use standards with known molecule numbers

  • Example protocol: PCR with 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds, 58°C for 30 seconds, and 72°C for 20 seconds, with subsequent melting curve analysis

  • Calculate molecule numbers based on standard curves plotting threshold cycles against the natural log of molecule numbers

• Protein detection:

  • Western blotting typically requires specific antibodies against pro-IL-36α

  • Consider that processing may affect epitope recognition

• Functional assays:

  • Include appropriate positive and negative controls

  • Account for potential LPS contamination effects

  • Consider the influence of cell culture conditions on IL-36R expression

• Transcriptional studies:

  • For promoter analyses, transient transfections should be conducted with appropriate controls (e.g., Renilla luciferase vector)

  • Cell treatments should include medium-only controls alongside stimulations