IL36A Human

What is IL36A and what is its role in human biology?

IL36A (Interleukin-36 alpha, also known as IL-1F6) is a pro-inflammatory cytokine belonging to the IL-1 superfamily. Human IL36A protein consists of 158 amino acids, with the active form comprising amino acids Lys6-Phe158 after N-terminal processing . IL36A is primarily expressed in epithelial barrier tissues such as skin, bronchial, intestinal, and renal epithelium, where it plays a crucial role in bridging innate and adaptive immune responses . It functions as a pro-inflammatory mediator that induces the production of inflammatory cytokines and chemokines. Research demonstrates that IL36A can induce IL-8 secretion in human epithelial carcinoma cells with an ED50 of 4-24 ng/mL, confirming its pro-inflammatory activity .

How does IL36A signaling operate at the molecular level?

IL36A signaling follows a multistep pathway:

• Activation: IL36A is secreted in an inactive form requiring N-terminal cleavage by proteases (cathepsin G and elastase) for activation, increasing its activity approximately 500-fold .

• Receptor binding: Active IL36A binds to IL-1R6 (IL-36 receptor).

• Complex formation: Upon binding, IL-1R6 recruits IL-1 receptor accessory protein (IL-1RAcP) to form a functional heterodimeric receptor complex .

• Signal transduction: This receptor complex initiates intracellular signaling cascades leading to pro-inflammatory gene expression.

• Regulation: IL36A signaling is naturally inhibited by IL-36Ra (receptor antagonist) and IL-38, which bind to IL-1R6 but do not recruit IL-1RAcP, thus preventing signaling .

Where is IL36A primarily expressed in the human body?

IL36A expression is strategically localized to epithelial barrier tissues that serve as first-line defenses against pathogens. Primary expression sites include:

• Skin (keratinocytes)

• Bronchial epithelium (respiratory tract)

• Intestinal epithelium (particularly colonic mucosa)

• Renal epithelium (kidney)

Studies have specifically identified elevated expression of IL36A in the colonic mucosa of ulcerative colitis patients . Activated keratinocytes are also a significant source of IL-36 cytokines in inflamed skin tissues, indicating their importance in cutaneous inflammatory conditions .

How is IL36A transcription regulated?

IL36A transcription is regulated by specific transcription factors and signaling pathways. Research indicates that C/EBPβ (CCAAT/enhancer-binding protein beta) plays a crucial role in IL36A transcriptional activation via binding to a half-CRE (cAMP response element) site . In murine macrophages, LPS-induced IL36A mRNA expression is essentially mediated through this mechanism . Experimental approaches to study this regulation include:

• Transient transfections with IL36A promoter constructs

• Luciferase reporter assays to measure promoter activity

• qRT-PCR to quantify IL36A mRNA levels

The standard protocol for measuring IL36A transcriptional activation involves transfecting cells (e.g., RAW264.7) with an IL36A promoter-luciferase construct, stimulating with appropriate agonists (e.g., LPS), and measuring luciferase activity to assess promoter activation .

How is inactive IL36A processed to its biologically active form?

IL36A undergoes essential post-translational processing for full biological activity:

• N-terminal cleavage: Specific proteases remove a portion of the N-terminus to generate the active form.

• Proteases involved: The neutrophil-derived proteases cathepsin G and elastase have been identified as activators of IL36A .

• Activation mechanisms: These proteases can process IL36A either as free proteases or as neutrophil extracellular trap (NET)-bound proteases .

• Activity enhancement: N-terminal cleavage results in a dramatic (~500-fold) increase in biological activity .

The human recombinant active form of IL36A typically encompasses amino acids Lys6-Phe158 of the full protein . This activation process represents a crucial regulatory checkpoint and potential therapeutic target in IL36A-driven inflammatory conditions.

What role does IL36A play in inflammatory bowel disease pathogenesis?

IL36A contributes significantly to inflammatory bowel disease (IBD) pathogenesis, particularly in ulcerative colitis:

Expression in IBD:

• Increased IL36A expression is detected in colonic mucosa of newly diagnosed pediatric IBD patients

• Expression is specifically elevated in ulcerative colitis patients compared to controls

• This elevated expression pattern is mirrored in mouse models of colitis

Mechanisms of Pathogenesis:

• Promoting innate immune cell recruitment: IL36A signaling drives inflammatory cell infiltration to the colon lamina propria

• Modulating T helper cell responses: IL36A influences intestinal T cell populations, with IL-36R deficiency leading to enhanced Th17 and reduced Th1 responses

• Compromising barrier function: IL36A may impair intestinal epithelial integrity

Experimental Evidence:
In DSS-induced colitis models, Il36r−/− mice exhibit reduced disease severity with decreased innate inflammatory cell infiltration to the colon lamina propria . During Citrobacter rodentium infection, IL-36R deficiency results in reduced inflammatory cell recruitment but increased bacterial colonization, suggesting a dual role in host defense and inflammatory pathology .

How does IL36A interact with other inflammatory pathways?

IL36A participates in a complex network of inflammatory pathway interactions:

Interactions with Innate Immune Pathways:

• Neutrophil Recruitment and Activation:

  • IL36A promotes neutrophil recruitment to inflammatory sites

  • Neutrophil-derived proteases activate IL36A, creating a positive feedback loop

• Pattern Recognition Receptor Crosstalk:

  • Pathogen-associated molecular patterns induce IL36A expression

  • LPS stimulation increases IL36A transcription via C/EBPβ

Interactions with Adaptive Immune Pathways:

What experimental models are most appropriate for studying IL36A function?

Multiple experimental models have proven valuable for investigating IL36A function:

In Vivo Models:

• IL-36R Knockout Mice (Il36r−/−):

  • Show reduced disease severity in DSS-induced colitis

  • Exhibit decreased innate inflammatory cell infiltration in colonic lamina propria

  • Display altered T helper cell responses in intestinal inflammation models

• Citrobacter rodentium Infection Model:

  • Allows study of IL36A in infectious colitis

  • In Il36r−/− mice, reveals reduced inflammatory cell recruitment and increased bacterial colonization

In Vitro Models:

• Human Epithelial Cell Lines (e.g., A431 cells):

  • Suitable for studying IL36A-induced cytokine production

  • Can measure IL-8 secretion in response to recombinant IL36A

• Primary Human Skin Biopsies:

  • Allow ex vivo testing of IL36A function and inhibitors

• RAW264.7 Macrophage Cell Line:

  • Useful for studying IL36A transcriptional regulation via reporter assays

How can researchers effectively measure IL36A expression and activity?

Several methodologies are available for measuring IL36A expression and activity:

Measuring Expression:

• Quantitative RT-PCR:

  • Can quantify IL36A mRNA levels using specific primers

  • Reference plasmid standards (like pJet-Il36A) enable absolute quantification

  • Protocol example: 95°C for 20 min, 40 cycles of 95°C for 20s, 58°C for 30s, and 72°C for 20s

• ELISA:

  • Commercial sandwich ELISA kits (such as ImmunotagTM Human IL36A ELISA)

  • Typical sensitivity: 0.094 ng/ml

  • Range: 0.156-10 ng/ml

Measuring Activity:

• Bioactivity Assays:

  • Cell-based assays measuring IL-8 secretion from responsive cell lines (e.g., A431 cells)

  • ED50 for this effect is typically 4-24 ng/mL

• Luciferase Reporter Assays:

  • Transient transfection with IL36A-responsive promoter constructs

  • Example protocol: RAW264.7 cells (1.0 × 10^5/well) transfected with 550 ng DNA, followed by stimulation and luciferase activity measurement

What are the current approaches to targeting IL36A therapeutically?

Recent advances have demonstrated progress in developing IL-36 pathway inhibitors:

Recent Developments:

• Macrocyclic peptide (36R-P138) and its optimized analog (36R-P192) have been shown to block IL-36R signaling effectively

• Small molecule 36R-D481 (<1000 Da) effectively binds IL-36R with high affinity, inhibiting IL-36 signaling

• X-ray crystallography reveals these compounds bind to the D1 domain of IL-36R, potentially disrupting cytokine binding

Challenges in IL36A Targeting:

• Pathway Redundancy:

  • Multiple IL-36 family members signal through the same receptor

  • Targeting IL36A alone may not be sufficient to block pathway activity

• Receptor Targeting:

  • Cytokine receptor targeting with small molecules has historically been difficult

  • The IL-36 receptor has a large binding surface area for cytokine interaction

  • Recent advances using encoded library technologies represent a significant breakthrough in this area

• Balancing Inhibition:

  • Avoiding disruption of beneficial immune functions while targeting pathological inflammation

What is the relationship between IL36A and other IL-36 family members?

The IL-36 family consists of four members within the IL-1 superfamily:

All IL-36 family members:

• Signal through the same receptor (IL-1R6)

• Require N-terminal processing for full activity

• Are primarily expressed at epithelial barriers

IL-36Ra acts as a natural inhibitor by binding to IL-1R6 without recruiting IL-1RAcP. Similarly, IL-38 (though not formally an IL-36 family member) has anti-inflammatory effects comparable to IL-36Ra . The balance between pro-inflammatory IL-36 agonists and anti-inflammatory antagonists is critical for tissue homeostasis.

What are emerging research areas in IL36A biology?

Several promising research directions are emerging in IL36A biology:

• Small Molecule Inhibitors:

  • The development of low molecular weight compounds targeting IL-36R represents a significant breakthrough

  • Recent work demonstrating that it's possible to target this cytokine receptor with small molecules (<1000 Da) opens new therapeutic possibilities

• Understanding Tissue-Specific Functions:

  • Differential roles of IL36A in various barrier tissues (skin vs. gut vs. lung)

  • Tissue-specific regulation of IL36A expression and processing

• IL36A in Host-Microbiome Interactions:

  • The role of IL36A in responding to commensal and pathogenic microbes

  • How microbiome composition influences IL36A expression and activation

  • Potential microbial regulation of IL36A processing enzymes

• Biomarker Development:

  • Exploring IL36A as a biomarker for inflammatory diseases

  • Correlating IL36A levels with disease activity and treatment response

• Combination Therapies:

  • Investigating synergistic effects of targeting IL36A alongside other inflammatory pathways

  • Personalized approaches based on individual patient IL36A expression profiles