IL36A 158 a.a. Human

What is the molecular structure of human IL36A and how does it compare to other IL-1 family members?

Human IL36A (IL-36 alpha) is a pro-inflammatory cytokine comprising 158 amino acid residues that forms a single nonglycosylated polypeptide with a molecular weight of approximately 17.7 kDa . It belongs to the IL-1 superfamily and shares homology with several family members: 30% with IL1ra, 27% with IL1G, 31% with IL36Ra/IL1F5, 36% with IL37/IL1F7, 46% with IL36G/IL1F8, 57% with IL36H/IL1F9, and 28% with IL1F10 .

The protein spans from Met1 to Phe158 (accession number Q9UHA7) . Unlike many other cytokines, IL36A lacks glycosylation, which has implications for its stability and binding kinetics in experimental systems. When designing experiments, researchers should consider this property, particularly when comparing IL36A with other cytokines that may exhibit post-translational modifications.

What is the expression profile of IL36A in human tissues and how should this inform experimental design?

IL36A exhibits a tissue-specific expression pattern that researchers should consider when designing physiologically relevant experiments. It is predominantly expressed in skin and lymphoid tissues, with notable expression also detected in fetal brain, trachea, stomach, and intestine . Within the immune system, IL36A is uniquely expressed on T cells, distinguishing it from other novel IL-1 family members .

The epithelial barrier tissues (skin, lung, kidney, and colon) are major sites of IL36A action, where IL-36R is predominantly expressed . When designing experiments, researchers should:

• Select appropriate cell types that naturally express IL36A or its receptor

• Consider tissue-specific co-factors that may modulate IL36A activity

• Account for potential differences between normal and inflamed tissue expression levels

• Utilize controls that reflect the appropriate tissue context

For immunohistochemistry studies, researchers should carefully validate antibodies against human samples from tissues with known expression profiles to establish specificity.

How does the IL36A signaling cascade function, and what methodologies are most appropriate for studying pathway activation?

IL36A signals through binding to the IL-36 receptor (IL-36R/IL1RL2), which then recruits the IL-1 receptor accessory protein (IL-1RAcP) to form a functional signaling complex . This interaction activates downstream signaling pathways mediated by nuclear transcription factor kappa B and mitogen-activated protein kinase signaling cascades .

For studying IL36A signaling, researchers should consider these methodological approaches:

• Receptor-ligand binding assays: Surface plasmon resonance or ELISA-based techniques to quantify binding kinetics between IL36A and IL-36R.

• Signaling pathway activation: Western blotting to detect phosphorylation of critical intermediates (IκB, p38, JNK, ERK) following IL36A stimulation. Timing is crucial; researchers should perform time-course experiments (5-120 minutes) to capture both early and sustained signaling events.

• Transcriptional readouts: qRT-PCR analysis of known IL-36A-responsive genes or RNA-seq to capture the broader transcriptional landscape. Researchers should include IL-1β stimulation controls to distinguish IL-36A-specific effects from general IL-1 family responses.

• Reporter assays: NF-κB or AP-1 luciferase reporter constructs in relevant cell types can provide quantifiable readouts of pathway activation.

When interpreting results, researchers should be mindful that IL-36A requires proteolytic processing for full biological activity, which may affect experimental outcomes depending on the protein preparation used.

What are the molecular determinants of IL36A specificity compared to other IL-36 family members, and how can researchers experimentally differentiate their functions?

The IL-36 family consists of four members: three agonists (IL-36α, IL-36β, IL-36γ) and one antagonist (IL-36Ra), all binding to the same IL-36R . Despite sharing the same receptor, these cytokines exhibit differential activities and expression patterns.

To experimentally differentiate IL36A functions from other family members:

• Comparative binding studies: Using recombinant proteins and binding competition assays to determine relative affinities for IL-36R.

• Isoform-specific knockdown: siRNA or CRISPR-Cas9 approaches targeting each isoform individually in relevant cell types, followed by functional assays.

• Isoform-specific antibody neutralization: Using validated antibodies that specifically block individual IL-36 family members.

• Expression analysis: Precise quantification of isoform expression in different tissues or disease states using isoform-specific primers for qRT-PCR.

• Domain swapping experiments: Creating chimeric proteins between IL-36 family members to map functional domains that confer specificity.

A particularly useful experimental approach is to combine neutralizing antibodies against IL36A with recombinant IL36R-Fc fusion proteins as competitive inhibitors, allowing researchers to determine the relative contribution of IL36A versus other IL-36 cytokines in complex biological systems.

What are the critical considerations when designing in vitro experiments with recombinant IL36A?

When designing experiments with recombinant human IL36A, researchers should address several critical factors:

• Protein preparation: Commercially available IL36A is typically produced in E. coli as a nonglycosylated protein . Ensure proper folding and bioactivity validation before use. N-terminal processing significantly affects activity; truncated forms typically display higher biological activity than full-length proteins .

• Concentration range: Titrate IL36A concentrations (typically 1-100 ng/mL) to establish dose-response relationships. Include positive controls (e.g., IL-1β) at established concentrations to benchmark responses.

• Temporal considerations: IL36A responses may differ temporally from other cytokines. Design time-course experiments (2, 6, 12, 24, 48 hours) to capture both immediate and delayed effects.

• Cell type selection: Choose physiologically relevant cells that express IL-36R, such as keratinocytes, epithelial cells, or specific immune cell subsets. Verify receptor expression before stimulation experiments.

• Readout selection: Select appropriate readouts based on expected biology. For IL36A, these might include:

  • Chemokine production (particularly CCL20, CCL11, CCL24, and CXCL-1)

  • NF-κB activation

  • Cell migration

  • Expression of other inflammatory mediators

• Antagonist controls: Include IL-36Ra or blocking antibodies against IL-36R as negative controls to confirm specificity.

When interpreting results, remember that in vitro potency may not directly translate to in vivo efficacy due to the complexity of the tissue microenvironment.

How should researchers design in vivo experiments to study IL36A function, and what models are most appropriate?

Based on the current understanding of IL36A biology, researchers should consider these approaches for in vivo experimentation:

• Model selection: Choose models aligned with tissues where IL36A plays significant physiological roles. Skin inflammation models are particularly relevant given IL36A's prominence in dermatological conditions . Consider:

  • Imiquimod-induced psoriasiform dermatitis

  • IL-23-induced skin inflammation

  • Chemical-induced skin inflammation (e.g., TPA application)

• Intervention strategies:

  • Blocking antibodies: Anti-IL-36R antibodies have been validated for in vivo use (e.g., intranasal administration prior to IL-36γ challenge effectively blocks neutrophil infiltration and chemokine production) .

  • Genetic models: IL-36R knockout mice allow for assessment of complete pathway ablation.

  • Recombinant protein administration: Delivery of recombinant IL36A to specific tissues can identify direct effects.

• Administration route: Match the route to the biology being studied. For lung inflammation, intranasal delivery has been validated . For skin conditions, topical or intradermal administration may be more appropriate.

• Readouts to assess:

  • Tissue histology (inflammation, infiltrating cell populations)

  • Cytokine/chemokine production

  • Neutrophil recruitment (particularly in lung models where IL36A induces significant neutrophilic infiltration)

  • Tissue-specific functional assessments

• Controls and comparisons:

  • Include IL-1 family interventions as comparators (anti-IL-1RI antibodies have shown efficacy in similar models)

  • Use isotype control antibodies for neutralization studies

Notably, while IL36A has been implicated in various inflammatory conditions, its role appears tissue-specific. For example, IL-36R signaling was not crucial in experimental arthritis models, unlike IL-1RI signaling . This highlights the importance of validating IL36A's relevance in each specific disease context.

How does IL36A contribute to inflammatory skin diseases, and what methodological approaches reveal its disease-specific functions?

IL36A plays a significant role in several inflammatory skin conditions, most notably pustular psoriasis . When investigating IL36A in disease contexts, researchers should employ these methodological approaches:

• Patient sample analysis:

  • Quantify IL36A expression in lesional versus non-lesional skin biopsies using qRT-PCR and immunohistochemistry

  • Analyze IL36A levels in patient serum as potential biomarkers

  • Perform single-cell RNA sequencing to identify cell-specific expression patterns

• Ex vivo tissue models:

  • Human skin explant cultures treated with recombinant IL36A can demonstrate direct tissue effects

  • Measure marker gene expression induced by IL-36 in human skin biopsies to assess pathway activation

• Genetic analysis:

  • Screen for IL36A pathway mutations in patients with inflammatory skin diseases

  • Correlate genetic variants with disease severity or treatment response

• Therapeutic targeting validation:

  • Test IL-36R blocking antibodies or small molecule inhibitors in pre-clinical models

  • Recent advances have demonstrated that small molecules (<1000 Da) can effectively target IL-36R , providing new experimental tools

The central role of IL36A in generalized pustular psoriasis (GPP) pathogenesis has been established, with evidence suggesting that targeting IL-36-mediated signaling can improve clinical outcomes . When designing studies, researchers should stratify patients based on disease subtype, severity, and genetic background to identify IL36A-driven disease endotypes.

What are the emerging roles of IL36A in non-dermatological conditions, and how should researchers approach these investigations?

While IL36A was initially characterized in skin inflammation, emerging evidence suggests roles in multiple organ systems that warrant investigation:

• Respiratory system:

  • IL-36R signaling in lung epithelial cells contributes to inflammation

  • IL-36γ administration increases neutrophil infiltration and chemokine production in bronchoalveolar lavage fluids

  • Methodological approach: Analyze IL36A expression in bronchial biopsies from patients with inflammatory lung diseases; employ air-liquid interface cultures of primary bronchial epithelial cells

• Gastrointestinal system:

  • IL-36 signaling has been implicated in intestinal fibrosis and inflammatory bowel disease

  • Methodological approach: Utilize intestinal organoid cultures to study IL36A effects on epithelial integrity and inflammatory responses

• Renal system:

  • IL-36 may contribute to renal tubulointerstitial lesions

  • Methodological approach: Examine IL36A expression in kidney biopsy specimens from patients with various nephropathies

• Infectious diseases:

  • IL-36 signaling appears important in host responses to various pathogens, including during COVID-19

  • Methodological approach: Study IL36A induction in response to pathogen-associated molecular patterns in relevant cell types

When investigating these non-dermatological roles, researchers should:

• Confirm expression of both IL36A and IL-36R in the tissue of interest

• Consider tissue-specific processing enzymes that may activate IL36A locally

• Account for potential compensatory mechanisms from other IL-36 family members

• Compare IL36A responses with canonical inflammatory mediators relevant to each tissue

Interestingly, IL36A's role appears tissue-specific—while crucial in certain inflammatory conditions, it was found dispensable in experimental arthritis models, unlike IL-1 signaling . This highlights the importance of tissue-specific validation of IL36A function.

What are the most effective strategies for detecting and quantifying active IL36A in experimental systems?

Detection and quantification of IL36A presents unique challenges requiring specific methodological considerations:

• Activity-based detection:

  • Bioassays using reporter cell lines expressing IL-36R can measure functional IL36A

  • Measure downstream signaling events (NF-κB activation) or cytokine production (e.g., IL-8 from keratinocytes) as functional readouts

  • Include IL-36Ra as a negative control to confirm specificity

• Protein detection techniques:

  • Western blot using validated antibodies (such as anti-human IL-36α/IL-1F6)

  • ELISA for quantification in biological fluids

  • Mass spectrometry for detection of different IL36A processing forms

• Processing considerations:

  • N-terminal truncation significantly enhances IL36A activity

  • Detect both full-length and processed forms using antibodies recognizing different epitopes

  • When working with recombinant protein, verify whether you're using full-length or truncated versions

• Troubleshooting detection issues:

  • Low signal: Ensure appropriate sample processing to release cell-associated IL36A

  • Non-specific binding: Use samples from IL36A knockout models as negative controls

  • Cross-reactivity: Validate antibody specificity against recombinant IL-36α, β, and γ

When quantifying IL36A in complex biological samples, remember that local concentrations in tissues may differ substantially from those in circulation. For tissue analysis, consider laser capture microdissection to assess IL36A expression in specific microenvironments.

How can researchers effectively study the therapeutic targeting of IL36A, and what are the considerations for developing IL-36R antagonists?

Recent advances have demonstrated the feasibility of targeting IL-36R with both biological and small molecule approaches . Researchers investigating therapeutic targeting should consider:

• Target validation approaches:

  • Genetic validation: IL-36R knockout models or siRNA knockdown

  • Antibody neutralization: Anti-IL-36R antibodies can be used to test pathway inhibition in vitro and in vivo

  • Competitive inhibition: Recombinant IL-36Ra can be employed as a natural pathway antagonist

• Small molecule discovery:

  • Recent work has successfully identified low molecular weight (<1000 Da) compounds targeting IL-36R

  • Screening approaches: DNA-encoded libraries (DEL) and mRNA-based display techniques have proven effective

  • Binding sites: X-ray crystallography has revealed that compounds binding to the D1 domain of IL-36R can disrupt cytokine binding

• Biological therapeutics development:

  • Monoclonal antibodies against IL-36R: Consider epitope selection based on structural insights

  • Fc-fusion proteins: IL-36Ra-Fc fusion proteins may provide extended half-life

  • Bispecific approaches: Targeting both IL-36R and complementary inflammatory pathways

• Efficacy assessment:

  • Ex vivo human skin models: Test compounds on skin biopsies to assess suppression of IL-36-induced marker genes

  • Animal models: Select appropriate disease models where IL-36 signaling is implicated

  • Biomarker development: Identify and validate biomarkers of IL-36 pathway activation

When developing therapeutic strategies, researchers should consider the tissue-specific roles of IL-36 signaling. While targeting this pathway shows promise for inflammatory skin diseases like generalized pustular psoriasis , the same approach may not be effective for conditions like arthritis where IL-36R signaling appears dispensable .

How do the different IL-36 agonists (α, β, γ) differ functionally, and what experimental approaches can elucidate their unique biological roles?

The IL-36 family consists of three agonists (IL-36α, IL-36β, IL-36γ) that all signal through the IL-36 receptor . Despite sharing the same receptor, these cytokines may have distinct biological functions based on:

• Expression patterns:

  • IL-36α is uniquely expressed on T cells, distinguishing it from other IL-1 family members

  • IL-36γ is prominently expressed in activated keratinocytes

  • Methodological approach: Comprehensive tissue expression mapping using qRT-PCR with isoform-specific primers

• Receptor binding characteristics:

  • Differential binding affinity to IL-36R may contribute to functional differences

  • Methodological approach: Surface plasmon resonance studies with each purified agonist to compare binding kinetics

• Downstream signaling variations:

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

  • Methodological approach: Time-course phosphorylation studies of signaling intermediates using phospho-specific antibodies

• Differential regulation:

  • Each agonist may be processed by different proteases or regulated by distinct mechanisms

  • Methodological approach: Protease inhibitor screens to identify specific enzymes involved in processing each isoform

To experimentally differentiate their roles, researchers can employ:

• Selective neutralization using isoform-specific antibodies

• CRISPR-mediated knockout of individual isoforms

• Reconstitution experiments in cell lines lacking all IL-36 agonists

• Transcriptomic comparison of responses to each purified agonist

When designing these studies, researchers should particularly focus on tissues where multiple agonists are co-expressed to determine whether they function redundantly or have specialized roles in different contexts or disease states.

What is the interplay between IL36A and IL-36Ra, and how can researchers effectively study this regulatory mechanism?

The IL-36 system includes a natural antagonist, IL-36Ra, which binds IL-36R but does not recruit the IL-1RAcP co-receptor, thereby inhibiting signaling . This regulatory mechanism is critical for maintaining immune homeostasis.

To study this regulatory axis:

• Binding competition studies:

  • Measure displacement of labeled IL-36Ra by increasing concentrations of IL36A

  • Determine binding sites and affinities through mutagenesis studies

  • Methodological approach: Develop a competitive binding assay using labeled recombinant proteins

• Expression ratio analysis:

  • The balance between agonist and antagonist likely determines net pathway activation

  • Methodological approach: Simultaneous quantification of IL36A and IL-36Ra in tissues using multiplexed qRT-PCR or digital droplet PCR

• Functional antagonism assays:

  • Pre-treatment with IL-36Ra before IL36A stimulation can establish antagonist potency

  • Dose-response curves with fixed IL36A concentration and titrated IL-36Ra

  • Methodological approach: NF-κB reporter assays with controlled agonist/antagonist ratios

• Genetic association studies:

  • Mutations in IL-36Ra have been linked to pustular psoriasis

  • Methodological approach: Screen for variants in both IL36A and IL-36Ra in patient cohorts to identify functional interactions

A particularly informative experimental approach is to develop a mathematical model of the IL-36 signaling system, incorporating binding kinetics, expression levels, and downstream signal transduction parameters. This can help predict how varying ratios of agonist to antagonist affect pathway activation in different tissues or disease states.