IL36B 153 a.a. Human

What is IL36B and what is its molecular structure?

IL36B (IL-36β) is a pro-inflammatory cytokine that belongs to the IL-1 family of cytokines. The human IL36B protein consists of 153 amino acids with a molecular weight of approximately 17.2 kDa . Structurally, IL36B shares sequence and structural homology with other IL-36 family members (IL-36α, IL-36γ, and IL-36Ra) . The protein contains β-strands that are critical for its stability, with studies showing that residues in these structures are particularly intolerant to substitutions . The IL36B gene is located on chromosome 2 in humans, between the IL1B and IL1BR loci .

From a functional perspective, IL36B signals through the IL-36 receptor (IL-36R, also termed IL1RL2) and the co-receptor IL-1RAcP . This interaction initiates downstream inflammatory signaling cascades that regulate immune responses in epithelial tissues.

How does IL36B differ from other IL-36 family cytokines?

IL36B is one of three agonistic isoforms (IL-36α, IL-36β, and IL-36γ) in the IL-36 cytokine group, with IL-36Ra functioning as an antagonist . While all three agonists signal through the same receptor, they exhibit different expression patterns and can be induced by different stimuli.

For example, research shows that while IL-36γ production is upregulated early after cigarette smoke stimulation and decreases over time, IL-36α production requires a longer duration of exposure . IL36B has been reported to be expressed at higher levels in psoriatic plaques compared to unaffected skin or healthy control skin . These differential expression patterns suggest non-redundant roles for the different IL-36 cytokines in inflammatory processes.

What is the expression pattern of IL36B in normal and disease states?

In pathological conditions:

• Psoriasis: IL36B is expressed at higher levels in psoriatic plaques compared to symptomless psoriatic skin or healthy control skin .

• COPD and smoking-related inflammation: IL-36 cytokines show altered production in long-term smokers with and without COPD, contributing to a pro-inflammatory environment in the lungs .

• Neutrophilic inflammation: IL36B contributes to neutrophilic inflammation in various tissues, particularly in the lung .

How is IL36B activation regulated in inflammatory conditions?

IL36B, like other IL-36 cytokines, requires post-translational processing for full activity. While the exact mechanisms for IL36B processing are still being investigated, studies suggest that neutrophil-derived proteases may be involved in this activation process.

The activity of IL36B is naturally regulated by IL-36Ra (IL-36 receptor antagonist), which is encoded by the IL36RN gene . IL-36Ra competes with IL-36 agonists for binding to the IL-36 receptor but does not induce signaling, thereby downregulating the activity of IL-36 cytokines including IL36B . Mutations in IL36RN that affect the function of IL-36Ra can lead to dysregulated IL-36 signaling and contribute to inflammatory conditions such as generalized pustular psoriasis .

How does IL36B signal transduction work at the molecular level?

IL36B initiates signaling by binding to the IL-36 receptor (IL-36R/IL1RL2) and recruiting the co-receptor IL-1RAcP . This receptor engagement activates intracellular signaling pathways that depend on the adaptor protein MyD88, as demonstrated by in vitro studies showing that IL-36α and IL-36γ induce proinflammatory cytokines in a manner that requires both IL-36R and MyD88 .

The downstream signaling cascade involves:

• Activation of MAP kinases

• Nuclear factor-κB (NF-κB) signaling

• Transcription of proinflammatory genes

Recent crystallography analysis has revealed that compounds targeting IL-36R bind to its D1 domain, potentially disrupting IL-36 cytokine binding and preventing signal transduction .

What cellular responses does IL36B stimulate in target tissues?

IL36B exerts various pro-inflammatory effects on different cell types:

• Synovial fibroblasts, articular chondrocytes, and mature adipocytes: IL36B stimulates production of IL6 and IL8 .

• Keratinocytes: Activates inflammatory responses, with keratinocytes being both a source and target of IL-36 cytokines in inflamed skin tissues .

• Alveolar macrophages: Exposure to IL-36 cytokines significantly increases mRNA expression of IL1A, IL1B, IL36G, and CXCL1, indicating that alveolar macrophages can be a source of CXCL1 and IL-36γ in response to IL36B stimulation .

• Neutrophils: IL-36 cytokines including IL36B promote neutrophil recruitment and activation .

What are the optimal conditions for working with recombinant human IL36B in vitro?

When working with recombinant human IL36B (153 a.a.) in research applications, the following conditions should be considered:

Storage and handling:

• Store lyophilized protein desiccated at -20°C .

• After reconstitution, maintain sterile conditions and store according to manufacturer recommendations.

Reconstitution protocol:

• Reconstitute in water or buffer containing a carrier protein (e.g., 0.1% BSA).

• Allow the protein to sit for 10 minutes at room temperature with gentle agitation.

• Avoid repeated freeze-thaw cycles.

Working concentration ranges:

• For cell stimulation experiments: 10-100 ng/mL (concentration should be optimized for specific cell types).

• For in vivo studies: typically 0.5-5 μg per injection (depends on model organism and route of administration).

How can researchers measure IL36B expression and activity?

Detection methods for IL36B expression:

Functional assays for IL36B activity:

• Cytokine induction assay: Measure IL-6, IL-8, or CXCL1 production in responsive cells (e.g., keratinocytes, fibroblasts) following IL36B stimulation .

• NF-κB reporter assay: Use cells transfected with an NF-κB responsive reporter to measure IL36B-induced signaling.

• Cell migration assays: Assess neutrophil recruitment in response to IL36B stimulation.

• Receptor binding assays: Evaluate the interaction between IL36B and IL-36R using surface plasmon resonance or similar techniques.

What is the evidence for IL36B involvement in psoriasis?

IL36B plays a significant role in psoriasis pathogenesis, with several lines of evidence:

• Increased expression: IL36B is expressed at higher levels in psoriatic plaques compared to unaffected skin or healthy control skin .

• Inflammatory amplification: IL36B stimulates the production of pro-inflammatory mediators like IL-6 and IL-8, which are elevated in psoriatic lesions .

• Genetic association: Mutations in IL36RN, which encodes the IL-36 receptor antagonist, are associated with generalized pustular psoriasis, highlighting the importance of regulated IL-36 signaling in skin homeostasis .

The IL-36 pathway creates an inflammatory feedback loop in psoriasis, where activated keratinocytes produce IL-36 cytokines that further stimulate inflammatory responses in keratinocytes and immune cells, perpetuating the inflammatory state .

How does IL36B contribute to lung inflammation?

IL36B and other IL-36 family members have been identified as critical mediators in various lung inflammatory conditions:

• COPD and smoking-related lung damage: IL-36 cytokine production is altered in long-term smokers with and without COPD and contributes to a pro-inflammatory environment in the lungs . Local IL-36α concentrations show a positive correlation with declining ventilatory lung function and increasing pro-inflammatory cytokine concentrations .

• Neutrophilic lung inflammation: IL-36 acts as a key upstream amplifier of neutrophilic lung inflammation by promoting activation of neutrophils, macrophages, and fibroblasts through cooperation with GM-CSF and viral stimuli (like poly(I:C)) .

• Infectious lung diseases: Studies using mouse models have shown that IL-36 signaling is important in lung inflammatory conditions associated with high neutrophil numbers. For example, IL-36 receptor-deficient mice exposed to cigarette smoke or cigarette smoke plus H1N1 influenza virus had attenuated lung inflammation compared to wild-type controls .

What therapeutic approaches target IL36B or its receptor?

Several therapeutic approaches targeting the IL-36 pathway are being developed:

• Small molecule inhibitors: Recent research has demonstrated the possibility of targeting the IL-36 receptor with small molecules (<1000 Da) . For example, DEL screening identified 36R-D481, a high-affinity low molecular weight IL-36R binder that effectively inhibits IL-36 signaling .

• Macrocyclic peptides: The mRNA-based display technique identified 36R-P138, a macrocyclic peptide blocking IL-36R signaling. Its optimized analog (36R-P192) effectively suppresses the expression of marker genes induced by IL-36 in human skin biopsies .

• X-ray crystallography insights: Structural studies have revealed that both cyclic peptides and small molecules bind to the IL-36R's D1 domain, potentially disrupting IL-36 cytokine binding . This structural information provides a basis for rational drug design targeting the IL-36 pathway.

These approaches show promise for treating conditions with aberrant IL-36 signaling, such as psoriasis, and potentially various lung inflammatory diseases where IL-36 plays a role .

What are the critical residues in IL36B structure-function relationship?

Research into the structure-function relationship of IL-36 family proteins has identified several critical amino acid residues:

• β-strand residues: Studies using in silico saturation mutagenesis have shown that the IL-36Ra residues most intolerant to substitutions are more likely to map to β-strands (80.0% vs. 47.8%, P = 0.027) . While this was specifically studied for IL-36Ra, the structural similarity between IL-36 family members suggests that β-strand residues may also be important for IL36B function.

• IL-36R binding interface: X-ray crystallography analysis reveals that IL-36 antagonists bind to the IL-36R's D1 domain, disrupting IL-36 cytokine binding . This suggests that residues in IL36B that interact with this domain are critical for receptor engagement and signaling.

• Solvent accessibility: Critical functional residues tend to be less accessible to solvents compared to other amino acids (average fraction of solvent accessible surface area: 0.21 vs. 0.38, P < 0.05) . This characteristic may help identify potentially important residues in IL36B structure.

Further research combining computational approaches with experimental validation is needed to fully map the critical residues in IL36B that determine its binding specificity, activity, and regulation.

What experimental models are most suitable for studying IL36B function?

Several experimental models have been validated for investigating IL36B function:

In vitro models:

• Primary human keratinocytes: Ideal for studying IL36B's role in skin inflammation and psoriasis .

• Alveolar macrophages: Useful for investigating IL36B's effects on lung inflammation .

• Synovial fibroblasts and articular chondrocytes: Appropriate for studying IL36B's role in joint inflammation .

In vivo models:

• IL-36R knockout mice: Valuable for assessing the contribution of IL-36 signaling to inflammatory diseases. These mice show attenuated lung inflammation when exposed to cigarette smoke or cigarette smoke plus H1N1 influenza virus .

• Cigarette smoke exposure models: Used to study IL-36's role in smoking-related lung inflammation and COPD .

• Psoriasis-like skin inflammation models: Useful for investigating IL36B's contribution to skin pathology.

Ex vivo models:

• Human skin biopsies: Effective for validating IL-36 pathway inhibitors and studying IL36B-induced gene expression .

• Precision-cut lung slices: Maintain the structural complexity of lung tissue while allowing for controlled experimental conditions.

How can interactions between IL36B and other inflammatory mediators be studied?

Understanding the interactions between IL36B and other inflammatory mediators requires integrated approaches:

• Co-stimulation experiments: Treat cells with IL36B in combination with other cytokines (e.g., GM-CSF, IL-1α/β) to identify synergistic or antagonistic effects. For example, research has shown that IL-36 cooperates with GM-CSF and viral mimics like poly(I:C) to promote activation of neutrophils, macrophages, and fibroblasts .

• Pathway inhibition studies: Use specific inhibitors of various signaling pathways to dissect the mechanisms of IL36B-mediated effects and their interaction with other inflammatory pathways.

• Multi-omics approaches: Combine transcriptomics, proteomics, and metabolomics to comprehensively map the effects of IL36B on cellular responses and identify points of intersection with other inflammatory mediators.

• Systems biology modeling: Develop computational models of inflammatory networks that incorporate IL36B signaling to predict emergent properties and interactions.

• In vivo studies with conditional knockouts: Use tissue-specific or inducible knockout models to investigate the role of IL36B in complex inflammatory environments where multiple mediators are present.