Recombinant Human Interleukin-36 beta (IL36B) (Active)

What is the structural composition of human IL-36β?

Human IL-36β is a 157 amino acid protein belonging to the IL-1 family of cytokines. It is characterized by a 12 beta-stranded beta-trefoil configuration, typical of IL-1 family members. Unlike many cytokines, IL-36β lacks a canonical signal peptide or prosegment and exists as two distinct isoforms that differ in their C-terminal 70 amino acids. Notably, the IL-36β1 isoform lacks four of the conserved beta-strands commonly found in the IL-1 family. Human IL-36β2 shares variable sequence identity with other mammalian orthologs: 62% with mouse, 67% with canine, 63% with bovine, and 59% with equine IL-36β isoforms .

How does IL-36β differ from other members of the IL-36 cytokine family?

IL-36β is one of several members of the IL-36 cytokine subfamily within the larger IL-1 family. This subfamily includes the pro-inflammatory agonists IL-36α, IL-36β, and IL-36γ, as well as the anti-inflammatory antagonist IL-36Ra. While all IL-36 cytokines share the IL-1 family's beta-trefoil structure and utilize the same receptor complex, they differ in their tissue distribution, regulation, and specific biological effects. IL-36β has distinct expression patterns in keratinocytes, naïve CD4+ T cells, neurons, and glial cells that differentiate it from other IL-36 cytokines. Additionally, IL-36β undergoes unique post-translational processing—cleavage of its first four N-terminal amino acids significantly increases its biological potency, a characteristic that may vary among IL-36 family members .

What are the primary cellular sources of IL-36β?

The primary cellular sources of IL-36β include keratinocytes, naïve CD4+ T cells, neurons, and glial cells. Expression is particularly notable in the skin, where keratinocytes produce IL-36β under both baseline and inflammatory conditions. In inflammatory states, especially in psoriatic lesions, IL-36β expression is significantly upregulated in keratinocytes. Additionally, synovial fibroblasts have been identified as important producers of IL-36β during inflammatory conditions. The expression pattern suggests IL-36β has important roles in both cutaneous and articular inflammatory responses, as well as potential neuroimmune functions given its presence in neural tissues .

What factors regulate the expression of IL-36β in different cell types?

IL-36β expression is subject to complex regulatory mechanisms that differ by cell type. In keratinocytes and synovial fibroblasts, inflammatory stimuli significantly upregulate IL-36β production. Pro-inflammatory cytokines including IL-1β, TNF-α, and IL-17A are potent inducers of IL-36 family cytokines, including IL-36β. These cytokines can act synergistically to amplify IL-36β expression. Additionally, IL-36 cytokines themselves can enhance their own expression, creating positive feedback loops. For example, IL-36β stimulation of bone marrow-derived dendritic cells (BMDCs) increases expression of both il36a and il36g genes, suggesting cross-regulation within the family. Mechanistically, the induction of IL-36 cytokines appears to involve multiple signaling pathways including ERK1/2, JNK1/2, p38, c-Jun, and NF-κB signaling cascades .

How is the bioactivity of IL-36β regulated post-translationally?

Post-translational regulation of IL-36β bioactivity occurs primarily through proteolytic processing. The full-length IL-36β protein exhibits relatively low biological activity, but its potency is dramatically increased following cleavage of the first four N-terminal amino acids (resulting in a truncated form starting at Arg5). This processing is mediated by neutrophil-derived serine proteases, particularly neutrophil elastase and cathepsin G. In vitro studies have demonstrated that supernatants from PMA-activated neutrophils can cleave and enhance the bioactivity of IL-36 cytokines, including IL-36β. This mechanism likely represents an important regulatory checkpoint that links neutrophil recruitment to amplification of IL-36-mediated inflammatory responses. Inhibitors of neutrophil elastase and cathepsin G diminish this activation, suggesting potential therapeutic targets for modulating IL-36β activity .

What is the relationship between IL-36β and other inflammatory cytokines?

IL-36β functions within a complex network of inflammatory cytokines with significant cross-regulation and functional overlap. IL-1β and TNF-α are potent inducers of IL-36 family cytokines in multiple cell types, including colonic myoblasts and pulmonary macrophages. Conversely, IL-36β itself can enhance the production of multiple pro-inflammatory mediators, including IL-6, IL-8, and various chemokines by keratinocytes, synovial fibroblasts, and articular chondrocytes. IL-36β also promotes Th1 polarization of T cells and enhances dendritic cell activation, therefore amplifying adaptive immune responses. This bidirectional relationship creates inflammatory circuits where IL-36β both responds to and contributes to the inflammatory milieu. Additionally, IL-36β expression is closely associated with IL-17-mediated inflammation, particularly in psoriatic lesions, suggesting functional relationships in IL-17-driven pathologies .

What is the receptor complex for IL-36β and how does signal transduction occur?

IL-36β signals through a heterodimeric receptor complex consisting of the IL-36 receptor (IL-36R, formerly known as IL-1Rrp2) and the IL-1 receptor accessory protein (IL-1RAcP). Upon binding of IL-36β to IL-36R, conformational changes facilitate recruitment of IL-1RAcP, forming a ternary complex that initiates downstream signaling. This receptor complex activation triggers intracellular signaling cascades primarily through two major pathways: the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and the Mitogen-Activated Protein Kinase (MAPK) pathway. These pathways culminate in the activation of transcription factors that regulate genes involved in inflammatory responses. The requirement for both IL-36R and IL-1RAcP for signal transduction has been firmly established, as deficiency in either component abrogates the biological effects of IL-36β stimulation .

How does IL-36Ra antagonize IL-36β signaling at the molecular level?

IL-36 receptor antagonist (IL-36Ra) functions as a selective inhibitor of IL-36 signaling through competitive binding to the IL-36 receptor. Mechanistically, IL-36Ra binds to IL-36R with high affinity but, unlike the agonists (IL-36α, IL-36β, and IL-36γ), it does not induce the recruitment of IL-1RAcP to form a signaling-competent complex. This binding effectively blocks the interaction of IL-36β and other agonists with IL-36R. In some cases, IL-36Ra binding may lead to non-signaling IL-36R homodimerization. In bone marrow-derived dendritic cells (BMDCs), IL-36Ra acts as a dose-dependent inhibitor of all three IL-36 agonists. Transgenic mouse models have demonstrated that IL-36Ra can inhibit IL-36γ-induced NF-κB activation, and similar mechanisms apply to antagonism of IL-36β signaling. This competitive inhibition represents an important endogenous regulatory mechanism for controlling IL-36-mediated inflammation .

What concentrations of IL-36β are required for biological activity in different experimental models?

The concentration of IL-36β required for biological activity varies depending on the experimental model and the specific biological readout being measured. For recombinant human IL-36β (aa 5-157), the effective dose that induces 50% of maximal response (ED50) for IL-8 secretion in A431 human epithelial carcinoma cells ranges from 0.8 to 4.8 ng/mL. This relatively low concentration range indicates that the truncated, activated form of IL-36β is a potent inducer of inflammatory responses. It's important to note that full-length IL-36β typically requires significantly higher concentrations to elicit comparable biological effects, highlighting the importance of N-terminal processing for optimal activity. When designing experiments with IL-36β, researchers should consider these concentration ranges and the activation state of the recombinant protein to ensure physiologically relevant conditions are established .

What are the optimal methods for producing biologically active recombinant IL-36β?

Production of optimally active recombinant IL-36β requires careful consideration of the protein form. Most commercially available and research-grade recombinant IL-36β preparations are produced in E. coli expression systems, typically generating the truncated form (Arg5-Glu157) that exhibits enhanced biological activity compared to the full-length protein. When producing recombinant IL-36β, researchers should consider:

• Expression system: E. coli systems are commonly used but may lack post-translational modifications; mammalian expression systems may provide proteins with more native-like modifications.

• Purification strategy: Multi-step chromatography approaches combining affinity, ion-exchange, and size-exclusion methods help ensure high purity.

• Truncation considerations: Expressing the protein without the first four amino acids (starting at Arg5) yields significantly higher biological activity.

• Quality control: Validation should include purity assessment by SDS-PAGE, endotoxin testing (critical for immunological assays), and biological activity testing (e.g., IL-8 induction in responsive cell lines).

For experiments requiring maximal biological activity, the truncated form (Arg5-Glu157) should be used, with typical working concentrations between 0.8-50 ng/mL depending on the cell type and readout .

What bioassays are most reliable for measuring IL-36β biological activity?

Several bioassays have been validated for measuring IL-36β biological activity, with reliability varying based on the biological context being investigated. The most commonly used and reliable assays include:

• IL-8 secretion assay in epithelial carcinoma cells (e.g., A431 cells): This assay measures IL-8 production following IL-36β stimulation, with typical ED50 values of 0.8-4.8 ng/mL.

• NF-κB reporter assays: Cell lines transfected with IL-36R and NF-κB-responsive reporters provide quantitative measurement of signaling activation.

• Cytokine induction in primary cells: Measuring production of IL-6, IL-8, or CXCL1 in primary keratinocytes, synovial fibroblasts, or articular chondrocytes following IL-36β stimulation.

• Dendritic cell activation assays: Assessing upregulation of costimulatory molecules or cytokine production in dendritic cells.

• T cell polarization assays: Evaluating the ability of IL-36β to enhance Th1 responses in CD4+ T cells.

For optimal reliability, positive controls (other IL-36 family members or established inducers like IL-1β) and negative controls (media alone or IL-36Ra co-treatment) should be included. Additionally, validation with neutralizing antibodies against IL-36R or signaling inhibitors provides specificity confirmation .

How can researchers effectively measure IL-36β expression in different tissue samples?

Effective measurement of IL-36β expression in tissue samples requires a multi-modal approach depending on the research question and sample type. Recommended methodologies include:

• Quantitative RT-PCR: For measuring IL-36β mRNA expression with high sensitivity. Primer design should account for the two isoforms of IL-36β and potentially include melt curve analysis to distinguish between them.

• ELISA: Commercial or custom-developed sandwich ELISAs can quantify IL-36β protein levels in tissue homogenates, cell culture supernatants, or biological fluids. Typical detection ranges for commercial ELISAs are 30-2000 pg/mL.

• Immunohistochemistry/Immunofluorescence: For localizing IL-36β expression within tissue architecture, identifying producing cell types, and assessing spatial relationships with receptors or responding cells.

• Western blotting: For semi-quantitative protein detection and distinguishing between full-length and processed forms.

• Single-cell RNA sequencing: For comprehensive analysis of cell-specific expression patterns within heterogeneous tissues.

In human studies, serum IL-36β levels may be measured by ELISA as part of disease association studies. For example, studies of inflammatory conditions like type 2 diabetes mellitus have employed this approach to compare cytokine levels between patients and healthy controls. Important considerations include sample storage conditions to prevent degradation and the potential need for protease inhibitors to preserve native protein forms .

What is the role of IL-36β in inflammatory skin diseases?

IL-36β plays a significant role in inflammatory skin diseases, particularly psoriasis. In psoriatic lesions, IL-36β expression is markedly upregulated in keratinocytes compared to healthy skin. This overexpression contributes to the inflammatory cascade characteristic of psoriasis through several mechanisms:

• Amplification of local inflammation: IL-36β induces production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides by keratinocytes, creating a self-sustaining inflammatory loop.

• Recruitment of immune cells: The chemokines induced by IL-36β promote infiltration of neutrophils, T cells, and dendritic cells into the skin.

• Enhancement of Th17 responses: IL-36β promotes IL-23 production by dendritic cells, which in turn supports Th17 cell differentiation and IL-17 production, a critical pathway in psoriasis pathogenesis.

• Keratinocyte hyperproliferation: IL-36β contributes to the characteristic epidermal hyperplasia seen in psoriasis by promoting keratinocyte proliferation.

The pathogenic role of IL-36 signaling in psoriasis is further supported by the identification of loss-of-function mutations in the IL-36Ra gene (IL36RN) in patients with generalized pustular psoriasis, resulting in unopposed IL-36 agonist activity. This pathophysiological understanding has led to consideration of IL-36 pathway blockade as a therapeutic strategy for psoriasis and other inflammatory skin conditions .

What evidence links IL-36β to metabolic disorders like type 2 diabetes?

Emerging evidence suggests a potential role for IL-36β and other IL-36 family cytokines in metabolic disorders, particularly type 2 diabetes mellitus (T2DM). A case-control study revealed altered IL-36 cytokine profiles in T2DM patients compared to healthy controls. Specifically:

• Serum levels of IL-36 agonists (IL-36α and IL-36γ) were significantly elevated in T2DM patients.

• Conversely, serum levels of the IL-36 receptor antagonist (IL-36Ra) were decreased in T2DM patients.

• These alterations correlated with markers of inflammation, including high-sensitivity C-reactive protein (hsCRP), suggesting a connection between IL-36 dysregulation and the chronic inflammation characteristic of T2DM.

How does IL-36β contribute to inflammatory responses in the lung?

IL-36β contributes to inflammatory responses in the lung through multiple mechanisms, participating in both protective immunity and potentially pathological inflammation. In pulmonary contexts, IL-36β functions include:

• Enhancement of epithelial defense: IL-36β stimulates bronchial epithelial cells to produce antimicrobial peptides and pro-inflammatory cytokines, bolstering barrier immunity against respiratory pathogens.

• Promotion of neutrophil recruitment: By inducing chemokine production (particularly IL-8/CXCL8), IL-36β facilitates neutrophil infiltration into the lung during infection or injury.

• Modulation of antigen-presenting cell function: IL-36β activates dendritic cells and alveolar macrophages, enhancing their ability to initiate adaptive immune responses against pulmonary pathogens.

• Amplification of inflammatory cascades: Through activation of NF-κB and MAPK pathways in lung structural and immune cells, IL-36β propagates inflammatory signaling networks.

This involvement in pulmonary inflammation suggests potential roles for IL-36β in respiratory conditions such as asthma, COPD, and infectious pneumonia. Notably, a complex interplay exists between neutrophil recruitment and IL-36β activation in the lung: neutrophil proteases can cleave and activate IL-36β, which in turn promotes further neutrophil recruitment, potentially creating a self-amplifying inflammatory circuit in pulmonary diseases .

How do post-translational modifications affect IL-36β structure-function relationships?

Post-translational modifications, particularly N-terminal processing, fundamentally alter IL-36β structure-function relationships. The truncated form of IL-36β (starting at Arg5) exhibits dramatically enhanced receptor binding affinity and biological activity compared to the full-length protein. This functional transformation likely results from conformational changes that optimize interaction with the IL-36 receptor.

Research in this area should address several key questions:

• Structural basis: Crystallographic or cryo-EM studies comparing full-length versus truncated IL-36β bound to IL-36R would reveal the molecular basis for enhanced receptor engagement.

• Alternative processing: Beyond the established cleavage after Lys4, are there other proteolytic processing events that generate IL-36β variants with distinct functional properties?

• Cell-specific processing: Do different cellular contexts (e.g., skin vs. lung) employ distinct proteolytic mechanisms for IL-36β activation?

• Endogenous regulation: Are there natural inhibitors of the proteases that activate IL-36β, representing an additional layer of regulatory control?

• Therapeutic implications: Could engineered forms of IL-36β with enhanced or attenuated activity serve as therapeutic agents for specific conditions?

Methodologically, site-directed mutagenesis approaches combined with functional bioassays would help identify critical residues that mediate the enhanced activity of processed IL-36β and potentially reveal new therapeutic strategies targeting this activation mechanism .

What are the methodological challenges in studying IL-36β interactions with its receptor complex?

Studying IL-36β interactions with its receptor complex presents several methodological challenges that researchers must address:

• Receptor expression heterogeneity: IL-36R expression varies widely across cell types and can be modulated by inflammatory stimuli, requiring careful characterization of receptor expression in experimental systems.

• Competition with endogenous ligands: In primary cell systems, endogenously produced IL-36 family members may compete with exogenously added IL-36β, necessitating careful controls or genetic approaches to isolate specific effects.

• Shared accessory protein: IL-1RAcP is utilized by multiple IL-1 family receptors, complicating the specificity of observed effects, particularly in systems where multiple IL-1 family members are active.

• Receptor complex assembly dynamics: The kinetics and stability of the ternary complex formation (IL-36β/IL-36R/IL-1RAcP) are challenging to measure in real-time.

• Post-receptor signaling complexity: Overlapping downstream signaling pathways between IL-36 and other inflammatory mediators make it difficult to attribute specific cellular responses to IL-36β.

Advanced methodological approaches to address these challenges include:

• Surface plasmon resonance or biolayer interferometry for quantitative binding analyses

• FRET-based approaches to monitor receptor complex assembly in living cells

• Proximity ligation assays to visualize receptor interactions in situ

• CRISPR-based receptor editing for clean genetic models

• Phosphoproteomic analyses to resolve signaling pathway specificity

These technical considerations are crucial for researchers designing experiments to elucidate the specific contributions of IL-36β signaling in complex biological systems .

How might IL-36β signaling be therapeutically targeted in inflammatory diseases?

Therapeutic targeting of IL-36β signaling presents promising opportunities for treating inflammatory diseases. Several strategic approaches could be pursued:

• Receptor antagonism: Development of optimized IL-36Ra variants or monoclonal antibodies against IL-36R could block IL-36β and other agonist signaling. This approach would be particularly relevant for conditions where IL-36Ra is deficient or where IL-36 agonists are overexpressed.

• Protease inhibition: Since neutrophil elastase and cathepsin G are critical for IL-36β activation, selective inhibitors of these proteases could reduce IL-36β-mediated inflammation while preserving other neutrophil functions.

• Signaling pathway modulation: Small molecule inhibitors targeting the specific nodes in NF-κB or MAPK signaling pathways that are critical for IL-36β effects could provide selective therapeutic benefits.

• Combination approaches: Dual targeting of IL-36β alongside complementary inflammatory pathways (e.g., IL-17 or TNF) might yield synergistic benefits in complex inflammatory diseases like psoriasis.

• Tissue-specific delivery: For localized inflammatory conditions, topical or inhalational delivery of IL-36 pathway inhibitors could maximize therapeutic efficacy while minimizing systemic effects.

Translational considerations include:

• Biomarker development to identify patients with IL-36-driven pathology

• Animal models that accurately recapitulate human IL-36 biology

• Careful assessment of potential immunosuppressive effects

These therapeutic strategies hold particular promise for inflammatory skin diseases like psoriasis, where IL-36 dysregulation is well-established, and potentially for metabolic inflammation in type 2 diabetes, where early evidence suggests IL-36 involvement .