IL 1 beta Human, His

What is IL-1β and what are its primary biological functions?

IL-1β (Interleukin-1 beta) is a proinflammatory cytokine encoded by the IL1B gene in humans. It serves as a critical mediator of inflammatory responses and is primarily produced by activated macrophages, monocytes, and a subset of dendritic cells known as slanDC . IL-1β exhibits multiple biological functions, including:

• Mediation of inflammatory responses

• Involvement in cellular proliferation, differentiation, and apoptosis

• Induction of cyclooxygenase-2 in the central nervous system, contributing to inflammatory pain hypersensitivity

• Amplification of B and T lymphocyte proliferation in response to antigens and mitogens

• Stimulation of neutrophilia and acute phase protein production

• Upregulation of chemokines and other inflammatory cytokines

• Regulation of zinc and iron redistribution in tissues

• Modulation of corticosteroid and glucose homeostasis

• Functioning as a potent adjuvant in antigen-specific antibody responses

IL-1β is exceptionally potent, functioning at picomolar concentrations to elicit downstream effects as part of both innate and adaptive immunity .

What is the molecular structure of human IL-1β?

Human IL-1β possesses a conserved β-trefoil conformation characteristic of the IL-1 family of cytokines. Its structure includes:

• 12 β-sheets forming a β-trefoil architecture

• A central hydrophobic core

• Six β-sheets (β1, β4, β5, β8, β9, and β12) arranged in an anti-parallel β-barrel configuration

• Six β-hairpins with consecutive β-sheet naming starting from the N-terminus

• Key functional loops, including β4/5 and β11/12, which are crucial for interactions with the IL-1 receptor accessory protein (IL-1RAcP)

The three-dimensional structure of IL-1β has been determined at high resolution using X-ray crystallography. Despite sharing structural similarity with other IL-1 family members, IL-1β has relatively low sequence identity with these proteins .

How is IL-1β produced and activated in human cells?

Unlike constitutively expressed IL-1α, IL-1β expression is highly regulated and limited to specific cell types:

• Transcription initiation: IL-1β is transcribed following activation of monocytes, macrophages, and dendritic cells through:

  • Toll-like receptor (TLR) stimulation by pathogen-associated molecular patterns (PAMPs)

  • Cytokine signaling

  • Auto-inflammatory induction by IL-1β itself

• Production as inactive precursor: IL-1β is initially synthesized as an inactive precursor protein (pro-IL-1β)

• Proteolytic activation: The precursor requires proteolytic processing by caspase-1 (also known as interleukin-1β converting enzyme or ICE) to generate the bioactive form

• Inflammasome involvement: Caspase-1 activation requires inflammasome assembly, which is triggered by danger-associated molecular patterns (DAMPs)

This two-step regulation (transcriptional control plus proteolytic activation) provides tight control over IL-1β activity, which is crucial given its potent inflammatory effects.

What are the receptor binding mechanisms of IL-1β?

IL-1β binding to its receptors involves a complex multi-step process:

• Primary receptor binding: IL-1β binds to IL-1 receptor type I (IL-1RI), which contains three immunoglobulin-like domains (D1, D2, and D3) forming two distinct binding sites (A and B)

• Interface characteristics:

  • Total buried surface area: 1932 Ų over 47 residues

  • Site A (formed by D1/D2): ~1000 Ų over 25 amino acids

  • Site B (formed by D3): Nearly equivalent sized interface over 21 amino acids

  • Five of six β-sheets from the Ig fold of D3 participate in the interface

  • A hydrogen bond forms between IL-1β and the linker between D1/2 and D3

• Accessory protein recruitment: Following IL-1β binding to IL-1RI, the IL-1 receptor accessory protein (IL-1RAcP) is recruited to form a functional signaling complex

• Critical structural elements: The β4/5 and β11/12 loops of IL-1β are essential for IL-1RAcP recruitment and subsequent signaling

Understanding these binding mechanisms has been crucial for developing therapeutics targeting IL-1β signaling, including the design of receptor antagonists with enhanced potency.

How does chronic IL-1β exposure influence epithelial-to-mesenchymal transition in cancer?

Research has revealed that chronic IL-1β exposure can induce epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer cells, a phenomenon with significant implications for cancer progression:

• Gradual EMT progression: A subset of non-small cell lung cancer cells undergoes a gradually progressing EMT phenotype following 21-day exposure to IL-1β

• EMT memory: The EMT and associated phenotypes (enhanced cell invasion, PD-L1 upregulation, chemoresistance) are sustained even after IL-1β withdrawal, a phenomenon termed "EMT memory"

• SLUG-dependence: The transcription factor SLUG is indispensable for establishing EMT memory, and high SLUG expression in lung cancer patients correlates with poor survival

• Epigenetic regulation: Chromatin immunoprecipitation and methylation-specific PCR revealed SLUG-mediated temporal regulation of epigenetic modifications, including:

  • Accumulation of H3K27 methylation marks

  • Accumulation of H3K9 methylation marks

  • DNA methylation in the CDH1 (E-cadherin) promoter

• Therapeutic implications: Chemical inhibition of DNA methylation restored E-cadherin expression in EMT memory cells and increased their susceptibility to chemotherapy-induced apoptosis

These findings highlight the complex role of IL-1β in cancer progression and suggest potential therapeutic strategies targeting EMT memory in tumors.

What are the considerations when designing IL-1β inhibition strategies?

Dysregulation of IL-1β signaling contributes to numerous pathological conditions, including sepsis, rheumatoid arthritis, inflammatory bowel disease, acute and chronic myelogenous leukemia, insulin-dependent diabetes mellitus, atherosclerosis, neuronal injury, and aging-related diseases . Several approaches for IL-1β inhibition have been developed:

• Receptor antagonists: Anakinra, a recombinant version of the natural IL-1 receptor antagonist (IL-1Ra), was the first FDA-approved IL-1 inhibitor (2001). IL-1Ra binds IL-1RI with high affinity but cannot recruit IL-1RAcP, thereby preventing signal transduction

• Engineered antagonists: Structural knowledge of IL-1β/IL-1RI interactions has enabled the development of more potent inhibitors:

  • EBI-005: A chimeric protein combining site A of IL-1Ra with site B of IL-1β

  • Binding kinetics: Dissociation constant of 6.3×10⁻⁶ s⁻¹ compared to 3.0×10⁻⁵ s⁻¹ for IL-1Ra

  • Theoretical half-life: 31 hours versus 6.4 hours for IL-1Ra

  • In vivo potency: 100-fold increase compared to IL-1Ra

• Targeting processing: Inhibition of caspase-1 to prevent pro-IL-1β cleavage represents another approach to limiting IL-1β activity

• Neutralizing antibodies: Antibodies targeting IL-1β directly can prevent receptor binding

When designing IL-1β inhibition strategies, researchers should consider:

• The specific disease context and relative contribution of IL-1β

• Potential compensatory mechanisms through other cytokines

• Systemic effects of IL-1β blockade on immune responses

• Binding site preferences and kinetics of inhibitory molecules

What are the optimal methods for producing recombinant His-tagged IL-1β?

Production of high-quality recombinant human IL-1β with a histidine tag requires careful consideration of expression systems and purification strategies:

• Expression system selection:

  • E. coli: Most commonly used for IL-1β expression due to simplicity and high yield

  • Mammalian cells: Preferred when post-translational modifications are required

  • Baculovirus-infected insect cells: Offers intermediate complexity with higher protein folding fidelity than bacteria

• Construct design considerations:

  • Position of His-tag: N-terminal tags are generally preferred as they minimize interference with the IL-1β receptor binding domains

  • Linker sequence: Including a flexible linker (e.g., GGGGS) between the His-tag and IL-1β sequence can reduce steric hindrance

  • Protease cleavage site: Incorporating a TEV or enterokinase site allows tag removal if needed for functional studies

  • Codon optimization: Adjust codons based on the expression system for improved protein yield

• Purification protocol:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

  • Secondary purification: Size exclusion chromatography to remove aggregates and ensure monomeric protein

  • Endotoxin removal: Critical for experiments where endotoxin contamination could confound IL-1β effects

  • Quality control: SDS-PAGE, Western blot, and functional assays to confirm identity and activity

• Optimizing solubility:

  • Expression temperature: Lower temperatures (16-25°C) often improve proper folding

  • Lysis buffer composition: Including mild detergents and appropriate salt concentrations

  • Avoiding aggregation: Addition of stabilizing agents like glycerol or arginine

• Activity verification:

  • IL-1β receptor binding assays

  • Cell-based assays measuring downstream signaling activation

  • Comparison with commercial non-tagged IL-1β standards

How does the His-tag affect IL-1β structure and function?

The addition of a histidine tag to IL-1β can potentially impact its structural integrity and biological activity:

What are the best methods for measuring IL-1β activity in cellular assays?

Several robust methods can be employed to assess IL-1β activity in research settings:

• NF-κB activation assays:

  • Reporter cell lines: Cells transfected with an NF-κB responsive element driving luciferase or GFP expression

  • EMSA (Electrophoretic Mobility Shift Assay): Detects NF-κB translocation to nucleus

  • Immunofluorescence: Visualizes p65 nuclear translocation

  • Typical detection range: 0.1-10 ng/ml IL-1β

• Cytokine induction:

  • ELISA/MSD assays: Measure downstream cytokines (IL-6, IL-8, TNF-α) induced by IL-1β

  • qPCR: Quantifies upregulation of inflammatory gene expression

  • Multiplex bead arrays: Simultaneously measures multiple cytokines

  • Most sensitive detection method, with responses at 10-100 pg/ml IL-1β

• Cellular phenotypic assays:

  • Proliferation assays in responsive cell types

  • Cell migration and invasion assays (particularly relevant for cancer research)

  • Adhesion molecule expression by flow cytometry

  • EMT marker analysis in epithelial cells exposed to chronic IL-1β

• Signaling pathway analysis:

  • Western blotting: Detects phosphorylation of pathway components (IKK, IκB, MAPKs)

  • Phospho-flow cytometry: Measures signaling at single-cell resolution

  • Inhibitor studies: Pharmacological dissection of IL-1β-activated pathways

• Considerations for His-tagged IL-1β:

  • Include appropriate controls (commercial non-tagged IL-1β)

  • Account for potential endotoxin contamination (include polymyxin B controls)

  • Test for tag-specific artifacts using anti-His antibodies as blocking controls

How can epigenetic changes induced by chronic IL-1β exposure be effectively measured?

Based on research showing IL-1β-induced epigenetic modifications in cancer cells , the following methodologies are recommended for studying these changes:

• DNA methylation analysis:

  • Methylation-specific PCR: Detects methylation status of specific promoters (e.g., CDH1/E-cadherin)

  • Bisulfite sequencing: Provides single-nucleotide resolution of methylation patterns

  • Genome-wide methylation arrays: Identifies global methylation changes across the genome

  • RRBS (Reduced Representation Bisulfite Sequencing): Cost-effective approach for genome-scale methylation analysis

• Histone modification profiling:

  • ChIP-qPCR: Measures specific histone marks (H3K27me3, H3K9me3) at regions of interest

  • ChIP-seq: Genome-wide mapping of histone modifications

  • Cut&Run or CUT&Tag: More sensitive alternatives to traditional ChIP

  • Western blotting: Global levels of specific histone modifications

• Chromatin accessibility:

  • ATAC-seq: Maps open chromatin regions genome-wide

  • DNase-seq: Identifies DNase I hypersensitive sites

  • MNase-seq: Determines nucleosome positioning

• Transcription factor binding:

  • ChIP-qPCR/ChIP-seq for SLUG and other EMT-associated transcription factors

  • DNA-protein interaction assays (e.g., electrophoretic mobility shift assay)

• Experimental design considerations:

  • Time-course experiments: Capture dynamic changes during chronic exposure

  • IL-1β withdrawal studies: Assess persistence of epigenetic modifications

  • Inhibitor studies: Use of DNMT inhibitors (5-azacytidine), histone methyltransferase inhibitors, or histone deacetylase inhibitors to reverse changes

  • Functional validation: Correlate epigenetic changes with phenotypic outcomes

How does IL-1β contribute to cancer progression, and what are the therapeutic implications?

IL-1β plays multifaceted roles in cancer biology, with significant implications for therapeutic strategies:

• Cancer-promoting mechanisms:

  • Chronic inflammation: IL-1β is a key mediator of inflammation, a recognized hallmark of cancer

  • EMT induction: Prolonged IL-1β exposure drives epithelial-to-mesenchymal transition in cancer cells, promoting invasiveness

  • PD-L1 upregulation: IL-1β-induced EMT is associated with increased PD-L1 expression, potentially contributing to immune evasion

  • Chemoresistance: Cancer cells exposed to chronic IL-1β develop resistance to chemotherapeutic agents

  • Epigenetic reprogramming: IL-1β induces stable epigenetic changes that persist even after cytokine withdrawal

• Therapeutic strategies targeting IL-1β in cancer:

  • Direct IL-1β inhibition: Antagonists and neutralizing antibodies

  • Epigenetic modifiers: DNA methyltransferase inhibitors can reverse IL-1β-induced epigenetic changes and restore chemosensitivity

  • SLUG inhibition: Preventing SLUG upregulation blocks IL-1β-induced EMT

  • Combination approaches: Pairing IL-1β inhibition with chemotherapy or immunotherapy

• Biomarker potential:

  • Circulating IL-1β levels as prognostic indicators

  • Tumor SLUG expression as a predictor of poor survival

  • EMT marker profiles for stratifying patients

  • Methylation signatures in cancer tissues

What is the evolutionary significance of IL-1β function across species?

IL-1β represents an evolutionarily conserved cytokine with fundamental roles in immune defense:

• Evolutionary conservation:

  • IL-1-like molecules are found across vertebrates and some invertebrates

  • The β-trefoil structure is maintained despite sequence divergence

  • Core functions in inflammatory response are preserved across species

• Fundamental defensive functions:

  • Amplification of immune responses to pathogens

  • Coordination of systemic responses (fever, acute phase protein production)

  • Metabolic adaptations during infection (zinc and iron redistribution)

  • Integration of innate and adaptive immunity

• Species-specific adaptations:

  • Variations in receptor binding interfaces

  • Differences in regulatory mechanisms

  • Species-specific inhibitory strategies

• Implications for research:

  • Animal models may not fully recapitulate human IL-1β biology

  • Species-specific antibodies and reagents are necessary for research

  • Understanding evolutionary context helps interpret experimental results

What are the latest approaches for controlling IL-1β activity in inflammatory diseases?

Several innovative approaches for modulating IL-1β activity in inflammatory conditions have emerged:

• Enhanced receptor antagonists:

  • Rationally designed IL-1Ra variants with improved affinity and half-life

  • EBI-005: A chimeric protein combining site A of IL-1Ra with site B of IL-1β, showing 100-fold increased potency compared to natural IL-1Ra

• Targeting IL-1β processing:

  • Inflammasome inhibitors preventing caspase-1 activation

  • Inhibitors of non-canonical processing pathways

• Receptor-targeted approaches:

  • Antibodies blocking IL-1RI

  • Soluble receptor decoys

  • Inhibitors of IL-1RAcP recruitment

• Downstream signaling inhibition:

  • Selective inhibitors of IL-1β-activated kinases

  • Novel NF-κB pathway modulators

• Cell type-specific delivery:

  • Nanoparticle-based targeting of IL-1β inhibitors to specific cell populations

  • Gene therapy approaches for localized IL-1Ra production

• Combination therapies:

  • IL-1β inhibition plus TNF-α blockade for synergistic anti-inflammatory effects

  • IL-1β antagonism with metabolic modulators for conditions like diabetes

How can batch-to-batch variability in recombinant IL-1β experiments be addressed?

Researchers working with recombinant IL-1β often encounter batch-to-batch variability that can confound experimental results:

• Standardization practices:

  • Activity normalization: Establish a bioactivity unit rather than relying solely on protein concentration

  • Reference standards: Maintain an internal reference standard to calibrate each new batch

  • Functional validation: Test each batch in a dose-response assay against a consistent cell line

  • Documentation: Maintain detailed records of source, lot numbers, and activity measurements

• Quality control measures:

  • Endotoxin testing: Use LAL assays to confirm acceptably low endotoxin levels

  • Purity assessment: SDS-PAGE and mass spectrometry to verify protein integrity

  • Aggregation analysis: Size exclusion chromatography or dynamic light scattering

  • Receptor binding assays: Surface plasmon resonance to confirm binding kinetics

• Experimental design considerations:

  • Single-batch experiments: Complete comparative studies using a single batch when possible

  • Internal controls: Include a consistent positive control in each experiment

  • Parallel testing: Run old and new batches side-by-side before switching

  • Calibration curves: Generate batch-specific dose-response curves

• Storage optimization:

  • Aliquoting: Prepare single-use aliquots to avoid freeze-thaw cycles

  • Stabilizing additives: Consider carrier proteins or stabilizers for dilute solutions

  • Temperature monitoring: Ensure consistent storage conditions

  • Stability testing: Periodically test stored aliquots for activity retention

What are the common pitfalls when studying IL-1β signaling in vitro?

Several methodological challenges can complicate the interpretation of IL-1β signaling studies:

• Endotoxin contamination:

  • Issue: Bacterial endotoxins can mimic or synergize with IL-1β effects

  • Solution: Rigorous endotoxin testing, inclusion of polymyxin B controls, endotoxin-removal protocols

• Receptor expression heterogeneity:

  • Issue: Variable IL-1RI and IL-1RAcP expression across cell types and passages

  • Solution: Characterize receptor expression, consider receptor transfection for consistent expression, include positive control cell lines

• Context-dependent responses:

  • Issue: IL-1β effects vary with cell density, serum conditions, and culture duration

  • Solution: Standardize culture conditions, perform time-course and dose-response studies, consider 3D culture systems

• Signal amplification and feedback:

  • Issue: IL-1β induces its own expression, complicating interpretation of primary vs. secondary effects

  • Solution: Include protein synthesis inhibitors, use short time points for primary effects, consider IL-1Ra to block secondary signaling

• His-tag specific considerations:

  • Issue: The His-tag may interfere with certain aspects of IL-1β function

  • Solution: Compare with untagged protein, consider tag removal, verify activity in multiple assay systems

• Cell type selection:

  • Issue: Not all cell types respond equivalently to IL-1β

  • Solution: Validate responsiveness, consider primary cells vs. cell lines, examine heterogeneity within populations

• Timing considerations:

  • Issue: Different signaling pathways and outcomes have distinct kinetics

  • Solution: Detailed time-course experiments, pulse-chase designs, consider both acute and chronic exposure models