il 18 Human

What is the structural basis for receptor recognition of human IL-18?

Human IL-18 features a characteristic β-trefoil fold typical of the IL-1 family of cytokines. Crystal structure analyses reveal that IL-18 forms a signaling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane. While the receptors' recognition mode for IL-18 is generally similar to IL-1β, notable differences exist in the architecture of the IL-18 receptor second domain (D2), which is unique among other IL-1R family members. This structural distinction presumably differentiates IL-18 receptors from other IL-1 receptors that exhibit a more promiscuous ligand recognition mode .

The mature IL-18 protein forms a barrel-like structure with its receptors interacting at the top of the β-barrel. The resolved crystal structures provide valuable insights for developing novel drugs to neutralize IL-18 activity in inflammatory conditions .

How is IL-18 produced and regulated in human cells?

IL-18 is synthesized as a biologically inactive precursor (proIL-18) following activation of certain receptors, including Toll-like receptors and proinflammatory cytokine receptors. This inactive form is stored in the cytosol until it matures via caspase-1, which is regulated by the inflammasome complex . Once processed, the mature IL-18 is secreted extracellularly where it can bind to its receptors.

A critical regulatory mechanism involves IL-18 Binding Protein (IL-18BP), a soluble decoy receptor that sequesters IL-18 with exceptionally high affinity (KD ~300 pM) , which is orders of magnitude stronger than the affinity of IL-18Rα for IL-18 (KD ~69 nM) . This prevents IL-18 from binding to cell surface receptors, thereby inhibiting inflammatory responses. Under homeostatic conditions, IL-18BP is present in serum at approximately 20-fold molar excess compared to IL-18 , ensuring most IL-18 is bound and inactive.

Interestingly, IL-18-induced IFN-γ upregulates IL-18BP expression, creating a negative feedback loop to dampen and resolve inflammation . This regulatory mechanism is crucial for maintaining immune homeostasis.

What are the primary biological effects of human IL-18?

Human IL-18 exhibits several key immunomodulatory effects that contribute to both healthy immune function and pathological inflammation:

• Strong augmentation of IFN-γ production in type-1 helper T (Th1) cells and natural killer (NK) cells

• Activation of NK-cell cytotoxicity

• Enhancement of proliferation in Th1 cells, NK cells, and CD8+ T cells

• Increased lytic activity of immune cells

• Induction of multiple inflammatory cytokines, including IFN-γ and granulocyte macrophage colony-stimulating factor (GM-CSF)

How does the mechanism of IL-18 sequestration by IL-18BP differ from viral IL-18BP orthologs?

The structure of human IL-18 in complex with IL-18BP has elucidated the molecular basis of IL-18 sequestration. Human IL-18BP covers the top of the IL-18 β-barrel with one side of its β-sandwich scaffold and protrudes loops into the barrel . This binding directly competes with IL-18Rα binding, explaining how IL-18BP effectively prevents receptor activation.

Viral IL-18BP orthologs identified in orthopoxviruses such as ectromelia virus (ectvIL-18BP) and Yaba-Like Disease Virus (yldvIL-18BP) act as virulence factors by attenuating IL-18-mediated immune responses . Structure-based comparisons reveal that while these viral proteins share functional homology with human IL-18BP, there are significant differences:

• The affinity of viral IL-18BPs for human IL-18 is considerably lower than human IL-18BP (KD ~1 nM vs. KD ~300 pM), though viral IL-18BPs display picomolar affinity toward mouse IL-18

• Major variations exist in the CD loop mediating IL-18 binding and in the AB loop

• Unlike human IL-18BP, viral IL-18BPs don't exploit additional interactions via side-chain-to-main-chain hydrogen bonds at binding site A

These differences represent evolutionary adaptations of viral proteins to subvert host immune responses and could inform the design of selective therapeutic agents .

What unique tetrameric structure has been observed in the IL-18:IL-18BP complex?

Recent structural studies have captured a fortuitous higher-order assembly between IL-18 and IL-18BP that had not been previously characterized. This assembly is coordinated by a disulfide bond distal to the binding surface, connecting IL-18 and IL-18BP molecules from different complexes and resulting in a novel tetramer with 2:2 stoichiometry .

This tetrapartite assembly was found to restrain IL-18 activity more effectively than the canonical 1:1 complex . The discovery of this structure provides new insights into potential mechanisms of IL-18 regulation and opens avenues for innovative therapeutic strategies targeting IL-18-mediated inflammation.

How can structural knowledge of IL-18 be leveraged for therapeutic development?

The detailed structural information about IL-18 complexes provides several opportunities for therapeutic development:

• The crystal structure of IL-18 with its receptors reveals unique features of the binding interface that can be targeted by small molecules or biologics to disrupt signaling

• Understanding how IL-18BP outcompetes IL-18Rα binding (due to ~230-fold higher affinity) enables the design of IL-18BP-based therapeutics with enhanced potency

• The unique CD loop structure in human IL-18BP that differs from viral orthologs could be exploited to design selective inhibitors

• The newly discovered tetrameric IL-18:IL-18BP complex with 2:2 stoichiometry offers a framework for developing novel multivalent inhibitors that may exhibit enhanced inhibitory effects

• The structure-driven understanding of binding site differences between human IL-18BP and viral IL-18BPs can guide the development of molecules that selectively modulate human IL-18 without interfering with viral defense mechanisms

These structural insights collectively provide a framework for innovative, structure-driven therapeutic strategies for IL-18-related disorders .

How is IL-18 involved in autoimmune and inflammatory diseases?

IL-18 plays significant roles in several autoimmune and inflammatory conditions through its potent proinflammatory effects:

• Systemic Lupus Erythematosus (SLE): Studies using MRLlpr mice (which develop SLE-like disease) genetically lacking IL-18 expression showed reduced renal pathogenesis compared to IL-18-competent mice. This indicates IL-18 is specifically involved in the kidney damage characteristic of SLE . Human studies have similarly found elevated IL-18 levels in SLE patients .

• Rheumatoid Arthritis (RA): Increased IL-18 levels have been documented in RA patients, contributing to pathogenesis through promotion of Th1 responses, stimulation of proinflammatory cytokine production, and activation of osteoclasts leading to bone erosion .

• Crohn's Disease: IL-18 levels are elevated in patients with Crohn's disease, where it likely contributes to intestinal inflammation through enhancement of inflammatory cytokine production and modulation of mucosal immune responses .

• Macrophage Activation Syndrome: IL-18BP-deficient mice developed more severe manifestations in models of this hyperinflammatory condition, highlighting the crucial role of IL-18:IL-18BP balance .

• COVID-19: Elevated IL-18 levels in blood and bronchoalveolar lavage fluid from coronavirus patients correlate with disease severity and worse clinical outcomes, suggesting a role for IL-18 in the cytokine storm associated with severe COVID-19 .

The common theme across these conditions is an imbalance between IL-18 and IL-18BP levels, resulting in excessive free IL-18 driving pathological inflammation .

What are the clinical applications of recombinant human IL-18 (rhIL-18)?

Recombinant human IL-18 (rhIL-18) has been evaluated in clinical settings, particularly for cancer treatment. A phase I study of rhIL-18 in patients with advanced cancer provided valuable insights:

• Dosing and Administration: Doses ranging from 3 to 1,000 μg/kg were administered as 2-hour intravenous infusions on 5 consecutive days

• Safety Profile: Common side effects included chills, fever, nausea, headache, and hypotension. Laboratory abnormalities included transient, asymptomatic grade 1-2 neutropenia, thrombocytopenia, anemia, hypoalbuminemia, hyponatremia, and elevated liver transaminases

• Pharmacokinetics: Plasma concentrations of rhIL-18 increased with dose, and 2.5-fold accumulation was observed with repeated dosing

• Biological Effects: Administration resulted in transient lymphopenia, increased expression of activation antigens on lymphocytes and monocytes, and increases in serum concentrations of IFN-γ, GM-CSF, IL-18BP, and soluble Fas ligand

• Clinical Outcomes: Two patients experienced unconfirmed partial responses after rhIL-18 treatment, suggesting potential anti-tumor activity

Importantly, a maximum tolerated dose was not determined, indicating rhIL-18 can be safely administered in biologically active doses . This safety profile, combined with immunostimulatory effects, supports further exploration of rhIL-18 in cancer immunotherapy and potentially other clinical applications.

How does the IL-18/IL-18BP ratio relate to disease severity?

The balance between IL-18 and its binding protein IL-18BP is critical for immune homeostasis, with the ratio of free IL-18 to total IL-18 serving as a more relevant indicator of inflammatory status than total IL-18 levels alone . Several research findings highlight this relationship:

• As IL-18BP has high IL-18 sequestration capacity, the concentration of free IL-18, rather than total IL-18, more accurately reflects inflammatory activity

• Elevated levels of free IL-18 have been identified in hyperinflammatory diseases such as macrophage activation syndrome and systemic juvenile idiopathic arthritis

• In mouse models of macrophage activation syndrome, IL-18BP-deficient mice developed more severe disease manifestations, demonstrating the crucial role of adequate IL-18 sequestration

• In COVID-19, elevated IL-18 levels correlate with disease severity and worse clinical outcomes, suggesting that IL-18/IL-18BP imbalance contributes to cytokine storm pathology

• The negative feedback loop where IL-18-induced IFN-γ upregulates IL-18BP expression helps resolve inflammation under normal conditions, but may become dysregulated in chronic inflammatory states

These findings suggest that therapeutic strategies targeting this balance, rather than simply inhibiting IL-18, might be more effective for treating inflammatory diseases .

What are the optimal methods for measuring human IL-18 in biological samples?

Accurate measurement of human IL-18 in biological samples requires specific, sensitive assays. Key methodological considerations include:

• Specific ELISA for human IL-18: A validated ELISA has been developed using two neutralizing monoclonal antibodies (#125-2H and #159-12B). This assay detects human IL-18 with a minimum detection limit of 10 pg/ml but does not react with heat-denatured IL-18. It shows no cross-reactivity with other cytokines and has been successfully used to measure IL-18 in patient plasma .

• Free vs. Total IL-18 Assessment: Since IL-18BP sequesters IL-18, measuring both total IL-18 and free IL-18 provides more clinically relevant information. This often requires:

  • Measuring total IL-18 via standard ELISA

  • Measuring IL-18BP via specific assays

  • Calculating free IL-18 based on the law of mass action and known binding affinity

• Sample Handling: Proper collection, processing, and storage of samples is critical. IL-18 may be present in different compartments (plasma, serum, tissue, etc.) with varying concentrations and stability.

• Reference Ranges: Establishing appropriate reference ranges is essential, as baseline IL-18 levels may vary across different populations and clinical contexts.

• Validation for Specific Applications: When investigating IL-18 in particular diseases or experimental settings, assay validation should confirm reliability in the specific context of use .

For research requiring comprehensive assessment of IL-18 status, combining multiple approaches provides the most complete picture of IL-18 biology in the system under study.

What experimental models are most appropriate for studying IL-18-mediated pathology?

Several experimental models have proven valuable for investigating IL-18's role in disease:

• Genetic Deletion Models: MRLlpr mice genetically devoid of IL-18 expression show reduced renal pathogenesis compared to IL-18-competent MRLlpr mice, while maintaining other lupus-like features such as lymphadenopathy and interferon-γ production. This model demonstrates IL-18's specific involvement in renal damage in SLE .

• IL-18BP-Deficient Models: These models develop more severe manifestations in hyperinflammatory conditions, highlighting the crucial regulatory role of IL-18BP .

• Human Cell Culture Systems: Primary human cells (NK cells, T cells, monocytes) or relevant cell lines can be used to study IL-18 signaling mechanisms, cytokine production, and cellular activation.

• Ex Vivo Tissue Analysis: Samples from patients with IL-18-associated diseases can provide direct evidence of IL-18's role in human pathology.

• Recombinant Protein Administration: Controlled administration of rhIL-18 in clinical trials has demonstrated dose-dependent biological effects including lymphocyte activation and cytokine induction .

When selecting experimental models, researchers should consider:

• The specific aspect of IL-18 biology being investigated

• Species-specific differences in IL-18 and IL-18BP interactions

• The availability of appropriate controls and reagents

• The translatability of findings to human disease

The combination of in vitro, ex vivo, and in vivo approaches typically provides the most comprehensive understanding of IL-18's role in specific pathological contexts .

How can neutralizing antibodies against human IL-18 be characterized?

Characterization of neutralizing antibodies against human IL-18 involves several complementary approaches:

• Generation and Initial Screening: Both murine and rat anti-human IL-18 monoclonal antibodies (mAbs) have been developed through immunization and hybridoma technology. From such efforts, 7 murine and 6 rat anti-human IL-18 mAbs have been established .

• Epitope Classification: Competitive binding ELISAs can classify antibodies into distinct groups based on their binding sites on the IL-18 molecule. In one study, antibodies were classified into 4 groups according to their epitope recognition patterns .

• Neutralization Assays: Functional characterization is critical, assessing the ability to block IL-18-induced biological effects. For example, 1 murine mAb and all 6 rat mAbs in one study neutralized IFN-γ production induced by IL-18 .

• Binding Affinity Determination: Techniques such as surface plasmon resonance can measure binding kinetics (kon and koff rates) and equilibrium dissociation constants (KD).

• Specificity Testing: Cross-reactivity testing against related cytokines and heat-denatured IL-18 ensures antibody specificity. Well-characterized antibodies should not show cross-reactivity with other cytokines .

• Application Development: Some neutralizing antibodies can be paired to develop sensitive detection assays. For instance, two neutralizing mAbs (#125-2H and #159-12B) were used to develop a specific human IL-18 ELISA with a minimum detection limit of 10 pg/ml .

These characterized antibodies become powerful tools for investigating IL-18's role in various diseases and analyzing the control mechanisms of IL-18 production and activity .

What therapeutic strategies target the IL-18 pathway?

Several therapeutic strategies have been developed or are under investigation for modulating IL-18 activity:

• Recombinant IL-18 Administration: rhIL-18 has been evaluated in cancer clinical trials, with doses ranging from 3 to 1,000 μg/kg administered as intravenous infusions. This approach aims to enhance anti-tumor immune responses through IL-18's immunostimulatory effects .

• IL-18BP-Based Therapies: Given IL-18BP's high affinity for IL-18, recombinant IL-18BP represents a promising approach for neutralizing excessive IL-18 in inflammatory conditions. The crystal structure of the IL-18:IL-18BP complex provides insights for optimizing such therapies .

• Anti-IL-18 Neutralizing Antibodies: Monoclonal antibodies that neutralize IL-18 activity have been developed and characterized. These could be developed as therapeutics for inflammatory diseases .

• Small Molecule Inhibitors: Targeting IL-18 processing (caspase-1 inhibitors) or downstream signaling pathways could provide alternative approaches for modulating IL-18 activity.

• Novel Complex-Based Strategies: The discovery of a tetrapartite assembly between IL-18 and IL-18BP with 2:2 stoichiometry, which restrains IL-18 activity more effectively than the canonical 1:1 complex, suggests potential for developing multivalent inhibitors with enhanced potency .

These strategies have different mechanisms of action and potential applications:

• IL-18 enhancement approaches (rhIL-18) for cancer immunotherapy

• IL-18 inhibition approaches for autoimmune and inflammatory diseases

The selection of strategy depends on the specific disease context and desired immunomodulatory effect.

What considerations are important when designing clinical trials for IL-18-targeted therapies?

Clinical trial design for IL-18-targeted therapies requires careful consideration of several factors:

• Patient Selection and Stratification:

  • Target populations where IL-18 imbalance has been documented (e.g., rheumatoid arthritis, Crohn's disease, SLE)

  • Consider biomarker-based stratification using measurements of free vs. bound IL-18

  • Evaluate comorbidities that might affect IL-18 biology or treatment response

• Dosing and Administration:

  • For rhIL-18, previous trials used doses of 3-1,000 μg/kg as 2-hour IV infusions on 5 consecutive days

  • Consider pharmacokinetic aspects, such as the 2.5-fold accumulation observed with repeated dosing

  • For IL-18 inhibitors, dosing should account for the high affinity of natural IL-18BP (KD ~300 pM)

• Safety Monitoring:

  • For rhIL-18, monitor for common side effects: chills, fever, nausea, headache, hypotension

  • Laboratory monitoring should include complete blood counts, liver function, and electrolytes

  • Special attention to inflammatory markers given IL-18's role in inflammation

• Efficacy Endpoints:

  • Disease-specific clinical outcomes

  • Biomarkers of IL-18 activity, including IFN-γ levels as pharmacodynamic markers

  • Biological effects such as changes in lymphocyte and monocyte activation

• Duration and Follow-up:

  • Consider both short-term biological effects and longer-term clinical outcomes

  • Monitor for delayed immune-related adverse events

  • Assess durability of response after treatment discontinuation

The finding that a maximum tolerated dose of rhIL-18 was not determined in previous trials suggests a potentially favorable safety profile, but careful monitoring remains essential, particularly when targeting novel patient populations.

How can the balance between IL-18 and IL-18BP be modulated for therapeutic benefit?

The IL-18/IL-18BP balance represents a critical regulatory mechanism and therapeutic target. Several approaches to modulate this balance include:

• Supplementing IL-18BP: In conditions with excess free IL-18, administering recombinant IL-18BP could restore immune homeostasis. The exceptionally high affinity of IL-18BP for IL-18 (KD ~300 pM) makes it an efficient sequestration agent .

• Targeting IL-18 Production: Inhibiting the inflammasome or caspase-1 can reduce IL-18 maturation and secretion, thereby decreasing free IL-18 levels.

• Enhancing Endogenous IL-18BP Production: Since IFN-γ upregulates IL-18BP expression as part of a negative feedback loop , controlled induction of this pathway could increase endogenous IL-18BP levels.

• Novel Formulations Based on Structural Insights: The tetrapartite assembly between IL-18 and IL-18BP (2:2 stoichiometry) restrains IL-18 activity more effectively than the canonical 1:1 complex . This finding suggests that multivalent IL-18 inhibitors might offer enhanced therapeutic potential.

• Targeting Specific Binding Sites: The detailed structural analysis of the IL-18:IL-18BP complex reveals specific interaction sites that could be targeted by small molecules or peptide mimetics .

The most appropriate strategy will depend on the specific disease context:

• In conditions like macrophage activation syndrome where IL-18BP-deficient mice develop more severe disease , supplementing IL-18BP may be beneficial

• In cancers where enhancing immune responses is desirable, selective inhibition of IL-18BP might increase IL-18 availability for anti-tumor effects

Research suggests that measuring the ratio of free to total IL-18, rather than total IL-18 alone, may better guide therapeutic decisions and monitor treatment efficacy .

What are the emerging research areas in IL-18 biology?

Several exciting frontiers in IL-18 research hold promise for advancing our understanding and therapeutic applications:

• Higher-Order IL-18 Complexes: The recent discovery of a novel tetramer with 2:2 IL-18:IL-18BP stoichiometry coordinated by disulfide bonds opens new avenues for understanding IL-18 regulation. This tetrapartite assembly more effectively restrains IL-18 activity than the canonical 1:1 complex, suggesting unexplored regulatory mechanisms .

• IL-18 in COVID-19 Pathogenesis: Elevated IL-18 levels in COVID-19 patients correlate with disease severity and worse clinical outcomes . Further research on IL-18's role in COVID-19 cytokine storms may yield insights applicable to other hyperinflammatory conditions.

• Tissue-Specific IL-18 Functions: Research showing IL-18's specific involvement in renal pathogenesis in lupus models, without affecting other disease parameters , suggests tissue-specific roles that warrant further investigation in various disease contexts.

• Viral IL-18BP Mechanisms: The structural and functional differences between human and viral IL-18BPs represent an evolving area of research at the intersection of virology and immunology.

• IL-18 in Cancer Immunotherapy: The partial responses observed in some cancer patients treated with rhIL-18 suggest potential for IL-18-based cancer immunotherapy approaches, particularly in combination with other immunomodulatory agents.

• IL-18/IL-18BP Balance in Precision Medicine: The critical importance of the IL-18/IL-18BP ratio rather than absolute IL-18 levels points toward more sophisticated biomarker approaches for personalized therapeutic strategies in inflammatory conditions.

These emerging areas highlight the continuing evolution of IL-18 research and its potential impact on understanding and treating diverse diseases.

What technical advances would facilitate IL-18 research?

Several technical advances would significantly advance IL-18 research:

• Improved Free IL-18 Measurement: Current approaches for measuring biologically active free IL-18 (versus IL-18 bound to IL-18BP) are indirect. Direct, high-throughput assays for free IL-18 would facilitate more accurate assessment of IL-18 activity in research and clinical settings .

• High-Resolution Imaging of IL-18 Receptor Complexes: Advanced imaging techniques to visualize IL-18 receptor complex formation and signaling in real-time within living cells would enhance our understanding of signal transduction.

• IL-18 Reporter Systems: Developing sensitive cellular reporter systems that respond specifically to IL-18 signaling would facilitate screening of modulators and investigation of pathway dynamics.

• Humanized Models: Better humanized mouse models expressing human IL-18, IL-18R, and IL-18BP would provide more translatable insights into IL-18 biology and therapeutic effects.

• Structure-Based Drug Design Tools: Computational tools specifically optimized for the unique structural features of IL-18 and its binding partners would accelerate the development of targeted therapeutics .

• Multiplexed Single-Cell Analysis: Technologies for simultaneously assessing IL-18 production, receptor expression, and downstream signaling at the single-cell level would reveal cellular heterogeneity in IL-18 responses.

These technological advances would collectively enhance our ability to understand IL-18 biology and develop more effective therapeutic strategies for IL-18-related diseases.

How might combination therapies involving IL-18 modulation prove beneficial?

Combination therapeutic approaches involving IL-18 modulation show particular promise in several contexts:

• Cancer Immunotherapy: Combining rhIL-18 with immune checkpoint inhibitors could enhance anti-tumor responses. IL-18's ability to increase IFN-γ production and NK cell activity could complement checkpoint inhibition by enhancing tumor immunogenicity and effector cell function.

• Autoimmune Disease Treatment: Combining IL-18BP or anti-IL-18 antibodies with existing standard-of-care treatments might provide synergistic benefits in conditions like rheumatoid arthritis and SLE, where IL-18 contributes to specific aspects of pathology .

• Cytokine Storm Management: In conditions characterized by hyperinflammation, such as macrophage activation syndrome or severe COVID-19, targeting multiple inflammatory mediators including IL-18 might be more effective than single-cytokine approaches .

• Viral Infection Therapies: Understanding the interplay between viral IL-18BPs and host IL-18 could inform combination strategies that both enhance antiviral immunity and limit immunopathology .

• Personalized Approaches Based on IL-18/IL-18BP Ratio: Therapies tailored to individual patients' IL-18/IL-18BP profiles might combine IL-18 pathway modulation with interventions targeting downstream effectors or complementary inflammatory pathways .

The design of these combination approaches should be guided by:

• Mechanistic understanding of pathway interactions

• Careful consideration of timing and sequencing

• Biomarker-based patient selection

• Comprehensive safety monitoring

The distinct biological effects and favorable safety profile observed in rhIL-18 clinical trials suggest that IL-18-based combination therapies may offer unique therapeutic opportunities across multiple disease settings.