IL 18 Mouse

What is IL-18 and how does it function in mice?

IL-18 is a member of the IL-1 family of cytokines that plays crucial roles in inflammatory responses. In mice, IL-18 increases significantly in response to various stimuli, particularly radiation exposure. IL-18 functions by binding to its receptor (IL-18R) composed of IL-18Rα and IL-18Rβ chains, activating downstream signaling pathways that lead to inflammatory responses, including induction of interferon-gamma (IFNγ) production. The biological activity of IL-18 is regulated by IL-18 binding protein (IL-18BP), which serves as its natural antagonist by preventing IL-18 from binding to its receptor . This balance between IL-18 and IL-18BP is critical for maintaining appropriate inflammatory responses in mice.

How does mouse IL-18 expression vary across different tissues?

Studies have demonstrated variable IL-18 expression across different mouse tissues following inflammatory stimuli. After total-body irradiation (TBI), IL-18 increases in mouse thymus, spleen, and bone marrow cells, though levels vary between tissues. Most notably, IL-18 shows significant and stable increases (2.5-24 fold) in mouse serum from day 1 after TBI for up to 13 days in a radiation dose-dependent manner . This tissue-specific expression pattern suggests that different cellular sources contribute to IL-18 production, with potential unique regulatory mechanisms in each tissue type. When designing experiments to measure IL-18, researchers should consider these tissue-specific variations to properly interpret results.

What are the common methods for measuring IL-18 in mouse samples?

Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring IL-18 in mouse serum, plasma, and tissue homogenates. Immunoblotting (Western blot) can also be used to detect IL-18 protein in tissue samples. Cytokine antibody arrays offer a multiplexed approach for simultaneous detection of IL-18 along with other cytokines . For gene expression analysis, quantitative PCR (qPCR) can measure IL-18 mRNA levels. When measuring IL-18 in experimental settings, it's important to consider that total IL-18 levels may not reflect bioactive IL-18, as free IL-18 (not bound to IL-18BP) represents the biologically active form. Therefore, measuring both IL-18 and IL-18BP provides more meaningful data regarding the functional status of this cytokine pathway .

How does IL-18 contribute to radiation injury in mouse models?

IL-18 plays a significant role in radiation-induced injury in mice, with serum levels increasing proportionally to radiation dose. Following total-body irradiation, IL-18 levels rise significantly (2.5-24 fold) and remain elevated for up to 13 days . This increase correlates with the severity of radiation injury and appears to be a key mediator of radiation-induced inflammation and tissue damage. Mechanistically, IL-18 activates downstream targets like interferon-gamma (IFN-γ), increases reactive oxygen species (ROS) production, and induces stress factors such as growth differentiation factor-15 (GDF-15) . These effects contribute to multiple organ injuries after radiation exposure. The consistent dose-dependent relationship between radiation exposure and IL-18 levels makes it a potential biomarker for radiation injury assessment and a target for therapeutic intervention.

What is the role of IL-18 in mouse models of atopic dermatitis?

In mouse models of atopic dermatitis (AD), IL-18 significantly contributes to inflammatory skin responses. Studies using MC903 (a vitamin D analog) to induce AD-like lesions have shown that IL-18 knockout mice develop less severe symptoms compared to wild-type mice. Specifically, IL-18 deficiency results in reduced clinical manifestations (erythema, edema, scaling, exudation, and crusts) and lower SCORAD values . Histologically, IL-18 knockout mice show decreased inflammatory cell infiltration, particularly mast cells, in AD-like lesions. At the molecular level, IL-18 knockout reduces pro-inflammatory cytokine production, including IL-4, a key Th2 cytokine involved in AD pathogenesis . These findings suggest that IL-18 promotes AD development by enhancing Th2-mediated inflammation and mast cell recruitment, making it a potential therapeutic target for treating AD.

How does IL-18 influence metabolic regulation in mouse models?

IL-18's role in metabolic regulation in mice appears paradoxical. While elevated IL-18 levels correlate with metabolic syndrome and type 2 diabetes in humans, IL-18 or IL-18Rα-deficient mice actually develop obesity and insulin resistance after 6 months on normal chow diets. This occurs due to hyperphagia and decreased energy consumption . Administration of exogenous IL-18 (intravenous, intracerebral, or intraperitoneal) corrects hyperphagia, increases catabolism (possibly via IL-18-induced IFNγ production), and decreases insulin resistance. Mechanistically, IL-18 may increase insulin sensitivity through phosphorylation of STAT3 in cells and possibly by activating AMP-activated kinase (AMPK) in muscles . These findings suggest that IL-18 functions similarly to leptin as an adipocytokine that controls both food intake and energy homeostasis. The increased IL-18 concentrations observed in metabolic disorders may represent a compensatory mechanism attempting to counteract insulin resistance induced by other pro-inflammatory factors.

How are IL-18 knockout mice generated and validated?

IL-18 knockout (IL-18−/−) mice are typically generated through targeted germline deletion of the IL-18 gene. The most widely used strain is B6.129P2-Il18tm1Aki/J, available from Jackson Laboratory . These mice are created by replacing part of the IL-18 gene with a neomycin resistance cassette, disrupting gene function. Validation of the knockout is performed through PCR genotyping according to standardized protocols provided by the producer . For experimental rigor, researchers should confirm the IL-18 deficiency at both genomic and protein levels. Genomic validation involves PCR analysis of tail DNA to confirm the presence of the mutant allele. Protein-level validation requires demonstrating absence of IL-18 production in tissues that normally express it, typically using ELISA, Western blot, or immunohistochemistry. Control experiments comparing wild-type and knockout mice in contexts known to induce IL-18 (such as LPS stimulation) are recommended to functionally validate the knockout.

What phenotypic characteristics distinguish IL-18 knockout mice from wild-type mice?

IL-18 knockout mice exhibit several distinctive phenotypic characteristics compared to wild-type mice. Under normal conditions, IL-18 knockout mice appear healthy but develop metabolic abnormalities over time, including obesity and insulin resistance after approximately 6 months on normal diets due to hyperphagia and reduced energy expenditure . In disease models, IL-18 knockout mice show striking differences from wild-type counterparts. In atopic dermatitis models induced by MC903, IL-18 knockout mice develop significantly milder skin lesions with reduced erythema, edema, scaling, exudation, and crusting. The SCORAD values in MC903-treated IL-18 knockout mice are significantly lower than in wild-type mice (3.4±0.5099 vs. 8.4±0.5099) . Histologically, IL-18 knockout mice show reduced inflammatory cell infiltration, particularly mast cells, in affected skin. At the molecular level, IL-18 knockout mice exhibit decreased expression of pro-inflammatory cytokines and chemokines, especially reduced Th2 cytokines like IL-4 .

What control groups are essential when designing experiments with IL-18 knockout mice?

When designing experiments with IL-18 knockout mice, several control groups are essential for proper interpretation of results. At minimum, four experimental groups should be considered: (1) wild-type control (untreated), (2) IL-18 knockout control (untreated), (3) wild-type treated/disease model, and (4) IL-18 knockout treated/disease model . The inclusion of both untreated wild-type and knockout controls is crucial to establish baseline differences attributable solely to IL-18 deficiency. Age and sex matching is critical, as IL-18 knockout mice develop metabolic abnormalities with age . Whenever possible, littermate controls should be used to minimize genetic background variations. For radiation or inflammatory challenge studies, multiple timepoints should be evaluated as IL-18 responses can vary temporally . Additionally, when studying specific pathways influenced by IL-18, consider including positive controls known to activate those pathways independently of IL-18 to demonstrate that downstream signaling remains intact in the knockout mice.

What are the methodological challenges in studying IL-18's role in bone marrow failure?

Studying IL-18's role in bone marrow failure presents several methodological challenges. Researchers have developed murine models for immune-mediated bone marrow failure by infusing allogeneic lymph node cells into sub-lethally irradiated recipients . When investigating IL-18's contribution, it's crucial to distinguish between its direct effects on hematopoietic stem cells versus indirect effects through immune modulation. One challenge is separating radiation-induced IL-18 production from immune-mediated IL-18 production, as radiation itself increases IL-18 levels . Another challenge is determining the cellular sources of IL-18 in the bone marrow microenvironment, which requires techniques like cell sorting followed by cytokine analysis or single-cell RNA sequencing. Additionally, the compensatory relationship between IL-18 and other cytokines means that knockout studies may be confounded by alterations in related pathways. Researchers must also consider the timing of IL-18 involvement, as its contribution may differ during initiation versus progression of bone marrow failure. Finally, translating findings from mouse models to human aplastic anemia requires careful validation, as increased circulating IL-18 is observed in severe aplastic anemia but may be dispensable for the actual disease process .

How can contradictory findings about IL-18 in mouse metabolism be reconciled?

The contradictory findings regarding IL-18's role in mouse metabolism present a fascinating research puzzle. While elevated IL-18 levels correlate with metabolic syndrome and type 2 diabetes in humans, IL-18 or IL-18Rα-deficient mice paradoxically develop obesity and insulin resistance . This apparent contradiction can be reconciled through several explanations. First, IL-18 resistance may develop in obesity and type 2 diabetes, similar to leptin resistance. Evidence shows that peripheral blood mononuclear cells from patients with obesity or type 2 diabetes exhibit decreased IL-18Rα and IL-18Rβ expression, rendering them resistant to IL-18 stimulation despite high circulating levels . Second, increased IL-18 in metabolic disorders likely represents a compensatory mechanism attempting to counteract insulin resistance induced by other inflammatory cytokines. Third, acute versus chronic IL-18 exposure may have different metabolic effects, with chronic elevation potentially triggering adaptive responses that alter receptor expression or downstream signaling. Fourth, tissue-specific IL-18 actions may differ from systemic effects, requiring tissue-specific knockout models for clarification. Finally, developmental compensation in germline knockout models might activate alternative pathways that confound interpretations. Research approaches to resolve these contradictions include conditional tissue-specific knockouts, time-controlled inducible systems, dose-response studies with recombinant IL-18, and investigation of IL-18 receptor regulation and signaling pathway adaptations under different metabolic conditions.

What are the optimal protocols for inducing IL-18-dependent disease models in mice?

Several well-established protocols exist for inducing IL-18-dependent disease models in mice. For atopic dermatitis models, the MC903 (calcipotriol) method involves shaving the dorsal skin surface to expose a 2x3 cm area and applying MC903 (4 nmol) diluted in ethanol topically once daily for 15 consecutive days . Control groups receive an identical volume of ethanol only. For radiation injury models, total-body irradiation at doses ranging from 5-12 Gy induces dose-dependent IL-18 responses, with 9 Gy commonly used to study survival outcomes . For metabolic studies examining IL-18's role, protocols involve either high-fat diet feeding (typically 60% calories from fat for 12-16 weeks) or normal chow feeding for extended periods (6+ months) in IL-18 knockout mice to observe the development of metabolic abnormalities . For bone marrow failure models, infusion of allogeneic lymph node cells (1-5×10^6 cells) into sub-lethally irradiated (5-6 Gy) recipients creates immune-mediated bone marrow destruction that can be analyzed in the context of IL-18 deficiency . Each model requires careful consideration of mouse strain, age, sex, housing conditions, and appropriate controls. Importantly, researchers should validate IL-18 involvement in their specific model by measuring IL-18 levels and responses to ensure the selected protocol appropriately engages the IL-18 pathway.

How should researchers interpret conflicting data on IL-18 function between different mouse strains?

Interpreting conflicting data on IL-18 function between different mouse strains requires careful consideration of several factors. Genetic background significantly influences cytokine responses, immune function, and disease susceptibility. When encountering strain-dependent differences in IL-18 function, researchers should first document strain-specific baseline IL-18 and IL-18BP expression levels, as well as receptor expression patterns. Different strains may have genetic polymorphisms affecting IL-18 production, processing, receptor binding, or downstream signaling. For example, C57BL/6 mice typically show stronger Th1 responses than BALB/c mice, which could impact IL-18-driven pathways . Additionally, compensatory mechanisms might differ between strains, with some strains upregulating alternative cytokines when IL-18 signaling is disrupted. To address these discrepancies, researchers should employ backcrossing to create congenic strains that differ only in the genes of interest, use multiple strains to validate findings, conduct side-by-side comparisons under identical conditions, and consider strain-specific differences when translating findings to human applications. Most importantly, researchers should view strain differences not as obstacles but as opportunities to uncover modifier genes and contextual factors that influence IL-18 biology, potentially revealing new therapeutic targets or personalized medicine approaches.

What techniques best measure the ratio of free versus bound IL-18 in mouse samples?

Accurately measuring the ratio of free (bioactive) versus bound (inactive) IL-18 in mouse samples is crucial for understanding IL-18's biological activity, as only unbound IL-18 can signal through its receptor. The gold standard approach involves a combination of techniques. First, total IL-18 should be measured using a standard ELISA that detects both free and bound forms. In parallel, IL-18BP should be quantified using a specific ELISA or immunoassay. Free IL-18 can then be calculated using the law of mass action based on the known binding affinity between IL-18 and IL-18BP . Alternatively, specialized ELISAs designed to detect only free IL-18 (using antibodies that recognize epitopes masked when IL-18 is bound to IL-18BP) can provide direct measurement. More sophisticated approaches include co-immunoprecipitation to physically separate IL-18/IL-18BP complexes from free IL-18, followed by quantification of each fraction. Functional bioassays that measure IL-18-dependent IFN-γ production by NK or T cells can complement these approaches by assessing biologically active IL-18. When interpreting results, researchers should consider that multiple splice variants of IL-18BP exist with different binding affinities, and other IL-18 binding partners besides IL-18BP may be present in complex biological samples. Standardization of sample collection, processing, and storage is critical, as these factors can affect the equilibrium between free and bound IL-18.

What biomarker potential does IL-18 have in mice compared to established markers?

IL-18 demonstrates considerable biomarker potential in mice, particularly for radiation exposure and inflammatory conditions. In radiation exposure, IL-18 shows a close correlation with established hematological radiation biomarkers including lymphocyte and neutrophil counts in blood of mice, minipigs, and nonhuman primates . The advantage of IL-18 as a radiation biomarker lies in its dose-dependent increase that proportionally reflects radiation severity and its relatively stable elevation (up to 13 days post-exposure), providing a longer detection window than rapidly changing cell counts. In atopic dermatitis models, IL-18 correlates with disease severity measures like SCORAD values, making it a potential biomarker for monitoring disease progression and treatment response . For metabolic conditions, the relationship is more complex, as IL-18 elevation may represent a compensatory response rather than a direct pathogenic factor . When evaluating IL-18 as a biomarker, researchers should consider both total IL-18 and the IL-18/IL-18BP ratio, as the latter better reflects bioactive IL-18 levels. Multiplexed approaches that measure IL-18 alongside related cytokines and established biomarkers provide the most comprehensive assessment. While singularly measured IL-18 may lack specificity for particular conditions, its integration into biomarker panels could enhance diagnostic and prognostic capabilities for various inflammatory and radiation-induced pathologies.

How might research on IL-18 in mice inform therapeutic development?

Research on IL-18 in mouse models has provided crucial insights for therapeutic development targeting this pathway. Perhaps the most promising application stems from studies of IL-18 binding protein (IL-18BP) as a radiation countermeasure. Administration of recombinant human IL-18BP (rhIL-18BP) at 1.5 mg/kg via subcutaneous injection increased 30-day survival of mice after 9 Gy total-body irradiation by 12.5-25% compared to vehicle controls . The therapeutic mechanisms involved neutralizing excess free IL-18, inhibiting downstream IFN-γ expression, decreasing reactive oxygen species, and protecting multiple organs from radiation-induced damage. This suggests IL-18BP could serve as a radiation mitigator in scenarios like nuclear accidents or radiotherapy complications. In atopic dermatitis models, IL-18 knockout mice showed significantly reduced disease severity, indicating that IL-18 inhibition might benefit inflammatory skin conditions . For metabolic disorders, the situation is more complex, as both excess and deficiency of IL-18 signaling can lead to metabolic abnormalities, suggesting that IL-18 pathway modulators rather than complete inhibitors might be needed . When developing IL-18-targeted therapeutics, researchers should consider temporal aspects of treatment (preventive vs. therapeutic), tissue-specific delivery methods, and potential compensatory mechanisms. The successful translation of mouse findings to larger animals (nonhuman primates and minipigs) for IL-18BP radiation countermeasures provides a promising model for developing other IL-18-targeted therapeutics.