IL36A 153 a.a. Mouse

What is IL36A 153 a.a. Mouse and what is its structural composition?

IL36A 153 a.a. Mouse is a recombinant protein belonging to the IL-1 cytokine family, produced as a single, non-glycosylated polypeptide chain containing 153 amino acids (residues 8-160) with a molecular mass of 17.0kDa. It lacks signal sequence, prosegment, and potential N-linked glycosylation sites. The protein is produced in E. coli expression systems and purified using proprietary chromatographic techniques . Like other IL-1 family members, it displays a characteristic 12 β-strand, β-trefoil configuration and is thought to have evolved from a mutual ancestral gene .

How does mouse IL36A compare to human IL36A?

Mouse and human IL36A share 54% amino acid sequence homology. This moderate level of conservation suggests functional similarities between species while highlighting potential differences in specific activities or potencies. When designing translational studies, researchers should consider these interspecies variations that might affect experimental outcomes and interpretations .

What is the tissue distribution pattern of IL36A in mice?

IL36A is predominantly expressed in skin and lymphoid tissues, but significant expression has also been detected in fetal brain, trachea, stomach, and intestine. This distribution pattern suggests tissue-specific roles in both immune function and epithelial barrier tissues. The protein exists both as an intracellular molecule and as a secreted factor, released in response to specific stimuli like lipopolysaccharide (LPS) and ATP-induced activation of the P2X7 receptor .

What is the optimal method for reconstituting lyophilized IL36A 153 a.a. Mouse?

For optimal reconstitution, the lyophilized protein should be dissolved in sterile 18MΩ-cm H₂O to a concentration of not less than 100μg/ml. Allow complete dissolution through gentle swirling rather than vigorous shaking, which may cause protein denaturation. Once reconstituted, the solution can be further diluted in appropriate aqueous buffers for experimental applications. The reconstituted protein should be aliquoted to avoid repeated freeze-thaw cycles, which can compromise biological activity .

How stable is IL36A 153 a.a. Mouse under different storage conditions?

While lyophilized IL36A is stable at room temperature for approximately three weeks, long-term storage at -20°C or -80°C is recommended to maintain maximum activity. The recombinant protein is typically formulated with 5% trehalose as a cryoprotectant to enhance stability during freeze-drying and reconstitution processes. Once reconstituted, the protein should be used immediately or stored in single-use aliquots at -80°C to prevent activity loss from repeated freeze-thaw cycles .

What are the molecular mechanisms through which IL36A induces neutrophil infiltration in the lungs?

IL36A induces neutrophil infiltration through several coordinated mechanisms:

• Chemokine induction: IL36A significantly upregulates the expression of neutrophil-specific chemokines CXCL1 and CXCL2 at both mRNA and protein levels in lung tissue .

• Cytokine cascade activation: Intratracheal instillation of IL36A triggers increased expression of early response cytokines including TNFα, IL-1α, and IL-1β, which amplify the inflammatory cascade .

• Receptor upregulation: IL36A induces the expression of its own receptor (IL-36R) in lung tissue, potentially creating a positive feedback loop that enhances and sustains inflammatory signaling .

• Direct effects on CD11c+ cells: IL36A directly stimulates CD11c+ cells to produce neutrophil chemokines and increases the expression of co-stimulatory molecules like CD40, enhancing their ability to activate T cells .

• NF-κB pathway activation: IL36A activates the NF-κB signaling pathway in macrophages, a key transcriptional program controlling inflammatory gene expression .

Flow cytometric analysis of bronchoalveolar lavage fluid following IL36A administration confirms that the majority of infiltrating cells are CD11c⁻CD11b⁺Ly6G⁺ neutrophils, with histological examination showing significant inflammatory cell recruitment to lung tissue .

How do IL36A-induced inflammatory responses differ between wild-type mice and those lacking IL-1α/β?

Comparative studies between wild-type C57BL/6 mice and IL-1αβ⁻/⁻ mice have revealed both similarities and differences in IL36A-induced responses:

The ability of IL36A to induce robust neutrophil recruitment and pro-inflammatory mediator expression in IL-1αβ⁻/⁻ mice demonstrates that IL36A can act independently of classical IL-1 cytokines . This functional redundancy has important implications for therapeutic approaches targeting inflammatory lung diseases, suggesting that inhibition of IL-1 alone may be insufficient to suppress inflammation driven by IL-36 family cytokines.

What experimental approaches can effectively measure IL36A-induced activation of immune cells?

Several methodological approaches have proven valuable for assessing IL36A effects on immune cell activation:

• Flow cytometric analysis: Multi-parameter flow cytometry using markers such as CD11c, CD11b, Ly6G, and CD40 allows for identification and characterization of specific cell populations responding to IL36A. This approach can be used to analyze cells from bronchoalveolar lavage fluid or single-cell suspensions of lung tissue .

• Ex vivo CD11c+ cell stimulation: Isolation of CD11c+ cells (dendritic cells and macrophages) followed by stimulation with defined concentrations of recombinant IL36A allows for direct assessment of cytokine/chemokine production and surface marker expression changes .

• T cell co-culture assays: Co-culturing IL36A-stimulated CD11c+ cells with CD4+ T cells provides a functional readout of antigen-presenting cell activation through measurement of T cell proliferation .

• Quantitative RT-PCR: Measurement of mRNA expression of cytokines, chemokines, and receptors in whole tissue or isolated cell populations provides sensitive detection of transcriptional changes induced by IL36A .

• NF-κB activation assays: Using reporter cell lines or biochemical approaches such as phospho-IκB detection can assess IL36A-induced activation of this key signaling pathway .

These complementary approaches enable comprehensive characterization of IL36A effects on immune cell activation and inflammatory responses.

How does IL36A self-regulate through modulation of its receptor expression?

A significant finding in IL36A research is its ability to induce the expression of its own receptor (IL-36R), creating a potential positive feedback loop:

• Intratracheal instillation of IL36A significantly increases IL-36R mRNA expression in lung tissue of both wild-type and IL-1αβ⁻/⁻ mice, while expression of the classical IL-1 receptor (IL-1RI) remains unchanged .

• This receptor upregulation occurs independently of IL-1α and IL-1β, as demonstrated in experiments using IL-1αβ⁻/⁻ mice .

• The mechanism may involve direct action of IL36A on structural or immune cells in the lung, or indirect effects through other induced mediators .

• This self-amplifying circuit potentially enhances tissue sensitivity to subsequent IL36A stimulation, creating a feed-forward amplification loop that could contribute to persistent inflammation .

• Targeting this regulatory mechanism could provide novel therapeutic approaches for controlling IL-36-mediated inflammation in pathological conditions.

What are the methodological considerations when designing in vivo experiments with IL36A in mouse models?

When designing in vivo experiments with IL36A, researchers should consider the following methodological aspects:

• Dosage optimization: Studies have shown that 10μg of recombinant IL36A administered intratracheally induces robust neutrophil infiltration and cytokine responses. Dose-response experiments should be conducted to determine optimal concentrations for specific experimental endpoints .

• Time course analysis: Peak neutrophil recruitment and cytokine/chemokine expression typically occur 24 hours after IL36A administration, but temporal dynamics may vary for different parameters .

• Endotoxin contamination control: Since IL36A is typically produced in E. coli, ensuring removal of endotoxin contamination is crucial. Using polymyxin treatment or including controls with LPS-unresponsive models (e.g., C3H/HeJ mice) helps distinguish IL36A-specific effects from those potentially caused by endotoxin contamination .

• Delivery method: For lung studies, intratracheal instillation provides direct delivery to the airways. Alternative routes such as intranasal or aerosolized delivery may be considered depending on the research question .

• Appropriate controls: Include vehicle controls (PBS with carrier proteins) and, where relevant, heat-inactivated protein controls to confirm specificity of observed effects .

• Complementary readouts: Combine multiple analytical approaches, including flow cytometry, histology, gene expression analysis, and protein quantification, to comprehensively characterize IL36A-induced responses .

What is the potential significance of IL36A research for inflammatory lung disease models?

IL36A research has several important implications for inflammatory lung disease models:

• Novel therapeutic target identification: The pro-inflammatory effects of IL36A in the lungs suggest that targeting this cytokine or its receptor could be beneficial in conditions characterized by excessive neutrophilic inflammation .

• Understanding mechanism redundancy: The finding that IL36A can induce neutrophil infiltration independently of IL-1α and IL-1β highlights redundancy in inflammatory pathways, potentially explaining why targeting individual cytokines sometimes produces limited clinical benefits .

• Biomarker development: IL36A or IL-36R expression levels could potentially serve as biomarkers for specific inflammatory phenotypes or for predicting response to certain therapies.

• Modeling neutrophilic inflammation: IL36A administration provides a model system for studying mechanisms of neutrophil recruitment and activation relevant to conditions like acute respiratory distress syndrome, severe asthma with neutrophilic inflammation, and certain infectious lung diseases .

• Understanding cytokine networks: IL36A research contributes to mapping the complex interactions within the IL-1 and IL-36 cytokine networks, including feedback mechanisms and compensatory pathways that maintain inflammatory responses even when individual components are blocked .

How do the structural features of IL36A 153 a.a. Mouse influence its functional activity?

The structural characteristics of IL36A 153 a.a. Mouse have direct implications for its functional activity:

• Amino acid composition: The recombinant protein contains amino acids 8-160 of the full-length protein, indicating that the N-terminal 7 amino acids and C-terminal portion beyond residue 160 are not included. Research suggests that specific N-terminal processing of IL-36 cytokines significantly enhances their biological activity, potentially affecting potency in experimental systems .

• Lack of glycosylation: As a bacterial expression product, the recombinant protein lacks post-translational modifications such as glycosylation that might be present in the native mouse protein. This could affect protein stability, receptor binding, or biological half-life in experimental systems .

• β-trefoil structure: The 12 β-strand, β-trefoil configuration characteristic of IL-1 family members is critical for receptor binding and biological activity. This structural feature is preserved in the recombinant protein .

• Solubility properties: The non-glycosylated nature of the recombinant protein influences its solubility characteristics, necessitating specific reconstitution procedures to maintain biological activity .

• Stability considerations: The lyophilized formulation with trehalose helps maintain protein stability during storage, but researchers should be aware that improper reconstitution or storage could affect structural integrity and thus functional activity .

Understanding these structure-function relationships is essential for proper experimental design and interpretation of results when using IL36A 153 a.a. Mouse in research applications.