Recombinant Mouse Interleukin-10 (Il10) (Active)

What is the molecular structure of mouse IL-10 and how does it compare to human IL-10?

Mouse IL-10 is a 178 amino acid molecule containing two intrachain disulfide bridges and is expressed as a 36 kDa non-covalently associated homodimer. The protein exists as a dimer that binds to IL-10 receptor chains, resulting in recruitment of additional receptor chains and activation of a signaling cascade involving JAK1, TYK2, and STAT3 . Mouse IL-10 shares 85% amino acid sequence identity with rat IL-10 and 71%-79% with other mammalian species including human IL-10 . A critical species-specific difference is that while human IL-10 can act on mouse cells, mouse IL-10 does not effectively interact with human IL-10 receptors and therefore cannot exert biological effects on human cells .

What are the key biological functions of mouse IL-10 in experimental systems?

Mouse IL-10, also known as cytokine synthesis inhibitory factor (CSIF), functions as a critical immunoregulatory molecule that controls inflammatory responses. In experimental systems, IL-10 primarily:

• Suppresses antigen presentation and Th1 proinflammatory responses

• Promotes phagocytic activity and Th2 responses

• Controls viral infections and allergic/autoimmune inflammation

• Inhibits the production of proinflammatory cytokines

In IL-10 knockout mouse models, these animals show high susceptibility to endotoxic shock due to overexpression of proinflammatory cytokines, and reconstitution with IL-10 can rescue them from LPS-induced lethality, demonstrating its crucial role in regulating inflammatory responses .

How is recombinant mouse IL-10 typically produced for research applications?

Recombinant mouse IL-10 is typically expressed in E. coli expression systems. The protein consists of amino acids 19-178 of the native sequence, with serine at position 19 as the N-terminal amino acid . After expression, the protein is purified through multiple chromatographic steps to ensure high purity. The lyophilized product is typically prepared from a 0.2 μm filtered solution in PBS . The bioactivity of recombinant mouse IL-10 is routinely measured using cell proliferation assays with MC/9-2 mouse mast cells, with effective doses (ED50) typically ranging from 0.12-0.72 ng/mL .

What are the optimal storage and handling conditions for maintaining recombinant mouse IL-10 activity?

To maintain the biological activity of recombinant mouse IL-10:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution in sterile water or buffer, aliquot the solution to minimize freeze-thaw cycles

• Reconstituted protein should be stored at -20°C for short-term use (1-2 weeks)

• Avoid repeated freeze-thaw cycles as they can significantly reduce protein activity

• For working solutions, maintain protein concentration above 10 μg/mL and include carrier protein (0.1-1% BSA) to prevent adsorption to plastic surfaces

• When designing experiments, account for potential loss of activity during storage and handling by conducting dose-response studies

Dimerization is critical for IL-10's biological function, and improper handling can disrupt the dimer structure, significantly reducing activity .

How should dose-response experiments be designed when studying IL-10's immunosuppressive effects?

When designing dose-response experiments with recombinant mouse IL-10:

• Begin with a broad concentration range (0.01-100 ng/mL) to establish the full response curve

• Include biological replicates (n≥3) to account for variability in cell responsiveness

• Establish appropriate time points for analysis, as IL-10 effects may vary temporally

• Include positive controls (such as dexamethasone) to benchmark immunosuppressive activity

• Measure multiple readouts, including:

  • STAT3 phosphorylation (early response)

  • Suppression of proinflammatory cytokine production (IL-1β, TNF-α, IL-6)

  • Changes in gene expression of IL-10-responsive genes

  • Functional outcomes in relevant cell types

The ED50 for many IL-10 biological effects typically falls in the range of 0.1-1 ng/mL, though this varies by experimental system and readout .

How do engineered IL-10 dimers with flexible linkers compare to natural IL-10 in experimental systems?

Engineered stable IL-10 dimers created by connecting two IL-10 monomers with flexible Gly-Ser linkers offer several advantages over natural IL-10:

• Enhanced stability: The covalently linked dimers show improved resistance to denaturation and dissociation compared to non-covalently linked natural IL-10 .

• Improved biological activity: Studies have demonstrated that stable IL-10 dimers can have enhanced biological activity as measured by ED50 values in functional assays. For instance, natural mouse IL-10 (Nm RRCHR) shows an ED50 of approximately 0.1 ng/mL, while some engineered variants have differing potencies depending on specific modifications to the RRCHR region .

• Maintained signaling properties: Despite structural modifications, engineered IL-10 dimers retain the ability to activate the IL-10 receptor complex and induce STAT3 phosphorylation, though possibly with altered dose-response characteristics .

• Potential therapeutic advantages: The improved stability and activity of engineered IL-10 dimers make them attractive candidates for future IL-10-based immunotherapy regimens .

When designing experiments comparing natural and engineered IL-10 variants, researchers should include comprehensive dose-response studies and multiple readouts of IL-10 activity.

What considerations are important when using mouse models to study human IL-10 biology?

When using mouse models to study human IL-10 biology, researchers should consider several important factors:

• Species-specific activity: Human IL-10 is biologically active in mouse cells, but mouse IL-10 does not act on human cells. This unidirectional cross-species activity creates unique opportunities for studying human IL-10 in mouse models .

• Transgenic models: Human IL-10 BAC transgenic mice represent valuable tools for studying human IL-10 regulation. These models contain the human IL-10 gene positioned centrally within its natural genomic context, allowing for assessment of tissue-specific and stimulus-specific expression patterns .

• Cell type-specific regulation: Human and mouse IL-10 may be differentially regulated in specific cell populations. For example, in human IL-10 BAC transgenic mice, IL-27 strongly induces mouse IL-10 in CD4+ T cells but has minimal effects on human IL-10 expression in the same cells, suggesting distinct regulatory mechanisms .

• Copy number independence: In transgenic models, human IL-10 expression appears to be regulated similarly regardless of transgene copy number, suggesting robust regulation by the surrounding genomic elements rather than a simple gene-dosage effect .

• Disease model relevance: The utility of human IL-10 transgenic mice may vary by disease model. For example, these mice effectively regulate LPS-induced inflammatory responses but may not recapitulate IL-10-dependent responses in specific infection models such as Leishmania donovani .

How can researchers effectively measure IL-10 signaling pathway activation in experimental systems?

To effectively measure IL-10 signaling pathway activation:

• STAT3 phosphorylation: The primary downstream mediator of IL-10 signaling is STAT3. Researchers should assess phosphorylation at Tyr705 using:

  • Western blotting with phospho-specific antibodies

  • Flow cytometry for single-cell resolution

  • ELISA-based phospho-protein detection

  • In-cell Western assays for high-throughput applications

• Dose and time course considerations: IL-10 signaling is dynamic, with peak STAT3 phosphorylation typically occurring 15-30 minutes after stimulation. Comprehensive dose-response (0.01-100 ng/mL) and time-course (5 minutes to 24 hours) experiments should be conducted .

• Receptor expression analysis: IL-10 signals through a heterodimeric receptor complex consisting of IL-10Rα and IL-10Rβ chains. Assessment of receptor expression by flow cytometry or qPCR can help interpret variable responses to IL-10 across different cell types .

• Target gene expression: Measure expression of IL-10-responsive genes such as SOCS3, IL-1RA, and various anti-inflammatory mediators by qPCR or protein detection methods .

• Functional readouts: Complement signaling measurements with functional assays, such as suppression of LPS-induced TNF-α production in macrophages or modulation of T cell differentiation .

What techniques are most effective for validating recombinant IL-10 bioactivity?

To comprehensively validate recombinant mouse IL-10 bioactivity:

• Proliferation assays: The gold standard assay uses MC/9-2 mouse mast cells, which proliferate in response to IL-10. This assay typically yields ED50 values of 0.12-0.72 ng/mL for bioactive mouse IL-10 .

• Cytokine suppression assays: Measure IL-10's ability to suppress LPS-induced TNF-α, IL-6, or IL-1β production in macrophages or dendritic cells. This functional readout directly assesses IL-10's anti-inflammatory properties .

• STAT3 phosphorylation: Quantify the induction of STAT3 phosphorylation in responsive cells like macrophages, dendritic cells, or B cells using Western blotting or flow cytometry .

• Gene expression analysis: Measure the induction of IL-10-responsive genes such as SOCS3 or the suppression of proinflammatory genes using qPCR or RNA-seq approaches .

• In vivo validation: In more advanced settings, recombinant IL-10 can be validated by its ability to rescue IL-10-deficient mice from LPS-induced lethality or ameliorate inflammatory conditions in appropriate animal models .

For each validation approach, include both positive controls (commercially validated IL-10) and negative controls (heat-inactivated IL-10 or irrelevant cytokines) to ensure specificity.

How can researchers effectively study the tissue-specific regulation of IL-10 expression?

To effectively study tissue-specific regulation of IL-10 expression:

• Reporter systems: Utilize IL-10 promoter-reporter constructs or IL-10-GFP knock-in mice to visualize IL-10 expression patterns in different tissues and cell types.

• BAC transgenic models: Human IL-10 BAC transgenic mice (hIL10BAC) provide valuable tools for studying the tissue-specific control of IL-10 expression. These mice contain the human IL-10 gene in its natural genomic context, allowing for faithful recapitulation of tissue-specific regulatory elements .

• Cell-specific analysis: Isolate specific cell populations (macrophages, dendritic cells, T cells, B cells, etc.) from different tissues to compare IL-10 expression patterns and regulatory mechanisms.

• Stimulus-specific responses: Compare IL-10 induction across different stimuli, such as LPS, IL-4, IFN-γ, or IL-27, as these may reveal cell type-specific regulatory pathways. For example, in bone marrow-derived macrophages (BMMs), LPS induces both mouse and human IL-10, while IFN-γ inhibits IL-10 expression .

• Epigenetic analysis: Examine chromatin accessibility (ATAC-seq), histone modifications (ChIP-seq), and DNA methylation patterns at the IL-10 locus across different tissues and cell types to identify regulatory elements.

• Transcription factor binding: Determine the tissue-specific transcription factors that regulate IL-10 expression through ChIP-seq or reporter assays with mutated binding sites .

• Cross-species comparison: Compare the regulation of mouse and human IL-10 within the same cellular context, as this can reveal conserved and divergent regulatory mechanisms .

How should researchers address variability in IL-10 responses across different experimental systems?

When addressing variability in IL-10 responses:

• Cellular receptor expression: Quantify IL-10 receptor expression levels on target cells, as this can significantly impact responsiveness. Flow cytometry or qPCR assessment of IL-10Rα and IL-10Rβ expression can help explain variable responses.

• Signaling pathway integrity: Verify the functionality of downstream signaling components, particularly JAK1, TYK2, and STAT3, as defects in these pathways can alter IL-10 responsiveness.

• Protein quality: Ensure recombinant IL-10 maintains its dimeric structure, as monomeric IL-10 has significantly reduced activity. Size exclusion chromatography or native PAGE can assess dimer formation.

• Dose-response characterization: Establish complete dose-response curves (0.01-100 ng/mL) for each experimental system, as the window of effective concentration may vary across different cell types and readouts.

• Cell activation state: The responsiveness to IL-10 can depend on the activation state of target cells. Pre-activation with appropriate stimuli (e.g., LPS for macrophages) may be necessary to observe robust IL-10 effects.

• Cross-species considerations: Remember that human IL-10 is active on mouse cells, but mouse IL-10 does not act on human cells. This species-specific activity must be considered when designing experiments and interpreting results .

• Tissue-specific regulation: Different cell types may exhibit distinct regulatory mechanisms for IL-10 expression and responsiveness. For example, IL-27 strongly induces mouse IL-10 in CD4+ T cells but has minimal effects on human IL-10 expression in the same cells from hIL10BAC mice .

What are the critical considerations when comparing data from different IL-10 variants or engineered constructs?

When comparing data from different IL-10 variants or engineered constructs:

• Structural integrity: Verify that each variant maintains appropriate folding and dimerization using circular dichroism spectroscopy, size exclusion chromatography, or native PAGE.

• Standardized bioactivity assays: Use consistent, well-characterized bioassays to determine relative potencies. For example, the MC/9-2 cell proliferation assay provides quantitative ED50 values that can be directly compared across variants .

• Dose normalization: Compare constructs based on molar concentrations rather than mass to account for differences in molecular weight, particularly when comparing natural dimers to engineered fusion proteins.

• Multiple readouts: Assess activity using multiple independent methods (e.g., STAT3 phosphorylation, cytokine suppression, and gene expression) to develop a comprehensive activity profile.

• Pharmacokinetic differences: Consider potential differences in protein stability, half-life, and tissue distribution when interpreting in vivo data.

• Structure-function analysis: For engineered variants with specific mutations (e.g., in the RRCHR region), correlate structural changes with functional outcomes to understand the molecular basis for altered activity .

• Statistical analysis: Use appropriate statistical methods to determine whether observed differences between variants are significant, and report effect sizes along with p-values.

For example, studies comparing natural mouse IL-10 (Nm RRCHR) with variants containing substitutions in the RRCHR region showed that these modifications can significantly alter bioactivity, with ED50 values ranging from 0.1 ng/mL for the unmodified protein to 59.28 ng/mL for certain variants (Nm ARCHA) .