Recombinant Human Interleukin-31 protein (IL31) (Active)

What expression systems are commonly used for producing Recombinant Human IL-31?

Researchers have successfully produced Recombinant Human IL-31 using both eukaryotic and prokaryotic expression systems, each with distinct advantages depending on the intended application:

For eukaryotic expression, the recombinant protein is typically secreted into the culture medium and can be purified to ≥95% purity . For prokaryotic systems, the protein is usually expressed as inclusion bodies that require solubilization and refolding, followed by purification using techniques such as size-exclusion chromatography . The choice between these systems depends on the experimental requirements for protein authenticity, yield, and downstream applications.

How can researchers validate the identity and purity of Recombinant Human IL-31?

Validation of recombinant IL-31 identity and purity involves multiple analytical techniques:

• SDS-PAGE analysis: Should demonstrate a single band at approximately 15-16 kDa with ≥95% purity .

• Mass spectrometry (MS): ESI-TOF analysis should confirm a molecular weight close to the theoretical value (approximately 15.57 kDa for the mature protein) .

• High-Performance Liquid Chromatography (HPLC): Should show a single major peak, confirming homogeneity of the preparation .

• Circular dichroism (CD): Can verify proper secondary structure, predominantly alpha-helical configuration as expected for this cytokine family .

• Endotoxin testing: For applications involving cell culture or in vivo experiments, endotoxin levels should be below 0.005 EU/μg as determined by the LAL method .

• Functional validation: Binding to recombinant IL-31 receptor alpha (IL-31RA) and oncostatin M receptor (OSMR) should be demonstrated, along with activation of downstream signaling pathways (particularly STAT3 phosphorylation) .

What receptors does IL-31 interact with and what signaling pathways does it activate?

IL-31 signals through a heterodimeric receptor complex consisting of IL-31 receptor alpha (IL-31RA, also known as GPL or GLMR) and Oncostatin M receptor beta (OSMR-β) . This receptor complex is expressed on various cell types, with constitutive expression on keratinocytes and inducible expression on monocytes following interferon-gamma stimulation .

Upon binding to its receptor complex, IL-31 activates multiple signaling pathways:

• JAK-STAT pathway: IL-31 strongly activates STAT3 and STAT5, and can also activate STAT1 to a lesser extent . This activation occurs through the recruitment and phosphorylation of Janus kinases (primarily JAK1 and JAK2) .

• Other pathways: Depending on the cell type, IL-31 signaling may also involve activation of PI3K/AKT and MAPK pathways, contributing to its diverse cellular effects.

The binding affinity of recombinant human IL-31 to its receptor has been characterized, with an EC50 value of approximately 16.36 μg/mL in ELISA assays using recombinant human IL-31RA fused with human Fc fragment (rhIL-31RA-hFc) . Notably, recombinant IL-31 can bind independently to either IL-31RA or OSMR-β expressed on cell surfaces, although physiological signaling requires the heterodimeric complex .

How can researchers effectively measure IL-31-induced STAT phosphorylation?

To assess IL-31-induced STAT phosphorylation, researchers should follow these methodological guidelines:

• Cell selection: Choose a cell line that expresses both components of the IL-31 receptor complex (IL-31RA and OSMR). A549 cells have been validated for this purpose and show robust STAT3 phosphorylation in response to IL-31 stimulation . Primary cells expressing the receptor complex (such as keratinocytes) may also be used.

• Stimulation protocol:

  • Serum-starve cells for 4-6 hours prior to stimulation to reduce baseline STAT phosphorylation

  • Treat cells with recombinant IL-31 at concentrations ranging from 10-100 ng/mL

  • Include a time course (typically 5, 15, 30, 60 minutes) to capture the kinetics of phosphorylation

• Detection methods:

  • Western blotting using phospho-specific antibodies against p-STAT3 (Tyr705), p-STAT5 (Tyr694), and p-STAT1 (Tyr701)

  • Flow cytometry using fluorescently-labeled phospho-STAT antibodies

  • Phospho-ELISA kits specifically designed for STAT phosphorylation detection

• Controls:

  • Positive control: Cells treated with a known STAT activator (e.g., IL-6 for STAT3)

  • Negative control: Unstimulated cells

  • Specificity control: Pre-treatment with JAK inhibitors (e.g., ruxolitinib) should abolish IL-31-induced STAT phosphorylation

• Quantification: Normalize phospho-STAT signals to total STAT protein levels to account for potential variations in protein expression between samples.

The peak of STAT3 phosphorylation typically occurs within 15-30 minutes of IL-31 stimulation, with subsequent decline due to negative feedback mechanisms .

How does IL-31 contribute to fibrosis development and what experimental models are appropriate for studying this function?

IL-31 has been implicated in fibrotic processes, particularly in the context of systemic sclerosis (SSc) . The mechanisms through which IL-31 promotes fibrosis include:

• Direct effects on fibroblasts: IL-31 significantly increases collagen production in dermal fibroblasts, with particularly pronounced effects in fibroblasts isolated from SSc patients .

• Cytokine modulation: IL-31 upregulates the expression of pro-fibrotic cytokines, including IL-4, IL-6, IL-10, and TGF-β1, while having minimal effects on Th1 cytokines like IFN-γ or Th17 cytokines such as IL-17A .

• Extracellular matrix remodeling: IL-31 decreases the expression of matrix metalloproteinases (MMPs) including MMP3, MMP9, and MMP13, while increasing tissue inhibitors of metalloproteinases (TIMPs) such as TIMP1, TIMP2, and TIMP3, thereby promoting collagen accumulation .

Experimental models for studying IL-31 in fibrosis:

In the BLM-SSc mouse model, administration of recombinant mouse IL-31 significantly exacerbates skin and lung fibrosis, with increased expression of Col1a1, Col1a2, and type I collagen compared to controls . This effect is more pronounced in bleomycin-treated mice than in PBS-treated control mice, suggesting synergy between IL-31 and existing pro-fibrotic pathways .

What is the role of IL-31 in immune regulation and inflammatory skin conditions?

IL-31 plays a significant role in immune regulation, particularly in the context of skin inflammation and T helper 2 (Th2) immune responses:

• Cellular source: IL-31 is predominantly produced by activated T cells, with preferential expression by Th2 cells . This selective expression pattern links IL-31 to allergic and inflammatory conditions characterized by Th2 polarization.

• Pruritus induction: IL-31 is a potent inducer of pruritus (itching), as demonstrated in transgenic mice overexpressing IL-31, which develop severe pruritus and dermatitis . This function is particularly relevant to inflammatory skin conditions like atopic dermatitis.

• Effects on keratinocytes: Keratinocytes constitutively express the IL-31 receptor complex and respond to IL-31 stimulation with altered gene expression profiles, contributing to skin barrier dysfunction and inflammation .

• Myeloid effects: IL-31 enhances myeloid progenitor cell survival in vitro and induces RETNLA and serum amyloid A protein expression in macrophages, suggesting a role in regulating innate immune responses .

• Th2 polarization: IL-31 promotes the upregulation of Th2 cytokines (IL-4, IL-10) in multiple tissues, reinforcing Th2-dominant immune responses .

Research applications for studying IL-31 in skin inflammation:

• Transgenic models: IL-31 transgenic mice develop spontaneous dermatitis and pruritus, providing a model for studying IL-31-driven skin inflammation.

• Receptor blockade studies: Antibodies targeting IL-31 or its receptor components can be used to assess the therapeutic potential of interrupting IL-31 signaling in inflammatory conditions.

• Ex vivo skin models: Human skin explants treated with recombinant IL-31 can serve as translational models for studying the effects of IL-31 on human tissue.

• Cytokine profiling: Analysis of IL-31 levels in patient samples (serum, skin biopsies) can provide insights into its role in various inflammatory conditions. Significantly elevated serum IL-31 levels have been observed in SSc patients compared to healthy controls, with higher levels in diffuse cutaneous SSc than in limited cutaneous SSc .

What are the recommended protocols for using Recombinant Human IL-31 in cell culture experiments?

When incorporating recombinant human IL-31 into cell culture experiments, researchers should follow these methodological guidelines:

Reconstitution and Storage:

• Reconstitute lyophilized IL-31 in sterile buffer (typically PBS containing 0.1% BSA) to a stock concentration of 100-500 μg/mL

• Allow the protein to fully dissolve (15-30 minutes at room temperature with gentle agitation)

• Prepare working aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -20°C to -80°C for long-term storage, with stability for at least 6-12 months under proper conditions

Experimental Design Considerations:

Methodological Notes:

• Cell types: Ensure cells express the IL-31 receptor complex (IL-31RA and OSMR). A549 cells, dermal fibroblasts, and keratinocytes are validated models .

• Incubation conditions:

  • For signaling studies: Short-term incubation (5 minutes to 2 hours)

  • For gene expression studies: Medium-term incubation (4-24 hours)

  • For functional studies (collagen production, etc.): Long-term incubation (24-72 hours)

• Handling precautions: Recombinant IL-31 is an active protein that may elicit biological responses in vivo; handle with appropriate precautions, particularly for preparations with low endotoxin levels intended for in vivo use .

• Endotoxin considerations: For sensitive applications, use preparations with certified low endotoxin levels (<0.005 EU/μg) to avoid confounding inflammatory responses .

How can researchers design experiments to study cross-talk between IL-31 and other cytokine signaling pathways?

Investigating the cross-talk between IL-31 and other cytokine signaling pathways requires careful experimental design:

• Co-stimulation experiments:

  • Treat cells with IL-31 alone, another cytokine alone (e.g., IL-4, IL-13, TGF-β), or both cytokines together

  • Compare the effects on downstream signaling (STAT phosphorylation, gene expression) to identify synergistic, additive, or antagonistic interactions

  • Include time course analyses to detect temporal effects in signaling cross-talk

• Sequential stimulation:

  • Pre-treat cells with one cytokine before adding IL-31, or vice versa

  • This approach can reveal priming effects or receptor desensitization mechanisms

• Receptor expression analysis:

  • Examine whether IL-31 modulates the expression of receptors for other cytokines, and vice versa

  • Methods include qPCR for receptor mRNA and flow cytometry for surface receptor protein

• Signaling pathway inhibition:

  • Use specific inhibitors of signaling pathways (JAK inhibitors, MAPK inhibitors, etc.) to dissect the contributions of each pathway to observed effects

  • siRNA knockdown of specific signaling components can provide more specific pathway inhibition

• Transcriptomic analysis:

  • Compare gene expression profiles induced by IL-31, other cytokines, and combinations thereof

  • This approach can identify unique and shared gene signatures and potential pathway convergence points

For example, researchers studying IL-31's relationship with TGF-β signaling in fibrosis should examine whether these cytokines synergistically enhance collagen production and if either cytokine regulates the expression or signaling capacity of the other's receptor .

What are the differences in experimental approaches when studying IL-31 in human versus mouse models?

Working with IL-31 across species requires awareness of important differences:

Key methodological considerations:

• In vitro studies with human cells: Use human recombinant IL-31 protein for experiments with human cell lines or primary cells .

• Mouse model experiments: Use mouse recombinant IL-31 for in vivo administration to mice. The BLM-SSc mouse model has been validated for studying IL-31's pro-fibrotic effects .

• Cross-species validation: When translating findings between species, validate that the observed mechanisms are conserved. This may involve parallel experiments in both human and mouse systems or careful selection of conserved readouts.

• Binding and functional assays: Due to the limited sequence homology, binding assays and functional studies should use species-matched components (i.e., human IL-31 with human receptors, mouse IL-31 with mouse receptors) to ensure physiologically relevant results.

How can researchers quantitatively assess IL-31's effects on extracellular matrix remodeling?

To comprehensively evaluate IL-31's impact on extracellular matrix (ECM) remodeling, researchers should employ multiple complementary approaches:

• Collagen production assessment:

  • mRNA quantification: qRT-PCR for collagen genes (COL1A1, COL1A2, COL3A1) with careful normalization to stable housekeeping genes

  • Protein quantification: Western blot, ELISA, or hydroxyproline assay for collagen protein levels in cell culture supernatants or tissue lysates

  • In situ visualization: Immunofluorescence or immunohistochemistry with collagen-specific antibodies

• Matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) expression:

  • mRNA analysis: qRT-PCR for multiple MMPs (MMP1, MMP3, MMP9, MMP13) and TIMPs (TIMP1, TIMP2, TIMP3)

  • Protein levels: ELISA or Western blot for secreted MMPs and TIMPs

  • Activity assays: Zymography to measure functional MMP activity

• ECM turnover dynamics:

  • Collagen degradation assays: Using fluorescently labeled collagen substrates

  • Matrix contraction assays: 3D collagen gel contraction by fibroblasts following IL-31 treatment

  • Real-time assessment: Live cell imaging of ECM remodeling

• Tissue-level analysis in animal models:

  • Histological staining: Masson's trichrome or picrosirius red for collagen visualization

  • Quantitative histomorphometry: Digital image analysis of stained sections

  • Second harmonic generation microscopy: Label-free imaging of collagen fibrils

In studies of IL-31's effects on ECM remodeling in the BLM-SSc mouse model, researchers observed that administration of recombinant IL-31 significantly decreased the mRNA levels of MMP3, MMP9, and MMP13 while increasing TIMP1, TIMP2, and TIMP3 expression in lung tissue . This altered MMP/TIMP balance favors collagen accumulation and is consistent with the observed enhancement of bleomycin-induced fibrosis in this model .

What are common challenges when working with Recombinant Human IL-31 and how can researchers address them?

Researchers working with recombinant IL-31 may encounter several technical challenges:

• Protein stability issues:

  • Challenge: Loss of activity during storage or experimental handling

  • Solution: Store reconstituted protein in small single-use aliquots at -80°C; add carrier protein (0.1% BSA) to dilute solutions; avoid repeated freeze-thaw cycles

• Batch-to-batch variability:

  • Challenge: Different protein preparations may show varying activity levels

  • Solution: Perform functional validation of each new batch; include internal controls for normalization; consider using the same batch for entire experimental series

• Endotoxin contamination:

  • Challenge: Bacterial expression systems may introduce endotoxin, confounding inflammatory readouts

  • Solution: Use low-endotoxin preparations (<0.005 EU/μg); include polymyxin B controls in sensitive assays; consider mammalian expression systems for critical applications

• Receptor expression heterogeneity:

  • Challenge: Variable expression of IL-31RA and OSMR among cell populations

  • Solution: Verify receptor expression before experiments; sort cells for homogeneous receptor expression; consider receptor transfection for mechanistic studies

• Non-specific binding:

  • Challenge: High concentrations of IL-31 may cause non-physiological effects

  • Solution: Perform dose-response studies; include competitive binding controls; validate with receptor blocking antibodies

• Limited detection sensitivity:

  • Challenge: Difficulty detecting low levels of endogenous IL-31 or subtle signaling changes

  • Solution: Use amplification methods; employ phospho-flow cytometry for single-cell resolution; consider digital ELISA platforms for enhanced sensitivity

How can researchers optimize experimental conditions for studying IL-31-induced cellular responses?

To maximize the reproducibility and physiological relevance of IL-31 experiments, consider these optimization strategies:

• Cell culture optimization:

  • Serum conditions: Reduce serum (0.5-2%) or use serum-free media during IL-31 stimulation to minimize interference from serum factors

  • Cell density: Maintain consistent cell density across experiments; optimize based on readout (typically 70-80% confluence for signaling studies)

  • Culture duration: For primary cells, use early passages to maintain consistent receptor expression

• Stimulation parameters:

  • Dose optimization: Perform full dose-response curves (1-500 ng/mL) to identify both threshold and saturation concentrations

  • Timing optimization: Conduct detailed time-course studies for each readout (minutes for signaling; hours for gene expression; days for functional outcomes)

  • Delivery method: For in vivo studies, compare different administration routes (subcutaneous, intraperitoneal, local injection)

• Readout optimization:

  • Signaling studies: Identify peak phosphorylation times for each STAT protein (typically 15-30 minutes for STAT3)

  • Gene expression: Determine optimal timepoints for early-response vs. late-response genes

  • Functional assays: Adjust cell type, density, and culture duration for maximal response dynamic range

• Control selection:

  • Positive controls: Include IL-6 family cytokines that share receptor components with IL-31

  • Negative controls: Use heat-inactivated IL-31 or irrelevant cytokines

  • Validation controls: Include JAK inhibitors to confirm signaling specificity

• Data normalization strategies:

  • Western blots: Normalize phospho-proteins to total protein rather than housekeeping genes

  • qPCR: Validate stability of reference genes under experimental conditions

  • Functional assays: Include internal standards and technical replicates

For studying IL-31's effects on fibroblasts, researchers have successfully used concentrations of 50-200 ng/mL with stimulation periods of 24-72 hours to observe significant increases in collagen production . For signaling studies in A549 cells, induction of STAT3 phosphorylation has been demonstrated at lower concentrations with shorter stimulation periods .