Recombinant Human Interleukin-17A (IL17A) (Active)

What is the molecular structure and basic characteristics of recombinant human IL-17A?

Recombinant human IL-17A is a disulfide-linked homodimeric secreted glycoprotein comprising 155 amino acid residues. The mature human IL-17A protein sequence typically spans from Gly24/Ile20 to Ala155, depending on the specific recombinant product . Under non-reducing conditions, IL-17A appears as a 28-38 kDa protein due to glycosylation and dimerization . When expressed in human cell lines, IL-17A exhibits proper glycosylation patterns with N-linked oligosaccharides that contribute to its stability and biological activity . Under reducing conditions, IL-17A can migrate as two bands at approximately 16 and 22 kDa on SDS-PAGE, reflecting different post-translational modifications . The protein has a predicted isoelectric point (pI) of approximately 8.62, indicating its slightly basic nature .

IL-17A adopts a conserved cystine knot fold shared among the IL-17 family cytokines. Besides forming homodimers, IL-17A can also form heterodimers with IL-17F, which may exhibit slightly different biological activities compared to IL-17A homodimers .

How does the expression system affect recombinant human IL-17A properties?

The expression system significantly impacts the structural and functional properties of recombinant human IL-17A. Two primary expression systems are commonly used:

Human cell-expressed IL-17A typically exhibits ED50 values of 0.12-1.2 ng/mL in IL-6 induction assays, while E. coli-derived protein shows ED50 values of 0.4-4 ng/mL, indicating potential differences in specific activity . For experiments requiring physiologically relevant post-translational modifications, human cell-expressed IL-17A is preferred, while E. coli-derived protein may be sufficient for applications where glycosylation is not critical .

What are the recommended reconstitution and storage conditions for maintaining IL-17A activity?

Proper reconstitution and storage are crucial for maintaining the biological activity of recombinant IL-17A. The following methodological approach is recommended:

Reconstitution Protocol:

• For carrier-containing formulations: Reconstitute at 100 μg/mL in PBS containing at least 0.1% human or bovine serum albumin .

• For carrier-free formulations: Reconstitute at 100 μg/mL in sterile PBS without additives .

• Allow the lyophilized protein to reach room temperature before reconstitution.

• Gently swirl until completely dissolved; avoid vigorous vortexing that can cause protein denaturation.

Storage Recommendations:

• Store lyophilized protein at 2-8°C before reconstitution .

• For short-term use (≤1 month), store reconstituted protein at 4°C.

• For long-term storage, prepare small aliquots and store at -18 to -20°C .

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles as they significantly reduce biological activity .

• When preparing working solutions for experiments, dilute in buffers containing a carrier protein (0.1-0.5% BSA or HSA) to prevent adsorption to labware surfaces.

For HSA-containing formulations, the reconstituted solution typically contains 1% human serum albumin and 10% trehalose, which enhances stability during freeze-thaw cycles .

How can I design experiments to assess IL-17A signaling pathways in different cell types?

Experimental design for IL-17A signaling studies should consider cell type-specific responses and the temporal dynamics of downstream pathway activation. A comprehensive approach includes:

Cell Selection and Preparation:

• Primary cells relevant to research question (e.g., aortic endothelial cells for vascular studies, fibroblasts for tissue remodeling) .

• Culture cells to 70-80% confluence in appropriate serum-reduced medium for 12-24 hours before IL-17A treatment to reduce background signaling.

• Include appropriate positive controls (e.g., TNF-α, IL-1β) and negative controls (vehicle only).

Concentration and Time-Course Optimization:

• Perform dose-response experiments ranging from 0.1-100 ng/mL IL-17A.

• For signaling studies, examine early timepoints (5, 15, 30, 60 minutes) for MAPK activation and later timepoints (1, 3, 6, 24 hours) for transcriptional responses .

Pathway Analysis Methods:

• Western blotting for phosphorylated p38 MAPK, which is specifically activated by IL-17A in endothelial cells .

• qRT-PCR to measure expression of IL-17A-responsive genes: IL-6, GM-CSF, CXCL1, and CXCL2 .

• ELISA to quantify secreted inflammatory mediators.

• Pathway inhibitors (e.g., p38 MAPK inhibitor) to confirm specific signaling mechanisms .

Functional Readouts:

• Cell adhesion assays measuring monocyte adhesion to IL-17A-activated endothelial cells .

• Intercellular adhesion molecule 1 (ICAM-1) expression by flow cytometry .

• Transwell migration assays to assess chemotactic responses.

When investigating IL-17A signaling, it's important to address the potential heterodimeric interactions with IL-17F, as both IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC) are required for responsiveness to IL-17A/F heterodimers .

What are the key methodological considerations for evaluating IL-17A bioactivity in experimental systems?

Accurate assessment of IL-17A bioactivity requires carefully standardized assays that reflect its physiological functions. Several methodological approaches are recommended:

Standard Bioactivity Assays:

• IL-6 induction assay in NIH-3T3 mouse embryonic fibroblasts - a standard measure of IL-17A activity (typical ED50: 0.12-1.2 ng/mL for human cell-expressed; 0.4-4 ng/mL for E. coli-derived) .

• IL-6 production in normal human dermal fibroblasts (NHDF) - measures physiologically relevant activity (typical ED50: 0.5-1.5 ng/mL) .

Critical Experimental Controls:

• Heat-inactivated IL-17A (95°C for 5 minutes) as negative control.

• Species-matched IL-17A for animal studies to account for species specificity.

• Blocking antibodies against IL-17RA to confirm receptor specificity.

• Inclusion of IL-17F and IL-17A/F heterodimers for comparative analyses.

Measuring Endothelial Cell Activation:

• Flow cytometry for cell surface adhesion molecules (ICAM-1).

• Monocyte adhesion assays under static or flow conditions.

• Quantification of chemokines CXCL1 and CXCL2 by ELISA or multiplex assay .

Technical Considerations:

• Use low-binding microcentrifuge tubes and pipette tips to prevent protein adsorption.

• Prepare fresh working solutions for each experiment.

• Include appropriate vehicle controls that match the IL-17A formulation buffer.

• For long-term studies, perform stability assessment of the reconstituted protein at experimental conditions.

When comparing results across different recombinant IL-17A preparations, standardize activity units rather than absolute concentrations to account for batch-to-batch and source-to-source variations in specific activity .

How does IL-17A interact with other inflammatory mediators in experimental disease models?

IL-17A functions within a complex network of inflammatory mediators, requiring careful experimental design to elucidate its specific contributions. Key methodological approaches include:

Co-stimulation Experiments:

• Combine IL-17A with TNF-α, IL-1β, or IFN-γ at submaximal concentrations to assess synergistic effects on target cell responses.

• Compare sequential vs. simultaneous cytokine treatments to model priming effects.

• Measure both transcriptional responses (mRNA levels) and protein outputs (secreted cytokines/chemokines).

In Vivo Models:

• Using genetic approaches such as IL-17A knockout mice (e.g., ApoE−/−/IL-17A−/− double knockout) to study atherosclerosis progression .

• Intravital microscopy to directly visualize leukocyte adhesion to endothelium in response to IL-17A .

• Tissue-specific conditional knockout or overexpression systems to distinguish local vs. systemic IL-17A effects.

Mechanistic Pathway Analysis:

• Specific inhibition of p38 MAPK pathway in endothelial cells to attenuate IL-17A-mediated activation, which regulates expression of proinflammatory cytokines, chemokines, and adhesion molecules .

• Analysis of IL-17 receptor complex formation (IL-17RA-IL-17RC heterodimers) and TRAF3IP2 adapter recruitment in response to different inflammatory stimuli .

• Investigation of transcriptional regulators downstream of IL-17A signaling.

Experimental Readouts:

• Quantification of neutrophil chemotaxis and activation, a hallmark of IL-17A activity .

• Assessment of antimicrobial peptide production in epithelial cells.

• Measurement of tissue-specific inflammatory markers.

• Evaluation of germinal center formation and B cell responses .

In cardiovascular disease models, IL-17A contributes to endothelial activation via transcriptional and post-translational mechanisms. Hyperlipidemic stress (e.g., oxidized LDL) up-regulates IL-17 receptors in aortic endothelial cells, enhancing their responsiveness to IL-17A and promoting leukocyte adhesion . The absence of IL-17A in mouse models reduces this endothelial activation and subsequent inflammatory responses.

What are common causes of variability in IL-17A activity assays and how can they be addressed?

Variability in IL-17A bioactivity assays can significantly impact experimental reproducibility. Researchers should consider these methodological solutions to common problems:

When transitioning from in vitro to in vivo experiments, consider:

• Differences in protein half-life and bioavailability in vivo.

• Endogenous IL-17A/F background levels.

• Compensatory mechanisms absent in simpler in vitro systems.

• Timing of IL-17A administration relative to disease onset in experimental models.

For increased reproducibility across laboratories, standardize reporting of:

• Specific recombinant IL-17A product and lot number.

• Detailed reconstitution and storage protocol.

• Cell types, passage numbers, and culture conditions.

• Complete assay methodology including incubation times and detection systems.

How can I distinguish between IL-17A homodimer and IL-17A/F heterodimer effects in experimental systems?

Distinguishing the specific contributions of IL-17A homodimers versus IL-17A/F heterodimers requires specialized experimental approaches:

Protein Selection Strategies:

• Use purified recombinant IL-17A homodimers and compare with equivalent molar concentrations of IL-17A/F heterodimers.

• Include IL-17F homodimers as additional control.

• Consider using site-directed mutants that preferentially form specific dimeric structures.

Receptor Targeting Approaches:

• Use receptor-specific blocking antibodies:

  • Anti-IL-17RA blocks both IL-17A and IL-17A/F signaling.

  • Combined anti-IL-17RA and anti-IL-17RC completely inhibits both cytokines.

  • Selective IL-17RC blockade may differentially affect responses.

• Employ siRNA knockdown of IL-17RA or IL-17RC to assess their relative contributions.

• Use cell lines with defined receptor expression profiles.

Functional Discrimination:

• Neutrophil migration assays - IL-17A induces neutrophil migration while IL-17F does not .

• Dose-response analyses - IL-17A and IL-17A/F may show different potencies.

• Kinetic studies - temporal differences in signaling activation.

• Gene expression profiling to identify distinct transcriptional signatures.

Analytical Methods:

• Western blotting under non-reducing conditions to visualize dimeric species.

• Immunoprecipitation with dimer-specific antibodies if available.

• Use of epitope-tagged recombinant proteins to track complex formation.

When interpreting results, consider that both IL-17RA and IL-17RC are required for responsiveness to IL-17A/F heterodimers, while IL-17A homodimers may signal through alternative receptor complexes including IL-17RA with either IL-17RC or IL-17RD .

What considerations are important when comparing different sources of recombinant IL-17A in research protocols?

Comparing results obtained with different recombinant IL-17A preparations requires careful consideration of several factors:

Source and Production Variables:

• Expression system differences:

  • Human cell-expressed (HEK293) IL-17A shows proper glycosylation and typically exhibits higher specific activity (ED50: 0.12-1.2 ng/mL) than E. coli-derived protein (ED50: 0.4-4 ng/mL) .

  • E. coli-derived proteins lack glycosylation but may be suitable for many applications .

• Sequence variations:

  • Some products include the signal peptide sequence while others start at mature protein sequence.

  • N-terminal methionine additions in E. coli-derived products .

  • Sequence boundaries vary slightly between products (Gly24-Ala155 vs. Ile20-Ala155) .

Formulation Differences:

• Carrier protein presence/absence:

  • Carrier-containing formulations (with BSA) enhance stability but may interfere with some applications .

  • Carrier-free versions (CF) are preferred for applications where BSA could interfere .

• Buffer composition differences between commercial sources.

• Presence of stabilizers like trehalose in some formulations .

Standardization Approaches:

• Normalize based on bioactivity rather than protein concentration:

  • Calculate EC50 values in standardized bioassay (e.g., IL-6 induction in NIH-3T3 cells).

  • Use biological activity units rather than absolute concentration.

• Include internal standards across experiments.

• Perform side-by-side comparisons when changing protein sources.

Application-Specific Considerations:

• For in vivo studies, carrier-free preparations may be preferred to avoid immune responses to BSA.

• For structural studies, glycosylation heterogeneity in human cell-expressed proteins may be problematic.

• For functional assays measuring downstream signaling, human cell-expressed IL-17A may more accurately reflect physiological activity.

• For ELISA standards, either formulation is typically suitable.

When transitioning between different IL-17A sources, validate the new product in your specific experimental system before collecting critical data. Document the specific product and lot number in publications to enhance reproducibility .

How does IL-17A contribute to tissue-specific inflammatory responses in different disease models?

IL-17A exhibits differential effects across tissues, contributing to disease pathogenesis through tissue-specific mechanisms:

Vascular System:

• IL-17A activates aortic endothelial cells via the p38 MAPK pathway, promoting monocyte adhesion and potentially contributing to atherosclerosis .

• Hyperlipidemic stress (oxidized LDL) up-regulates IL-17 receptors in aortic endothelial cells, enhancing their responsiveness to IL-17A .

• In ApoE−/−/IL-17A−/− double knockout mice, leukocyte adhesion to endothelium is reduced, suggesting a direct role in vascular inflammation .

• IL-17A induces expression of proinflammatory cytokines (IL-6, GM-CSF) and chemokines (CXCL1, CXCL2) in endothelial cells, creating a chemotactic gradient for neutrophil recruitment .

Mucosal Tissues:

• IL-17A produces protective mucosal inflammation against microbial infection by inducing antimicrobial peptide production .

• In intestinal inflammation, IL-17A can have dual roles:

  • Promoting neutrophil recruitment and antimicrobial defense.

  • Limiting chronic inflammation and protecting against colitis progression .

• Experimental approaches should include tissue-specific knockout models and localized cytokine delivery to distinguish protective versus pathogenic roles.

Lymphoid Tissues:

• IL-17A enhances germinal center formation by regulating B cell chemotaxis in response to CXCL12 and CXCL13 .

• It increases B cell retention within germinal centers and enhances somatic hypermutation rates .

• Research protocols should include B cell trafficking assays and analysis of affinity maturation in the presence of IL-17A.

Methodology for Tissue-Specific Studies:

• Tissue-specific conditional knockout models.

• Ex vivo tissue explant cultures with defined IL-17A exposure.

• Tissue-specific reporter systems to track IL-17A-responsive cells.

• Single-cell RNA sequencing to identify cell-specific responses within heterogeneous tissues.

When investigating IL-17A in tissue-specific inflammation, consider the temporal dynamics of acute versus chronic responses and the interaction with tissue resident cells versus recruited inflammatory cells .

What are the current methodological approaches for targeting IL-17A signaling in experimental disease models?

Research on IL-17A signaling inhibition employs various strategic approaches with distinct experimental considerations:

Direct IL-17A Neutralization:

• Anti-IL-17A neutralizing antibodies:

  • Provide high specificity but variable tissue penetration.

  • Dose titration critical for partial versus complete inhibition.

  • Consider potential immunogenicity in long-term studies.

• Soluble receptor constructs (IL-17RA-Fc):

  • Broader inhibition profile (blocks multiple IL-17 family members).

  • May better mimic therapeutic approaches being developed clinically.

Receptor Targeting Strategies:

• Anti-IL-17RA or anti-IL-17RC blocking antibodies:

  • Allows differential blockade of receptor components.

  • IL-17RA blockade affects multiple IL-17 family members.

• Genetic approaches:

  • Cell-type specific receptor knockout using Cre-lox systems.

  • Inducible knockout systems for temporal control.

  • CRISPR/Cas9 editing for precise receptor modifications.

Downstream Signaling Inhibition:

• p38 MAPK inhibitors specifically attenuate IL-17A-mediated endothelial cell activation by:

  • Reducing expression of proinflammatory cytokines and chemokines.

  • Decreasing adhesion molecule expression (ICAM-1).

  • Limiting monocyte adhesion to activated endothelium .

• Inhibitors of TRAF3IP2 adapter to disrupt IL-17R-mediated signaling .

• NF-κB pathway inhibitors to block downstream transcriptional responses.

Experimental Design Considerations:

• Preventive versus therapeutic intervention timing.

• Local versus systemic administration.

• Combination with other inflammatory pathway inhibitors.

• Assessment of both intended target inhibition and off-target effects.

• Verification of target engagement using phospho-specific antibodies for p38 MAPK or other pathway components .

For cardiovascular disease models, the p38 MAPK pathway represents a specific therapeutic target for IL-17A-mediated endothelial activation. Inhibition of this pathway ameliorates the expression of inflammatory cytokines, chemokines, and adhesion molecules, potentially reducing atherosclerotic progression .

How can transcriptomic and proteomic approaches enhance understanding of IL-17A signaling networks?

High-throughput molecular profiling technologies offer powerful tools for dissecting IL-17A signaling networks:

Transcriptomic Approaches:

• RNA-seq time-course experiments:

  • Capture early (1-3 hours) and late (6-24 hours) transcriptional responses.

  • Identify primary versus secondary response genes.

  • Compare profiles from different cell types (endothelial cells, fibroblasts, epithelial cells).

• Single-cell RNA-seq:

  • Resolve heterogeneous responses within cell populations.

  • Identify IL-17A-responsive cell subsets in complex tissues.

  • Map transcriptional trajectories during IL-17A stimulation.

• Comparative analysis workflows:

  • IL-17A vs. IL-17F vs. IL-17A/F heterodimer responses.

  • IL-17A alone vs. IL-17A combined with TNF-α or IL-1β.

  • Wild-type vs. signaling-deficient cells (p38 MAPK inhibited) .

Proteomic Methods:

• Phosphoproteomics to map IL-17A-induced signaling events:

  • Early phosphorylation events in p38 MAPK cascade .

  • Temporal dynamics of pathway activation.

  • Identification of novel signaling nodes.

• Secretome analysis:

  • Quantify changes in secreted inflammatory mediators.

  • Identify novel IL-17A-regulated factors.

• Proximity labeling approaches:

  • Map IL-17 receptor complex components.

  • Identify transient protein-protein interactions.

Integrated Multi-omics:

• Combined transcriptome-proteome correlation analysis.

• Network modeling of IL-17A signaling:

  • Identify critical nodes and potential feedback loops.

  • Map crosstalk with other inflammatory pathways.

  • Predict potential therapeutic targets.

• Validation strategies for omics findings:

  • CRISPR screening of candidate regulators.

  • Targeted inhibition of newly identified pathways.

  • In vivo confirmation in disease models.

Computational Analysis:

• Pathway enrichment analysis to identify IL-17A-regulated biological processes.

• Transcription factor binding site analysis to map transcriptional regulators.

• Interactome mapping to visualize protein interaction networks.

• Cross-species conservation analysis to identify evolutionarily conserved IL-17A responses.

Advanced transcriptomic and proteomic approaches can reveal how IL-17A specifically activates the p38 MAPK pathway in endothelial cells, both transcriptionally and post-translationally, leading to vascular inflammation and potential atherosclerotic progression .

What are the emerging research directions in IL-17A biology and experimental applications?

Current research on IL-17A is expanding in several promising directions that require sophisticated experimental approaches:

Tissue Microenvironment Interactions:

• Investigation of IL-17A's role in tissue-specific immune responses using organoid models and tissue-on-chip technologies.

• Analysis of how tissue metabolic state influences IL-17A signaling, particularly in hyperlipidemic environments where IL-17 receptor expression is upregulated .

• Development of spatial transcriptomics approaches to map IL-17A-responsive cellular niches within complex tissues.

Precision Targeting Strategies:

• Development of heterodimer-specific inhibitors that selectively target IL-17A/A homodimers versus IL-17A/F heterodimers.

• Cell type-selective delivery systems for IL-17A pathway modulators.

• Temporal control of IL-17A signaling using optogenetic or chemically-inducible systems.

• Selective inhibition of downstream pathways (e.g., p38 MAPK) in specific cell populations .

Systems Biology Approaches:

• Multi-scale modeling of IL-17A signaling from molecular interactions to tissue-level responses.

• Machine learning applications to predict IL-17A-dependent disease progression.

• Network analysis to identify critical regulatory nodes in IL-17A signaling.

• Quantitative systems pharmacology to optimize therapeutic targeting strategies.

Translational Applications:

• Development of more physiologically relevant assays to predict in vivo IL-17A function.

• Biomarker discovery for IL-17A pathway activation in clinical samples.

• Patient-derived models to assess personalized responses to IL-17A inhibition.

• Combinatorial targeting approaches addressing IL-17A along with synergistic pathways.

The burgeoning understanding of IL-17A's role in cardiovascular inflammation through specific activation of p38 MAPK in endothelial cells opens new avenues for targeting this pathway in atherosclerosis and related disorders. Future research will likely focus on integrating these molecular insights into therapeutic strategies that selectively modulate IL-17A signaling in disease-relevant tissues while preserving its protective functions in antimicrobial immunity .

How can researchers optimize experimental design when studying IL-17A in complex disease models?

Optimizing experimental design for IL-17A studies in complex disease models requires systematic consideration of multiple variables:

Model Selection and Refinement:

• Choose animal models that recapitulate human disease-relevant IL-17A pathways:

  • ApoE−/−/IL-17A−/− double knockout mice for atherosclerosis studies .

  • Tissue-specific IL-17 receptor conditional knockout models.

  • Humanized mouse models expressing human IL-17 receptors.

• Consider temporal aspects of disease progression:

  • Early inflammatory phase versus chronic disease.

  • Preventive versus therapeutic intervention timing.

• Account for sex differences in IL-17A responses and disease susceptibility.

Intervention Design:

• Dose optimization strategies:

  • Perform detailed dose-response studies (0.1-100 ng/mL range for in vitro, scaled appropriately for in vivo).

  • Consider pharmacokinetics and tissue distribution for in vivo studies.

• Targeting approaches:

  • Compare direct IL-17A neutralization versus receptor blockade.

  • Evaluate pathway-specific inhibition (e.g., p38 MAPK inhibitors for vascular studies) .

  • Test combination approaches targeting multiple inflammatory mediators.

Readout Selection:

• Multi-parameter assessment:

  • Molecular markers (phospho-p38 MAPK, cytokine/chemokine profiles) .

  • Cellular responses (adhesion molecule expression, leukocyte adhesion) .

  • Tissue pathology (inflammatory infiltration, tissue damage).

  • Functional outcomes (vascular function, tissue repair).

• Temporal analysis:

  • Early signaling events (minutes to hours).

  • Intermediate transcriptional responses (hours).

  • Late functional consequences (days to weeks).

Statistical and Experimental Design Considerations:

• Power analysis for appropriate sample sizing.

• Include both positive controls (known IL-17A-dependent models) and negative controls.

• Randomization and blinding procedures for in vivo studies.

• Consider factorial experimental designs to assess multiple variables simultaneously.

• Include genetic background controls for knockout models.