IL17A Human, Sf9

What is IL-17A and what are its primary cellular sources?

IL-17A is the prototypical member of the IL-17 family of cytokines (IL-17A through IL-17F). It functions primarily as a proinflammatory cytokine produced mainly by T helper 17 (TH17) cells. Other cellular sources include γδ T cells, innate lymphoid cells, and some reports suggest myeloid-lineage cells including neutrophils and microglia, though the latter remains somewhat controversial . IL-17A exists as a homodimer but can also form heterodimers with IL-17F (IL-17AF), with IL-17A homodimers inducing far more potent signaling compared to IL-17F homodimers .

How does IL-17A signal, and what are its main biological functions?

IL-17A signals through an obligate dimeric receptor complex consisting of IL-17RA and IL-17RC subunits. Upon binding, IL-17A initiates recruitment of the adaptor protein Act1 to IL-17RA and triggers downstream pathways including NF-κB activation and phosphorylation of MAPKs such as JNK, p38, and ERK1/2 . The main function of IL-17A is to coordinate local tissue inflammation by increasing production of proinflammatory and neutrophil-mobilizing cytokines and chemokines. IL-17A has evolved primarily for host protection, particularly at barrier surfaces, though its inflammatory activities can become problematic in autoimmune conditions .

What advantages does the Sf9 expression system offer for recombinant human IL-17A production?

Sf9 cells provide several advantages for IL-17A expression:

• Capacity to produce correctly folded disulfide-bonded proteins

• Higher yield compared to mammalian systems

• Post-translational modification capabilities appropriate for cytokine production

• Ability to express the properly dimerized form of IL-17A

• Lower contamination risk than bacterial systems

Sf9-expressed IL-17A maintains the structural features needed for receptor binding studies and functional assays, making it suitable for research applications requiring properly folded, biologically active cytokine.

What are critical quality control parameters for Sf9-expressed IL-17A?

For optimal research use, Sf9-expressed IL-17A should be validated for:

Researchers should verify dimer formation and biological activity by confirming the protein's ability to induce IL-6, IL-8, or CXCL1 production in responsive cell lines .

How can I verify the biological activity of Sf9-produced IL-17A?

The biological activity of Sf9-expressed human IL-17A can be verified by:

• Induction of downstream cytokines and chemokines in responsive cell lines:

  • Human cells: measure CXCL1 (GROα), IL-6, and IL-8 production

  • Mouse cells: measure CXCL1 (KC), IL-6, and CXCL2 (MIP-2) production

• Signaling pathway activation:

  • Assess recruitment of Act1 adaptor protein to IL-17RA

  • Measure phosphorylation of IκBα (NF-κB pathway)

  • Detect phosphorylation of MAPKs (JNK, p38, and ERK1/2)

• Receptor binding assays:

  • Flow cytometry using biotinylated IL-17A to measure binding to cells expressing IL-17RA

  • Competition assays with unlabeled IL-17A or neutralizing antibodies

What concentrations of IL-17A are appropriate for in vitro studies?

Typical working concentrations of IL-17A for different experimental applications:

Dose-response experiments should be performed for each cell type and experimental system to determine optimal concentrations, as responsiveness to IL-17A varies significantly between different cell types.

What methods can be used to study IL-17A/IL-17RA binding interactions?

Several validated methods are available for studying IL-17A/IL-17RA interactions:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified IL-17RA on a sensor chip

  • Measure binding kinetics of IL-17A in real-time

  • Calculate association (kon) and dissociation (koff) constants

• Cellular binding assays:

  • Incubate cells expressing IL-17RA with biotinylated IL-17A

  • Detect binding using avidin-fluorescein and flow cytometry

  • Competitive binding assays can be performed with unlabeled IL-17A or inhibitors

• Computational modeling:

  • The crystal structure of IL-17RA complexed with IL-17A homodimer reveals three major interaction sites

  • The deep binding pocket formed by specific amino acid residues (N89-E92, D121-E125, S257-D262, and T163-S167) is optimal for drug targeting

How can I screen for novel inhibitors of IL-17A?

To identify inhibitors of IL-17A/IL-17RA interaction:

• Structure-based virtual screening:

  • Target the deep binding pocket on IL-17RA that interacts with IL-17A residues 35-46

  • Computer-aided docking can predict compounds that may disrupt the interaction

• Biochemical screening assays:

  • ELISA-based competition assays measuring displacement of labeled IL-17A

  • AlphaScreen or FRET-based proximity assays for high-throughput screening

• Cell-based functional assays:

  • Measure inhibition of IL-17A-induced cytokine production (IL-6, IL-8, CXCL1)

  • Assess blockade of signaling events (Act1 recruitment, MAPK phosphorylation)

  • Flow cytometry to quantify inhibition of IL-17A binding to cell-surface receptors

The small molecule cyanidin (A18) has been identified as an effective inhibitor that blocks IL-17A binding to IL-17RA by forming contacts with specific residues in the binding pocket (D121, Q124, S168, and D262) .

What are the key signaling events triggered by IL-17A?

IL-17A binding to its receptor complex initiates a cascade of signaling events:

• Receptor complex formation:

  • IL-17A binds to the heterodimeric receptor composed of IL-17RA and IL-17RC subunits

  • This creates a signaling-competent complex at the cell surface

• Proximal signaling events:

  • Recruitment of the adaptor protein Act1 to the receptor

  • Act1 serves as a platform for subsequent signaling events

• Activation of downstream pathways:

  • NF-κB pathway: phosphorylation and degradation of IκBα, leading to nuclear translocation of NF-κB

  • MAPK pathways: phosphorylation of JNK, p38, and ERK1/2

  • These pathways collectively induce transcription of proinflammatory genes

• Gene expression changes:

  • Induction of cytokines (IL-6), chemokines (CXCL1, IL-8, CXCL2), and other inflammatory mediators

  • Expression of antimicrobial peptides at barrier surfaces

  • Upregulation of tissue remodeling factors

How do IL-17A homodimers differ functionally from IL-17A/F heterodimers?

IL-17A can exist as homodimers or heterodimers with IL-17F, each with distinct signaling properties:

All forms activate qualitatively similar signaling pathways, but IL-17A homodimers induce far more potent signals compared to IL-17F homodimers, with IL-17AF thought to be of intermediate signaling strength .

How is IL-17A implicated in autoimmune diseases?

IL-17A plays critical roles in several autoimmune and inflammatory conditions:

• Multiple sclerosis/EAE:

  • IL-17A-producing TH17 cells are critical in EAE pathogenesis

  • IL-17A promotes demyelination and recruits neutrophils to the CNS

  • Inhibition of IL-17A attenuates disease severity in EAE models

• Psoriasis:

  • High levels of IL-17A are found in psoriatic skin lesions

  • Anti-IL-17A antibody (Cosentyx/secukinumab) is FDA-approved for psoriasis treatment

  • IL-17A drives keratinocyte hyperproliferation and inflammatory gene expression

• Rheumatoid arthritis:

  • IL-17A is present in synovial fluids from arthritis patients

  • Contributes to bone and cartilage degradation

  • Works synergistically with other inflammatory cytokines

• Systemic sclerosis with pulmonary arterial hypertension:

  • IL-17A levels are significantly elevated in patients with high risk for PAH

  • IL-17A is predicted as an upstream regulator in proteomics analyses

  • IL-17A and IL-6 levels correlate with PAH risk in SSc patients

What therapeutic approaches target IL-17A?

Several strategies have been developed to inhibit IL-17A signaling:

• Monoclonal antibodies:

  • Secukinumab (Cosentyx): FDA-approved anti-IL-17A antibody for psoriasis

  • Currently used in over 50 clinical trials for various autoimmune diseases

  • High efficacy but limited to intravenous administration and high production costs

• Small-molecule inhibitors:

  • Cyanidin (A18): blocks IL-17A/IL-17RA interaction by targeting the binding pocket

  • Present in red berries and other fruits, particularly in fruit skin

  • Provides cost-effective alternative to antibody therapies

  • Demonstrates efficacy in reducing IL-17A-induced cytokine production and attenuating TH17-mediated EAE

• Receptor-targeting approaches:

  • Antibodies targeting IL-17RA to block binding of multiple IL-17 family members

  • Bispecific antibodies targeting both IL-17A and IL-17F

  • MT-6194: suppresses downstream IL-17A signaling molecules in systemic sclerosis models

What are common pitfalls when working with IL-17A in cell-based assays?

Researchers should be aware of these challenges when designing IL-17A experiments:

• Variable cell responsiveness:

  • Different cell types show widely varying sensitivity to IL-17A

  • Primary cells often respond differently than cell lines

  • Response may depend on expression levels of IL-17RA and IL-17RC

• Co-stimulation requirements:

  • Many cell types require co-stimulation with TNF-α or other cytokines for robust IL-17A responses

  • In isolation, IL-17A may produce weak signals in some experimental systems

• Species-specific differences:

  • Human and mouse IL-17A are not fully cross-reactive

  • While binding pocket residues are conserved between species, optimal activity is typically achieved with species-matched cytokines

• Storage and handling issues:

  • IL-17A activity can be compromised by repeated freeze-thaw cycles

  • Adsorption to plastic surfaces can reduce effective concentration

  • Carrier proteins may be needed for dilute solutions

How can I optimize experimental design for IL-17A inhibition studies?

For robust inhibition studies:

• Establish clear positive and negative controls:

  • Include anti-IL-17A antibody as positive control for inhibition

  • Use structurally similar but non-inhibitory compounds as negative controls

• Verify target engagement:

  • Confirm that inhibitors directly disrupt IL-17A/IL-17RA binding

  • Use multiple assay readouts (binding, signaling, gene expression)

• Assess specificity:

  • Test effects on other IL-17 family members (especially IL-17F)

  • Evaluate impact on other cytokine signaling pathways

  • The amino acid residues in the IL-17RA docking pocket are conserved between human and mouse receptors, allowing for cross-species inhibition studies

• Consider combination approaches:

  • IL-17A often works synergistically with other cytokines (TNF-α, IL-1β)

  • Some conditions may require targeting multiple pathways