Recombinant Human Interleukin-17F protein (IL17F) (Active)

What is the structural composition of recombinant human IL-17F protein?

Recombinant human IL-17F is a disulfide-linked homodimeric protein belonging to the IL-17 cytokine family. The full-length human IL-17F consists of 163 amino acids with a 30 amino acid signal peptide, resulting in a mature form spanning amino acids 31-163 . Among IL-17 family members, IL-17F shows the closest homology to IL-17A (approximately 44% amino acid sequence identity) but shares limited sequence homology (16-30%) with other family members (IL-17B, C, D, and E) . The protein features a conserved cystine-knot fold near the C-terminus while exhibiting considerable sequence divergence at the N-terminus . When expressed in mammalian expression systems like HEK293 cells, the recombinant protein typically demonstrates ≥95% purity with minimal endotoxin contamination (<0.005 EU/μg) .

How does IL-17F signal through its receptor complex?

IL-17F primarily signals through a heterodimeric receptor complex composed of IL-17RA and IL-17RC subunits, though it can also signal through IL-17RC homodimers . The signaling mechanism involves:

• Initial binding of IL-17F to IL-17RC with high affinity

• Formation of a heterodimeric receptor complex with IL-17RA

• Recruitment of the adapter protein TRAF3IP2 through SEFIR domain interactions

• Downstream activation of TRAF6-mediated pathways

• Activation of NF-κB and MAP kinase signaling cascades

• Transcriptional activation of target genes (cytokines, chemokines, antimicrobial peptides, matrix metalloproteinases)

This signaling cascade ultimately results in inflammatory responses characterized by neutrophil recruitment and activation . The IL-17F homodimer can also signal via IL-17RC homodimeric receptor complexes, triggering downstream activation of TRAF6 and NF-κB signaling .

What are the main biological activities of recombinant human IL-17F?

Recombinant human IL-17F exhibits several biological activities that impact immune regulation and tissue responses:

• Cytokine/Chemokine Induction: IL-17F stimulates the production of pro-inflammatory cytokines including IL-6, IL-8, and GM-CSF from various cell types .

• Tissue Remodeling: It regulates cartilage matrix turnover by increasing matrix release while simultaneously inhibiting new matrix synthesis .

• Vascular Effects: IL-17F inhibits angiogenesis and induces the expression of IL-2, TGF-β, and monocyte chemoattractant protein-1 (MCP-1) in endothelial cells .

• Antimicrobial Defense: As an effector cytokine of the innate and adaptive immune system, IL-17F contributes to antimicrobial host defense by stimulating the production of antimicrobial peptides like β-defensins (DEFB1, DEFB103A, and DEFB104A) by mucosal epithelial cells .

• Neutrophil Recruitment: IL-17F promotes neutrophilic inflammation, particularly at infection and inflammatory sites, acting primarily through the recruitment and activation of neutrophils .

Which cell types naturally express IL-17F?

IL-17F expression has been documented in several immune cell populations:

• T Helper 17 (Th17) Cells: These are the primary source of IL-17F and represent the signature effector cells producing this cytokine .

• Activated CD4+ T Cells: When stimulated, these cells upregulate IL-17F expression .

• γδ T Cells: These innate-like T cells produce IL-17F following stimulation with PMA and Ionomycin .

• Activated Monocytes: Upon activation, monocytes can express and secrete IL-17F .

• Memory CD4+ T Cells: These cells have been identified as sources of IL-17F in reporter mouse studies .

• NKT Cells: Natural killer T cells can also produce IL-17F under certain conditions .

• Lamina Propria T Cells: These tissue-resident T cells isolated from the intestinal mucosa express IL-17F, particularly in inflammatory conditions .

How do IL-17A and IL-17F differ in their role in inflammatory bowel disease (IBD)?

The differential roles of IL-17A and IL-17F in inflammatory bowel disease reveal complex immunoregulatory mechanisms:

In contrast, IL-17F serum concentrations do not exhibit significant differences between IBD patients (median: 15.11 pg/ml; range: 0.09–189.84 pg/ml) and healthy controls (median: 11.56 pg/ml; range: 0.19–32.49 pg/ml) (p=0.33) .

Tissue Expression Patterns:
Expression analysis reveals that colonic IL-17F levels are significantly higher in Crohn's disease compared to ulcerative colitis . This differential expression suggests distinct pathogenic mechanisms between these IBD subtypes.

Experimental Approaches to Study IL-17A/F in IBD:

• Serum protein measurement: Using ELISA or multiplex assays to quantify circulating levels.

• Intestinal tissue expression analysis: Employing qPCR, in situ hybridization, and immunohistochemistry to assess local expression.

• Functional ex vivo studies: Isolating lamina propria mononuclear cells to analyze cytokine production following stimulation.

• Animal models: Utilizing IL-17A or IL-17F knockout mice in DSS-colitis or TNBS-colitis models to assess their specific contributions.

These findings suggest IL-17A may serve as a potential biomarker for disease activity specifically in ulcerative colitis, while IL-17F might play a more prominent role in the pathophysiology of Crohn's disease through tissue-specific mechanisms rather than systemic effects.

What methodological considerations are critical when using recombinant IL-17F in in vitro experimental systems?

When designing experiments with recombinant IL-17F, several methodological considerations are essential for reliable results:

Expression Systems and Protein Quality:
Different expression systems yield recombinant IL-17F with varying properties:

• E. coli-derived IL-17F: Generally higher yield but may lack proper folding and post-translational modifications .

• Mammalian cell-derived IL-17F (HEK293): Better resembles native protein with appropriate glycosylation and folding patterns .

Protein Validation Protocol:

• Purity assessment: SDS-PAGE and HPLC analysis (target: ≥95% purity) .

• Endotoxin testing: LAL assay (acceptable level: ≤0.005 EU/μg) .

• Functional validation: Bioactivity assays measuring IL-6/IL-8 induction in responsive cell lines.

• Structural confirmation: Mass spectrometry to verify protein integrity.

Experimental Design Considerations:

• Concentration range: Typically effective between 10-100 ng/mL, but dose-response curves should be established for each system.

• Homodimers vs. heterodimers: Natural IL-17F exists as both IL-17F homodimers and IL-17A/F heterodimers with distinct potencies.

• Receptor saturation: Pre-testing with receptor-blocking antibodies to confirm specificity.

• Synergistic effects: Consider co-stimulation with other cytokines (TNF-α, IL-1β) that may potentiate IL-17F effects.

• Cell responsiveness: Not all cell types express adequate levels of IL-17RC; verify receptor expression before experiments.

Storage and Handling:

• Reconstitute in sterile, buffer-containing solutions (PBS with 0.1% BSA)

• Minimize freeze-thaw cycles (aliquot upon reconstitution)

• Maintain at -80°C for long-term storage

• Use carrier proteins for dilute solutions to prevent adhesion loss

These methodological considerations ensure experimental reproducibility and biological relevance when working with recombinant IL-17F.

How does IL-17F signaling interact with other inflammatory pathways in chronic inflammation models?

IL-17F signaling demonstrates complex interactions with other inflammatory pathways, creating unique signature patterns in different chronic inflammation models:

Interaction with TH2 Signaling in Allergic Inflammation:
In OVA-alum induced asthma models, IL-17F knockout mice exhibit significantly higher production of TH2 cytokines (IL-4, IL-5, IL-13) compared to wild-type or IL-17 knockout mice . This contrasts with IL-17 knockout mice, which show reduced TH2 responses, indicating IL-17F has a regulatory role in restricting allergic asthma development by suppressing TH2 responses .

Synergism with TNF-α Signaling:
IL-17F can synergize with TNF-α to amplify inflammatory responses through:

• Enhanced activation of NF-κB signaling

• Stabilization of target mRNAs encoding inflammatory mediators

• Augmented production of neutrophil-attracting chemokines

Crosstalk with Pattern Recognition Receptor Pathways:
Experimental data suggests IL-17F signaling can amplify responses to microbial products through:

• Upregulation of TLR expression on target cells

• Potentiation of NOD1/2 signaling responses

• Enhanced production of antimicrobial peptides following pathogen detection

Methodological Approaches to Study Pathway Interactions:

• Combined cytokine stimulation assays: Treating cells with IL-17F alone or in combination with TNF-α, IL-1β, or IFN-γ to detect synergistic gene induction.

• Pathway inhibitor studies: Using specific inhibitors of NF-κB, MAPK, or STAT signaling to dissect pathway contributions.

• Conditional knockout models: Tissue-specific deletion of IL-17RC combined with other pathway components to assess in vivo interactions.

• Phosphoproteomic analysis: Examining signaling node activation patterns after combined stimulation.

• Reporter gene assays: Using pathway-specific reporters to quantify cross-regulation.

Understanding these signaling interactions provides insights into potential therapeutic targets and explains the complex role of IL-17F in different inflammatory conditions.

What are the functional differences between IL-17F homodimers and IL-17A/IL-17F heterodimers?

IL-17F can exist as homodimers (IL-17F/F) or form heterodimers with IL-17A (IL-17A/F), with these different configurations exhibiting distinct functional properties:

Receptor Binding Affinities:

• IL-17F homodimers: Preferentially bind to IL-17RC with high affinity and can signal through IL-17RC homodimers

• IL-17A/F heterodimers: Bind with intermediate affinity to both IL-17RA and IL-17RC, requiring the heterodimeric receptor complex for signaling

Potency Comparison:

Tissue-Specific Effects:

• Lung tissue: Transgenic overexpression of IL-17F in lung epithelium results in lymphocyte and macrophage infiltration and mucus hyperplasia, similar to IL-17A overexpression, but with delayed kinetics .

• Intestinal tissue: IL-17F appears to have more prominent expression in Crohn's disease compared to ulcerative colitis, while IL-17A shows stronger association with UC activity .

Experimental Approaches to Distinguish Functions:

• Recombinant protein studies: Using purified IL-17F/F homodimers vs. engineered IL-17A/F heterodimers

• Selective neutralizing antibodies: Antibodies specifically targeting each configuration

• Receptor blockade: Selective blockade of IL-17RA vs. IL-17RC to differentiate signaling requirements

• Gene expression profiling: Comparative transcriptomics following stimulation with each dimer configuration

These functional differences explain why targeting specific configurations might yield different therapeutic outcomes in inflammatory diseases.

What are the current challenges in translating IL-17F research findings to therapeutic applications?

Despite advances in understanding IL-17F biology, several challenges remain in developing therapeutic approaches targeting this cytokine:

Differential Roles in Disease Pathogenesis:
IL-17F demonstrates complex and sometimes contradictory roles in different disease models:

• In IBD, IL-17F shows higher expression in Crohn's disease compared to ulcerative colitis, yet serum levels don't correlate with disease activity unlike IL-17A

• In allergic asthma, IL-17F knockout mice exhibit enhanced rather than reduced TH2 responses, suggesting a protective role

• In antimicrobial defense, IL-17F promotes protective neutrophil recruitment and antimicrobial peptide production

Technical Challenges in Targeting Specificity:

• Heterodimer complexity: Selectively targeting IL-17F/F homodimers without affecting IL-17A/F heterodimers remains difficult

• Receptor sharing: IL-17RC serves as a receptor for both IL-17A and IL-17F, complicating selective pathway inhibition

• Tissue-specific signaling: Differences in receptor expression and signaling outcomes across tissues require tailored therapeutic approaches

Methodological Approaches to Address These Challenges:

• Structure-guided antibody design: Developing antibodies that specifically recognize IL-17F epitopes not shared with IL-17A

• Tissue-targeted delivery: Creating delivery systems that localize IL-17F antagonists to specific disease sites

• Pathway-selective inhibition: Identifying downstream signaling components unique to IL-17F versus IL-17A

• Biomarker development: Establishing reliable biomarkers to identify patients likely to benefit from IL-17F-targeted therapy

• Combination approaches: Testing IL-17F modulation in combination with other cytokine-targeting strategies

Evaluation of Therapeutic Potential:
Clinical success will likely depend on:

• Patient stratification based on IL-17F expression patterns

• Disease-specific approach (inhibition in some contexts, augmentation in others)

• Distinguishing between local tissue and systemic effects

• Maintaining antimicrobial defense while reducing pathological inflammation

Addressing these challenges will be crucial for translating the growing body of IL-17F research into effective therapeutic strategies for inflammatory diseases.