IL 17A/F Human

What is the structural composition of the human IL-17A/F heterodimer?

The human IL-17A/F is an approximately 40 kDa secreted disulfide-linked heterodimeric glycoprotein comprised of IL-17A and IL-17F subunits . X-ray crystallography analysis reveals that IL-17A/F forms a "two-faced" cytokine that structurally mimics both IL-17A and IL-17F homodimers . The protein structure features a cystine-knot-like disulfide arrangement, although unlike most cysteine knot superfamily members that utilize three intrachain disulfide bonds, IL-17 family cytokines generate the same structural form with only two disulfide links .

The structural architecture of IL-17A/F can be visualized as a garment with:

• A collar (cystine-knot-like disulfides)

• Two sleeves (first, short β-hairpin)

• A body (second, long β-hairpin)

• A skirt (N-terminal region forming a small β-sheet with the tip of the second β-hairpin)

The inter-chain disulfide (Cys129A-Cys47F) forms a critical connection between the two subunits, which agrees with published mass spectrometry data . Compared to the respective homodimers, IL-17A/F shows more structural order, with only 13% of residues lacking electron density (primarily the first 13-15 residues of each chain and residues 58-63 of the flexible coil in the A-chain) .

How does IL-17A/F compare functionally to IL-17A and IL-17F homodimers?

IL-17A/F is a biologically active protein that induces chemokine production and airway neutrophilia with intermediate potency between IL-17A (most potent) and IL-17F (least potent) . This gradient of biological activity correlates with binding affinities toward the IL-17RA receptor .

In genome-wide expression studies using microarrays, IL-17A and IL-17F alone had similar regulatory effects on rheumatoid arthritis synoviocytes, with IL-17F being quantitatively less active . When comparing gene regulation patterns:

• IL-17A induced changes (≥2-fold) in 705 probe sets compared to control

• IL-17F induced changes (≥2-fold) in 438 probe sets compared to control

• A total of 680 distinct probe sets (601 genes) were induced by either IL-17A and/or IL-17F

The heterodimer shows intermediate binding affinity to IL-17RA between the high-affinity IL-17A and low-affinity IL-17F. Interestingly, this binding affinity directly correlates with biological potencies in inducing IL-6 and CXCL1 expression in cell-based assays .

What is the receptor binding mechanism of IL-17A/F?

IL-17A/F signals through the assembly of a heterotrimeric receptor complex comprising IL-17RA and IL-17RC . The binding interface in the IL-17A/F:IL-17RA complex is similar in size to those observed in IL-17A and IL-17F complexes (2,373 Ų of total buried surface compared to 2,219 Ų and 2,294 Ų for IL-17A and IL-17F complexes, respectively) .

The binding interface comprises three main interaction sites, referred to as Sites 1-3:

• Site 1: Located in the skirt region and largely concealed in free IL-17A and IL-17F structures; requires significant receptor-induced conformational changes for binding

• Site 2: A large, pre-formed cavity described as a "pocket in the garment"

• Site 3: Includes additional interfaces that contribute to receptor binding

Unexpectedly, crystallographic studies revealed that IL-17RA can bind to either the "A-face" or "F-face" of the heterodimer with similar affinity, despite IL-17RA showing much higher affinity toward IL-17A than IL-17F homodimers . Similarly, IL-17RC does not discriminate between the two faces of the cytokine heterodimer, enabling the formation of two topologically distinct heterotrimeric complexes with potentially different signaling properties .

How can researchers experimentally distinguish between effects of IL-17A/F and homodimeric forms?

To distinguish between the effects of IL-17A/F heterodimer and homodimeric IL-17A or IL-17F, researchers can employ several methodologies:

• Comparative stimulation experiments: Treat cells with equivalent concentrations of recombinant IL-17A, IL-17F, and IL-17A/F heterodimer, then measure biological responses. The heterodimer typically produces intermediate effects between IL-17A and IL-17F .

• Receptor neutralization: Use specific antibodies against IL-17RA or IL-17RC to determine which receptor components are essential for specific cellular responses. IL-17A/F requires both receptors for signaling .

• Gene expression profiling: Employ microarray or RNA-seq approaches to compare genome-wide expression patterns induced by each cytokine form. Statistical analysis using algorithms like MAS 5.0 can identify differentially regulated genes .

• Using defined recombinant proteins: Biotinylated recombinant proteins like the commercially available Recombinant Human IL-17A/F Heterodimer Biotinylated Protein allow for specific detection and functional studies of the heterodimer .

• Peptide-based approaches: Recently identified essential IL-17-derived peptides (nIL-17) that mimic the activity of full-length cytokines can be used to distinguish common mechanisms from form-specific effects .

What are the molecular mechanisms underlying the synergistic effects between IL-17A/F and TNF-α?

IL-17A/F and TNF-α demonstrate significant synergistic effects in inducing inflammatory responses. This synergy occurs through several mechanisms:

• Receptor upregulation: IL-17A and IL-17F both upregulate TNFRII expression without affecting TNFRI, IL-17RA, or IL-17RC levels. TNFRII blockade inhibits the synergistic induction of CCL20 by IL-17A or IL-17F and TNF-α .

• Gene expression synergy: Using a cooperation index (I') defined as I'(IL-17A, TNF) = H'TNF+IL-17A - (H'TNF + H'IL-17A), researchers identified 130 and 203 genes synergistically induced by IL-17A or IL-17F plus TNF-α, respectively .

• Novel target genes: Among the synergistically regulated genes, CXCR4, LPL, and IL-32 were validated by real-time RT-PCR as new target genes induced by the combination of IL-17A/F and TNF-α .

• Signaling pathway integration: IL-17A/F primarily signals through TRAF6-dependent activation of NF-κB and MAP kinase pathways through the SEFIR domain-containing adaptor protein Act1, which possesses E3 ligase activity . These pathways can integrate with TNF-α signaling to enhance inflammatory responses.

The synergistic interaction between IL-17A/F and TNF-α is particularly relevant in rheumatoid arthritis pathogenesis, where both cytokines are expressed in RA synovium and contribute to disease severity .

How does the nIL-17 peptide sequence contribute to the biological activity of IL-17A/F?

Recent research has identified an essential 20-mer IL-17-derived peptide (nIL-17) that is responsible for the bioactivity of IL-17A, IL-17F, and the IL-17A/F heterodimer . This peptide sequence:

• Mimics full-length cytokine activity: nIL-17 mimics a range of actions elicited by the full-length cytokines, including activation of IL-17RA/C-dependent intracellular signaling .

• Activates cellular responses: The peptide induces activation of NIH-3T3 mouse embryonic fibroblast cells and human dermal blood endothelial cells (HDBECs), leading to increased expression of cytokines, chemokines, and adhesion molecules .

• Promotes leukocyte recruitment: nIL-17 has been shown to promote leukocyte recruitment to pre-inflamed tissues in vivo (air pouch model) and in vitro (to inflamed endothelium) .

• Provides a therapeutic target: Researchers have generated a novel antibody (Ab-IPL-IL-17) that specifically targets the active nIL-17 peptide sequence. This antibody effectively reduces inflammatory processes in preclinical models of immune-mediated inflammatory diseases (IMIDs) and in human clinical samples from inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) .

The identification of this essential sequence has significant implications for understanding IL-17A/F biology/pathogenesis in both mouse and human systems, and offers new approaches for therapeutic intervention.

What is the role of IL-17A/F heterodimer in autoimmune and inflammatory diseases?

IL-17A/F heterodimer plays significant roles in several autoimmune and inflammatory diseases:

• Rheumatoid Arthritis (RA): Both IL-17A and IL-17F are expressed in plasma cell-like cells from RA synovium but not osteoarthritis synovium . The heterodimer is upregulated in immune cells during inflammatory arthritis and contributes to disease severity . In the presence of TNF-α, both IL-17A and IL-17F induce similar expression patterns in RA synoviocytes, suggesting that IL-17F is also a target in Th17-mediated diseases such as RA .

• Psoriasis: IL-17A has been implicated in psoriasis pathogenesis, with anti-IL-17A antibodies showing remarkable clinical efficacy in psoriasis and psoriatic arthritis patients . While IL-17A/F's specific role in psoriasis is less well-characterized than IL-17A, its intermediate potency suggests it may contribute to disease pathology .

• Inflammatory Bowel Disease (IBD): IL-17A/F appears to play a role in IBD, as demonstrated by the effectiveness of antibodies targeting the nIL-17 peptide in reducing inflammation in human clinical samples from IBD patients .

• Protective immunity: IL-17A and IL-17F protect the skin and mucosal barriers against infectious agents . Both cytokines act on fibroblasts, epithelial cells, and endothelial cells, playing a key role in the recruitment, activation, and migration of neutrophils .

How do current therapeutic approaches target IL-17A/F heterodimer?

Current and emerging therapeutic approaches that target IL-17A/F heterodimer include:

• Anti-IL-17A monoclonal antibodies: While primarily targeting IL-17A, these antibodies may also affect IL-17A/F heterodimer function. They have shown remarkable clinical efficacy in psoriasis and psoriatic arthritis patients .

• Peptide-specific antibodies: Novel approaches include developing antibodies like Ab-IPL-IL-17 that specifically target the essential peptide sequence (nIL-17) responsible for IL-17A/F biological activity . This antibody has shown effectiveness in reducing inflammatory processes in preclinical models of IMIDs and in human clinical samples from IBD and RA .

• Receptor targeting: Since IL-17A/F signals through IL-17RA and IL-17RC, therapies targeting these receptors could block signaling from multiple IL-17 family members, including the heterodimer .

• Pathway inhibition: Targeting downstream signaling components like Act1 or TRAF6 could inhibit the effects of IL-17A/F signaling .

Importantly, Ab-IPL-IL-17 has demonstrated effectiveness comparable to reference anti-IL-17 antibodies in reducing inflammatory processes, with potentially lower immunogenicity and fewer adverse hematological side effects in mouse models .

What are the challenges in experimental design when studying IL-17A/F heterodimer?

Researchers face several challenges when designing experiments to study the IL-17A/F heterodimer:

• Heterodimer production and purification: Generating pure IL-17A/F heterodimer without contamination by IL-17A or IL-17F homodimers requires specialized techniques. Biotinylated recombinant proteins can help ensure specificity .

• Distinguishing heterodimer from homodimer effects: Given the overlapping functions and receptor usage of IL-17A, IL-17F, and IL-17A/F, it can be challenging to attribute specific cellular responses to the heterodimer .

• Receptor complex heterogeneity: The ability of IL-17A/F to form two topologically distinct receptor complexes (binding IL-17RA to either the A-face or F-face) introduces complexity in understanding signaling outcomes .

• Context-dependent effects: The synergistic interactions with other cytokines like TNF-α and the tissue-specific responses to IL-17A/F add layers of complexity to experimental design and data interpretation .

• Species differences: While human and mouse IL-17A and IL-17F share substantial sequence homology (61% and 56% respectively), experiments must account for potential species-specific differences in IL-17A/F biology .

How do structural differences in IL-17A/F impact receptor binding and signaling outcomes?

The unique structural features of IL-17A/F heterodimer significantly impact receptor binding and downstream signaling:

• Dual receptor binding faces: Unlike homodimers, IL-17A/F presents two distinct faces ("A-face" and "F-face") for receptor binding. Surprisingly, IL-17RA can bind to either face with similar affinity, despite showing much higher affinity for IL-17A than IL-17F homodimers .

• Receptor-induced conformational changes: Significant receptor-induced conformational changes occur in the skirt region of IL-17A/F, affecting both subunits and enabling binding at Site 1 . In the free IL-17A/F structure, Site 2A is open while Site 2F is masked by the flexible coil, requiring a large shift of this structural element for receptor binding .

• Masked binding sites: In the X-ray structure of free IL-17A/F, Site 1F is blocked by several residues of the A-subunit (Lys39 to Arg43), notably Phe41, whose side-chain occupies the pocket where Trp62 of IL-17RA binds. These cytokine residues are displaced by the receptor in the complex .

• Non-conservative interface changes: Non-conservative amino acid changes at the heterodimer interface are tolerated through solvent exposure of the side chains or structural adaptations that exploit new opportunities for inter-chain contacts .

These structural features likely contribute to the intermediate binding affinity and biological potency of IL-17A/F compared to the respective homodimers, and may enable unique signaling outcomes through the formation of topologically distinct receptor complexes.

What emerging methodologies might advance our understanding of IL-17A/F biology?

Several emerging methodologies hold promise for advancing our understanding of IL-17A/F biology:

• Single-cell technologies: Single-cell RNA sequencing and mass cytometry could reveal cell-specific responses to IL-17A/F and identify previously unrecognized cellular sources of the heterodimer.

• CRISPR-Cas9 gene editing: Precise modification of IL-17A, IL-17F, and receptor genes could help dissect the specific contributions of each component to heterodimer function.

• Structural biology approaches: Advanced techniques like cryo-electron microscopy could further elucidate the conformational dynamics of IL-17A/F-receptor interactions.

• Biomarker development: The identification of specific biomarkers for IL-17A/F activity could improve the monitoring of therapeutic interventions in clinical settings.

• Peptide-based tools: The recently identified nIL-17 peptide sequence provides a new tool for studying essential aspects of IL-17A/F biology and developing targeted therapeutics with potentially fewer side effects .

How might targeting IL-17A/F specifically (versus pan-IL-17 approaches) impact therapeutic outcomes?

Selective targeting of IL-17A/F heterodimer compared to pan-IL-17 approaches could offer several advantages:

• Preserved beneficial immunity: Selective targeting might maintain protective functions of other IL-17 family members while inhibiting pathogenic IL-17A/F activity.

• Reduced side effects: More selective targeting could potentially reduce adverse effects associated with broader IL-17 inhibition, such as increased susceptibility to certain infections.

• Disease-specific intervention: Given the varying contributions of different IL-17 forms to specific diseases, selective IL-17A/F targeting might be particularly beneficial in conditions where the heterodimer plays a predominant role.

• Novel peptide-based approaches: The Ab-IPL-IL-17 approach targeting the essential nIL-17 peptide sequence has shown effectiveness comparable to reference anti-IL-17 antibodies, with lower immunogenicity and fewer adverse hematological side effects in mouse models .

• Dual receptor targeting: Given IL-17A/F's requirement for both IL-17RA and IL-17RC, selective targeting of the heterodimer might be achieved by interfering with the specific binding interfaces involved in heterodimer-receptor interactions .