Recombinant Mouse Interleukin-17A (Il17a), partial (Active)

What is mouse IL-17A and what are its key structural characteristics?

Mouse IL-17A is a proinflammatory cytokine belonging to the IL-17 family, with a predicted molecular weight of approximately 15.0 kDa. The protein consists of 133 amino acids with the sequence: AAIIPQSSAC PNTEAKDFLQ NVKVNLKVFN SLGAKVSSRR PSDYLNRSTS PWTLHRNEDP DRYPSVIWEA QCRHQRCVNA EGKLDHHMNS VLIQQEILVL KREPESCPFT FRVEKMLVGV GCTCVASIVR QAA . It functions as an effector cytokine in both innate and adaptive immune systems, playing crucial roles in antimicrobial host defense and maintenance of tissue integrity . The protein structure contributes to its ability to form homodimers and heterodimers with other IL-17 family members, particularly IL-17F, which affects its biological potency and receptor binding properties. Recombinant forms of the protein typically encompass amino acids 26-158, representing the mature secreted form of IL-17A without the signal peptide .

How does IL-17A signaling work at the molecular level?

IL-17A signals through a heterodimeric receptor complex composed of IL-17RA and IL-17RC subunits. Upon binding to this receptor complex, IL-17A triggers homotypic interaction of the receptor chains with the adapter protein TRAF3IP2 . This interaction initiates downstream signaling through TRAF6-mediated activation of both NF-kappa-B and MAP kinase pathways . The signaling cascade ultimately results in transcriptional activation of various inflammatory mediators, including cytokines, chemokines, antimicrobial peptides, and matrix metalloproteinases . Research indicates that IL-17A signaling demonstrates pathway redundancy, as inhibition of individual downstream components like p38 or NF-κB p65 did not significantly decrease IL-17A-induced gene expression in chondrocytes or synovial fibroblasts . This comprehensive signaling network explains IL-17A's potent proinflammatory effects and its involvement in numerous inflammatory conditions.

What cell types produce IL-17A and which cells respond to it?

IL-17A is predominantly produced by activated CD4+ and CD8+ T lymphocytes, particularly by T-helper 17 (Th17) cells, for which it serves as a signature effector cytokine . Additionally, a subset of gamma-delta T cells produces IL-17A as part of an inflammatory circuit downstream of IL-1β, TLR2, and IL-23A-IL-12B . In terms of responsive cells, IL-17A primarily acts on cells expressing the IL-17RA/IL-17RC receptor complex, which includes a wide range of cell types. In airway epithelium, IL-17A mediates neutrophil chemotaxis by inducing CXCL1 and CXCL5 chemokine production . It also stimulates fibroblasts, epithelial cells, and macrophages to release IL-8 and prostaglandins . Research on osteoarthritis has demonstrated that both chondrocytes and synovial fibroblasts express IL-17RA and IL-17RC and respond to IL-17A stimulation with significant transcriptional changes . This broad cellular responsiveness explains IL-17A's central role in coordinating inflammatory responses across multiple tissue types and disease states.

What are the optimal storage and reconstitution conditions for recombinant mouse IL-17A?

Recombinant mouse IL-17A is typically shipped in lyophilized form at room temperature and requires proper reconstitution for optimal activity . For reconstitution, researchers should use sterile phosphate-buffered saline (PBS) containing an appropriate carrier protein, such as bovine serum albumin (BSA) or directly in cell assay media . The addition of a carrier protein (minimum 0.1%) is critical for preventing protein loss through adsorption to tubes or plates and maintaining stability in solution . Once reconstituted, the protein should be aliquoted to avoid repeated freeze-thaw cycles, which can diminish biological activity. For short-term storage (1-2 weeks), the reconstituted protein can be kept at 2-8°C, while long-term storage requires -20°C to -80°C conditions with carrier protein present. It's important to note that reconstitution conditions may vary slightly between manufacturers, so researchers should always verify specific recommendations for their particular product. Proper handling ensures maintained biological activity for experimental applications and improves reproducibility across experiments.

How can researchers validate the activity of recombinant mouse IL-17A in vitro?

Validating the activity of recombinant mouse IL-17A requires multiple complementary approaches to ensure both structural integrity and functional competence. Structurally, researchers should verify protein purity (typically >95%) using SDS-PAGE analysis to confirm the expected molecular weight of approximately 15.0 kDa . For functional validation, several bioassays can be employed. The most common approach involves stimulating responsive cell lines (such as fibroblasts or epithelial cells) with the recombinant IL-17A and measuring the induction of downstream targets. Key readouts include quantification of induced cytokines (IL-6, TNF-α), chemokines (CXCL1, CXCL5), or matrix metalloproteinases (particularly MMP1) using ELISA or qPCR . Gene expression analysis following IL-17A treatment should demonstrate upregulation of canonical targets such as IL6, NFKBIZ, and MMP1, as these were identified as significantly upregulated in response to IL-17A stimulation . Additionally, researchers can validate signaling pathway activation by assessing phosphorylation of p38 MAPK or NF-κB p65 through Western blotting. Including appropriate positive controls (commercially validated IL-17A) and negative controls (vehicle treatment) is essential for proper validation.

What are recommended concentrations of recombinant mouse IL-17A for different experimental applications?

The optimal concentration of recombinant mouse IL-17A varies depending on the specific experimental application, target cells, and desired readout. For transcriptome analysis and general in vitro stimulation of primary cells (chondrocytes and synovial fibroblasts), a concentration of 10 ng/ml has been shown to induce significant transcriptional changes . This concentration effectively activated signaling pathways and induced expression of target genes like IL6, PDPN, and NFKBIZ . For in vivo protection studies against fungal pathogens like Candida albicans, IL-17A has demonstrated protective effects, though specific dosing must be carefully calibrated for the particular disease model and administration route . When using IL-17A inhibitors, such as the IL-17A antibody secukinumab, corresponding concentrations ranging from 0.5 to 50 μg/ml may be appropriate for in vitro neutralization experiments, with 50 μg/ml showing significant inhibition of IL-17A-induced gene expression . It's important to note that dose-response curves should be established for each experimental system, as cellular responsiveness can vary based on receptor expression levels and other factors influencing IL-17A sensitivity.

What controls should be included when using recombinant mouse IL-17A in experiments?

Proper experimental design with recombinant mouse IL-17A requires several critical controls to ensure valid and interpretable results. Vehicle controls (buffer with carrier protein identical to IL-17A reconstitution buffer) are essential baseline controls for all experiments to account for any effects from the diluent itself. Positive stimulation controls using well-characterized inflammatory mediators (such as TNF-α or IL-1β) help confirm cellular responsiveness in situations where IL-17A response is unexpectedly low. For functional studies, pathway inhibitor controls are valuable, including specific IL-17A neutralizing antibodies (like secukinumab at 5-50 μg/ml) to verify that observed effects are specifically attributable to IL-17A signaling . When studying gene expression, examining housekeeping genes such as GAPDH and ACTB is important for normalization and ensuring equal sample loading . Additionally, time-course experiments can serve as internal controls by demonstrating the expected temporal pattern of IL-17A response. For in vivo experiments, isotype-matched antibody controls should be used alongside IL-17A neutralizing antibodies. These comprehensive controls help distinguish direct IL-17A effects from experimental artifacts and provide confidence in the specificity and validity of observed responses.

How does IL-17A contribute to fungal defense mechanisms in mouse models?

IL-17A plays a critical role in host defense against fungal pathogens, particularly Candida albicans, as demonstrated in murine models of systemic candidiasis. Studies with IL-17A receptor knockout (IL-17AR-/-) mice revealed substantially reduced survival following systemic Candida challenge, with dramatically increased fungal burden in the kidneys (25-fold increase at 96 hours post-infection) . The protective mechanism involves IL-17A-mediated mobilization of peripheral neutrophils and their recruitment to infected tissues—processes significantly impaired and delayed in the absence of IL-17A signaling . In normal mice, expression of IL-17A provided protection against otherwise lethal doses of C. albicans, with 100% survival at day 7 and 65% survival at day 42 post-infection . Mechanistically, IL-17A interconnects adaptive and innate immunity, activating signaling cascades that induce antimicrobial peptides and inflammatory mediators crucial for pathogen clearance . These findings establish the mIL-17A/mIL-17AR system as essential for normal antifungal host defense in vivo, suggesting potential therapeutic applications for recombinant IL-17A in treating systemic fungal infections in immunocompromised patients with conditions such as cancer or advanced AIDS .

What is the role of IL-17A in osteoarthritis models and how does it affect joint tissue?

IL-17A exerts significant effects on joint tissues in osteoarthritis (OA) models by inducing transcriptional changes associated with inflammation and tissue degradation. In studies using primary cells derived from end-stage OA patients, both chondrocytes and synovial fibroblasts expressed IL-17 receptors (IL-17RA and IL-17RC) and responded robustly to IL-17A stimulation . Transcriptome analysis revealed that IL-17A treatment significantly altered the expression of numerous genes involved in inflammation, extracellular matrix degradation, and cellular signaling . The most prominently upregulated gene in both cell types was MMP1, which encodes a matrix metalloproteinase involved in cartilage degradation, suggesting a direct mechanistic link between IL-17A and joint destruction . IL-17A-induced transcriptional changes in these cells were associated with experimental arthritis, knee arthritis, and musculoskeletal disease gene sets, indicating broad relevance to joint pathology . The signaling pathways activated by IL-17A in joint cells include NF-κB and MAP kinase cascades, though inhibition studies suggest functional redundancy in these pathways. These findings suggest that IL-17A signaling may represent a potential therapeutic target in OA, with IL-17A neutralizing antibodies like secukinumab showing promise in inhibiting the IL-17A-induced inflammatory response in joint cells .

How does blocking IL-17A signaling affect immune responses in mouse models?

Blocking IL-17A signaling in mouse models produces significant and context-dependent effects on immune responses. In fungal infection models, disruption of IL-17A signaling through IL-17A receptor knockout substantially impairs antifungal immunity, compromising neutrophil mobilization and recruitment to infected tissues, resulting in increased pathogen burden and reduced survival . This underscores IL-17A's essential role in coordinating effective antimicrobial immune responses. In contrast, in inflammatory disease contexts like osteoarthritis, blocking IL-17A signaling with neutralizing antibodies such as secukinumab significantly inhibits the expression of inflammatory mediators including IL-6 and tissue-destructive enzymes like MMP1 in joint tissues . Specifically, 50 μg/ml secukinumab significantly inhibited IL-17A-induced gene expression in chondrocytes, while in synovial fibroblasts, even 5 μg/ml caused significant decreases in IL-17A-induced IL6, PDPN, and NFKBIZ expression . These divergent effects highlight the dual nature of IL-17A signaling—beneficial in infectious contexts but potentially harmful in chronic inflammatory conditions. The precise immunological consequences of IL-17A blockade depend on the disease model, tissue context, timing of intervention, and compensatory mechanisms involving other cytokines, making careful experimental design essential when studying IL-17A neutralization.

What disease models are appropriate for studying IL-17A function?

Several disease models are particularly suitable for investigating IL-17A function, each highlighting different aspects of this cytokine's roles in health and disease. Fungal infection models, especially systemic Candida albicans challenge, effectively demonstrate IL-17A's critical role in antifungal host defense by assessing survival, fungal burden, and neutrophil recruitment in wild-type versus IL-17AR knockout mice . For studying IL-17A in joint pathology, both in vitro models using primary chondrocytes and synovial fibroblasts from osteoarthritis patients and in vivo inflammatory arthritis models provide insights into IL-17A's contribution to joint inflammation and tissue destruction . Experimental autoimmune encephalomyelitis (EAE) models are valuable for examining IL-17A's role in autoimmune neuroinflammation, while psoriasis-like skin inflammation models highlight its function in dermatological conditions. Airway inflammation models can reveal IL-17A's contribution to neutrophilic recruitment via induction of CXCL1 and CXCL5 chemokines in respiratory pathologies . Additionally, germinal center formation in secondary lymphoid organs can be studied to understand IL-17A's influence on B cell responses, including their chemotactic response to CXCL12 and CXCL13, retention within germinal centers, somatic hypermutation, and plasma cell selection . These diverse models collectively provide a comprehensive understanding of IL-17A function across multiple physiological and pathological contexts.

How do post-translational modifications affect IL-17A function and signaling?

Post-translational modifications (PTMs) significantly influence IL-17A function and signaling efficacy, potentially altering receptor binding affinity, protein stability, and biological activity. The production system for recombinant IL-17A is particularly important in this regard—proteins expressed in yeast systems (like Pichia pastoris) undergo natural folding and post-translational modifications that more closely resemble native mammalian modifications compared to E. coli-derived proteins . This difference results in superior functionality of yeast-expressed IL-17A for research applications . While the specific PTMs of mouse IL-17A are not fully characterized in the provided search results, glycosylation patterns likely play a role in modulating protein half-life and receptor interactions. Researchers investigating IL-17A signaling should consider that differences in PTMs between recombinant and native IL-17A may influence experimental outcomes. When selecting recombinant IL-17A for experiments, preference should be given to preparations that maintain the protein's native conformation and modification state. Additionally, when interpreting contradictory findings between studies, differences in the source and modification state of IL-17A preparations should be considered as potential contributing factors to discrepancies in observed biological activities.

What are the key differences between yeast-derived and E. coli-derived recombinant mouse IL-17A?

The expression system used to produce recombinant mouse IL-17A significantly impacts its structural characteristics and functional properties. Yeast-derived IL-17A (from systems like Pichia pastoris) undergoes natural folding and post-translational modifications that more closely resemble those of native mammalian proteins, resulting in superior functionality compared to E. coli-derived proteins . This difference arises because prokaryotic systems like E. coli lack the cellular machinery for eukaryotic post-translational modifications and may produce proteins with incorrect folding, potentially compromising biological activity. High-quality recombinant mouse IL-17A preparations should be free from endotoxins, HIS-TAGS, and carriers, closely resembling the native form of the protein . The purification process also differs between expression systems, with yeast-derived IL-17A typically purified using ion-exchange chromatography to achieve >95% purity . When selecting recombinant IL-17A for experiments, researchers should consider these production differences and their potential impact on experimental outcomes. For applications requiring physiologically relevant activity profiles, yeast-derived IL-17A may be preferable, particularly for complex cellular assays or in vivo studies where proper protein folding and modification are critical for accurate modeling of IL-17A biology.

How can researchers distinguish between direct IL-17A effects and secondary inflammatory responses?

Distinguishing direct IL-17A effects from secondary inflammatory cascades requires strategic experimental approaches with appropriate controls and temporal considerations. To identify direct effects, researchers should implement short-term stimulation experiments (30 minutes to 4 hours) to capture immediate transcriptional changes and signaling events before secondary mediators accumulate. Comparing gene expression profiles in early versus late timepoints helps differentiate primary from secondary responses. Pathway inhibition studies using specific blockers of IL-17A signaling components, alongside IL-17A neutralizing antibodies like secukinumab, can help establish causal relationships between IL-17A signaling and observed outcomes . Experiments in conditioned media transfer systems, where media from IL-17A-stimulated cells is transferred to naive cells, with or without IL-17A neutralization, can identify effects mediated by secreted factors versus direct IL-17A signaling. Additionally, genetic approaches using cells with receptor knockdown/knockout (IL-17RA/IL-17RC) compared to wild-type cells provide definitive evidence for direct IL-17A dependence. RNA-sequencing analysis combined with bioinformatic pathway mapping can further distinguish primary IL-17A response genes from secondary inflammation signatures. These complementary strategies collectively enable more precise attribution of biological effects to direct IL-17A action versus downstream inflammatory cascades.

What approaches can be used to study IL-17A in the context of other IL-17 family members?

Studying IL-17A within the broader context of other IL-17 family members requires integrative approaches that address potential functional redundancy, synergy, and distinct signaling properties. Comparative transcriptome analysis represents a powerful strategy, as demonstrated by studies comparing the transcriptional responses induced by IL-17A, IL-17F, and IL-17AF heterodimer in chondrocytes and synovial fibroblasts . This approach revealed hierarchical potency (IL-17A > IL-17AF > IL-17F) and identified both shared and cytokine-specific gene expression patterns . Receptor expression profiling using techniques like RT-qPCR and immunohistochemistry to characterize IL-17RA and IL-17RC expression patterns across different tissues and disease states provides crucial context for interpreting IL-17 family member effects . Utilizing genetic models with selective knockout of individual IL-17 family members or their receptors enables assessment of non-redundant functions in vivo. For mechanistic studies, recombinant proteins of consistently high quality should be used at equimolar concentrations to enable valid comparisons of signaling potency and specificity. Additionally, neutralizing antibodies with defined specificities for individual IL-17 family members can help dissect their respective contributions to complex inflammatory phenotypes. Finally, multi-omics approaches integrating transcriptomics, proteomics, and metabolomics data can provide comprehensive views of how IL-17 family members collectively shape immune and inflammatory responses in different physiological and pathological contexts.