IL 17A/F Rat

What is IL-17A/F Rat and how does it differ structurally from IL-17A and IL-17F homodimers?

IL-17A/F Rat is a disulfide-linked heterodimeric glycoprotein composed of two distinct proteins from the IL-17 family: IL-17A and IL-17F. The heterodimer consists of one monomeric subunit of each protein, creating a unique cytokine with intermediate biological properties between the two parent proteins. Structurally, recombinant rat IL-17A/F contains 269 amino acids with a total molecular mass of 30.7 kDa when produced in E. coli .

The constituent proteins share 50% amino acid sequence identity with each other, and both contain a cysteine knot motif with two disulfide bonds that is characteristic of the IL-17 family . The rat IL-17A precursor consists of 155 amino acids (including a 23-amino acid signal sequence), while the rat IL-17F precursor consists of 153 amino acids (with a 20-amino acid signal sequence) . Both monomers contain N-linked glycosylation sites, though recombinant products from E. coli are non-glycosylated .

What are the biological activities of IL-17A/F Rat and how does its potency compare to homodimers?

IL-17A/F Rat exhibits biological activities that position it between IL-17A and IL-17F homodimers in terms of potency. Specifically, IL-17A/F induces chemokine production and airway neutrophilia with intermediate potency, with IL-17A being the most potent and IL-17F being the least potent of the three cytokine forms .

This intermediate activity profile makes IL-17A/F particularly useful in experimental systems where researchers need a cytokine with moderate inflammatory properties. The heterodimer binds to the IL-17RA receptor, which forms a heterodimeric complex with IL-17RC for signal transduction . This binding initiates downstream signaling cascades that promote inflammatory gene expression, particularly chemokines that attract neutrophils and other immune cells to sites of inflammation.

Which cell types produce IL-17A/F in rats and what stimuli regulate its expression?

IL-17A/F is primarily produced by activated CD4+ T cells, specifically the Th17 subset of helper T cells . The production of this heterodimer is regulated by various cytokines, with IL-23 being a particularly important stimulus that causes Th17 lymphocytes to manufacture IL-17A/F .

In pathological conditions such as heart failure, increased IL-17A expression has been observed in the paraventricular nucleus (PVN) of the brain, indicating that neuronal or glial cells may also be sources of IL-17 cytokines under certain conditions . Research has demonstrated that in rat models of heart failure induced by coronary artery ligation, IL-17A levels increase significantly in both plasma and cerebrospinal fluid, with peak levels observed approximately 4 weeks after induction of myocardial infarction .

What are the optimal methods for detecting IL-17A/F expression in rat experimental models?

When examining IL-17A/F expression in rat models, researchers should consider multiple detection approaches:

• mRNA expression analysis: Quantitative RT-PCR can be used to measure IL-17A and IL-17F mRNA expression in various tissues. In brain tissue studies, researchers have successfully quantified IL-17A, IL-17RA, and IL-17RC mRNA expression in specific regions such as the PVN .

• Protein quantification: IL-17A/F protein levels can be measured in bodily fluids using enzyme-linked immunosorbent assays (ELISAs). In cardiac dysfunction studies, researchers have detected IL-17A in both plasma and cerebrospinal fluid (CSF) .

• Immunohistochemistry/Immunofluorescence: For tissue localization, confocal immunofluorescent imaging has been employed to detect IL-17RA expression in brain tissue. This technique allows visualization of receptor distribution in specific cell types when combined with cell-specific markers like NeuN for neurons .

When correlating IL-17A/F levels between compartments, linear regression analysis has revealed positive correlations between plasma and CSF levels, suggesting that peripheral inflammation may influence central nervous system IL-17A levels .

How can genetic manipulation approaches be optimized for IL-17A/F pathway studies in rats?

For genetic manipulation of the IL-17A/F pathway in rats, adeno-associated virus (AAV) vector-mediated RNA interference has proven effective:

• AAV serotype selection: AAV9 vectors have been successfully used for central nervous system delivery, particularly for targeted delivery to specific brain nuclei such as the PVN .

• siRNA design considerations: When targeting IL-17RA, specific siRNA sequences can be designed to selectively reduce receptor expression without affecting related receptors like IL-17RC, demonstrating the specificity of this approach .

• Verification of knockdown efficacy:

  • Molecular verification: qRT-PCR measurement of target gene expression should be performed to confirm significant reduction of IL-17RA mRNA levels .

  • Functional verification: Downstream effects on inflammatory mediators can serve as functional readouts of successful knockdown .

  • Visualization of transduction: Including reporter genes such as GFP in the viral construct allows visual confirmation of successful transduction and accurate targeting .

• Stereotaxic delivery parameters: For PVN targeting, bilateral microinjections (0.3 μl of 10^12 viral particles/ml) have demonstrated good coverage of the target nucleus .

What are the key considerations when interpreting IL-17A/F-related data in neuroinflammation and cardiac dysfunction models?

When investigating IL-17A/F in models linking neuroinflammation to cardiac dysfunction, several interpretive frameworks should be considered:

• Temporal dynamics: IL-17A levels in plasma show a specific time course after cardiac injury, with progressive increases beginning within one week and peaking at approximately 4 weeks after coronary artery ligation . This temporal pattern should be considered when designing intervention studies.

• Regional specificity within the brain: IL-17RA expression varies across PVN subnuclear regions (dorsal parvocellular, medial parvocellular, ventrolateral parvocellular, and posterior magnocellular), with differential upregulation in heart failure . This necessitates precise anatomical targeting in experimental manipulations.

• Cell-type specificity: IL-17RA is expressed in both neuronal and non-neuronal elements of the PVN, requiring careful interpretation of which cell types mediate observed effects .

• Pathway interactions: When manipulating IL-17 signaling, researchers should monitor effects on multiple downstream inflammatory mediators, as IL-17A/F induces various proinflammatory cytokines and chemokines that may have distinct contributions to the observed phenotypes .

What physiological parameters should be assessed when studying IL-17A/F effects in rat cardiovascular models?

When evaluating IL-17A/F contributions to cardiovascular pathophysiology, comprehensive assessment should include:

• Cardiac function parameters:

  • Left ventricular end-diastolic pressure (LVEDP)

  • Left ventricular pressure-systolic peak (LVPSP)

  • Maximum rate of left ventricular pressure development (LV dP/dt max)

  • Systolic blood pressure (SBP)

  • Heart rate

• Anatomical measurements:

  • Body weight (BW)

  • Left ventricle-to-body weight ratio (LV/BW)

  • Heart weight-to-body weight ratio (HW/BW)

• Neuroinflammatory markers:

  • mRNA expression of proinflammatory cytokines in the PVN

  • Sympathetic nerve activity measurements

In experimental studies, IL-17RA knockdown in the PVN of heart failure rats has been shown to improve several cardiac parameters, particularly increasing LV dP/dt max and decreasing LVEDP compared to control rats, indicating improved cardiac contractility and reduced congestion .

How can correlation analyses between central and peripheral IL-17A/F levels inform experimental design?

Analysis of the relationship between central (CSF) and peripheral (plasma) IL-17A levels provides important insights for experimental design:

This correlation suggests several experimental considerations:

• Sampling timepoints: The peak elevation at 4 weeks post-cardiac injury indicates an optimal window for intervention studies .

• Biomarker potential: Plasma IL-17A may serve as an accessible biomarker reflecting central neuroinflammatory processes .

• Mechanistic hypotheses: The correlation raises questions about whether peripheral IL-17A crosses the blood-brain barrier or whether systemic inflammation triggers local IL-17A production in the CNS .

• Intervention timing: The progressive increase in IL-17A levels suggests early intervention might be necessary to prevent the establishment of neuroinflammation .

What are the optimal reconstitution and storage conditions for recombinant IL-17A/F Rat protein?

Recombinant IL-17A/F Rat protein is typically supplied as a sterile filtered white lyophilized (freeze-dried) powder. For optimal handling:

• Reconstitution: The lyophilized protein should be reconstituted from its concentrated form (1mg/ml), which contains no additives . While specific buffer recommendations were not provided in the search results, typical reconstitution buffers for cytokines include sterile PBS or other physiological buffers, potentially with carrier protein to prevent adhesion to tubes.

• Functional testing: After reconstitution, functional activity should be verified using appropriate bioassays, such as chemokine induction in target cells or neutrophil migration assays .

• Source considerations: The search results indicate recombinant rat IL-17A/F can be produced in E. coli expression systems, resulting in a non-glycosylated form of the protein that maintains biological activity but may differ from the native glycosylated form in certain properties .

What are the key experimental controls when studying IL-17A/F signaling in neuroinflammation models?

When investigating IL-17A/F signaling in neuroinflammation, particularly in the context of cardiac dysfunction models, several experimental controls are essential:

• Genetic manipulation controls:

  • Scrambled siRNA (Scr siRNA) controls should be used to account for non-specific effects of viral delivery and RNA interference .

  • Validation of knockdown specificity by demonstrating reduced expression of the target gene (e.g., IL-17RA) without affecting related genes (e.g., IL-17RC) .

• Surgical controls:

  • Sham-operated controls that undergo the same surgical procedures without the actual disease induction (e.g., sham operation versus coronary artery ligation) .

  • Age-matched naive controls to distinguish effects of surgical manipulation from disease-specific processes.

• Regional specificity controls:

  • When targeting specific brain nuclei such as the PVN, verification of injection site accuracy through reporter gene expression (e.g., GFP) or post-mortem histological analysis .

  • Analysis of multiple subnuclear regions within structures like the PVN to account for heterogeneity in receptor expression and response .

How can researchers effectively integrate IL-17A/F studies with broader neuroinflammatory pathway analysis?

To comprehensively understand IL-17A/F's role within the broader neuroinflammatory landscape:

• Multilevel analysis approach:

  • Molecular: Measure expression of multiple inflammatory mediators beyond IL-17A/F itself, including downstream cytokines and chemokines induced by IL-17 signaling .

  • Cellular: Identify the specific cell types expressing IL-17 receptors (using markers like NeuN for neurons) and those producing inflammatory mediators in response to IL-17A/F .

  • Systems: Evaluate physiological outcomes such as sympathetic nervous system activity and cardiac function parameters to link molecular changes to systemic effects .

• Temporal considerations:

  • Design longitudinal studies that capture the evolution of inflammatory processes, recognizing that IL-17A levels follow specific temporal patterns after initial injury .

  • Consider both acute and chronic phases of inflammation, as IL-17A/F may have different roles at different stages.

• Pathway interaction analysis:

  • Explore interactions between IL-17 signaling and other neuroinflammatory pathways.

  • Consider combined interventions targeting multiple inflammatory mediators to identify synergistic or redundant mechanisms.

How might comparative studies of IL-17A/F between species inform translational research?

The search results indicate that rat and human IL-17 family members show approximately 60% amino acid sequence homology . This partial conservation suggests:

• Cross-species validation requirements: Findings in rat models require careful validation before translation to human applications, particularly given the moderate degree of sequence homology.

• Comparative receptor binding studies: Differences in receptor binding properties between species could inform the development of species-specific therapeutic approaches targeting the IL-17A/F pathway.

• Evolutionary insights: Comparative studies across multiple species could reveal evolutionarily conserved versus species-specific aspects of IL-17 biology, potentially identifying core functions that are most likely to translate across species.

• Humanized models: Development of rat models expressing human IL-17 pathway components could bridge the translation gap and provide more directly relevant preclinical data.

What novel therapeutic approaches might emerge from targeting IL-17A/F signaling in neuroinflammatory conditions?

Based on findings that IL-17A/F signaling contributes to neuroinflammation and associated cardiac dysfunction, several therapeutic approaches warrant investigation:

• CNS-targeted IL-17 pathway interventions:

  • Region-specific delivery of IL-17RA antagonists to structures like the PVN could modulate neuroinflammation without systemic immunosuppression .

  • Development of small molecule inhibitors capable of crossing the blood-brain barrier to target IL-17 signaling in specific neural circuits.

• Combination approaches:

  • Targeting IL-17A/F signaling alongside other neuroinflammatory pathways could provide synergistic benefits.

  • Combining IL-17 pathway modulation with conventional heart failure therapies might address both peripheral and central contributions to disease progression.

• Biomarker development:

  • The correlation between plasma and CSF IL-17A levels suggests potential for using plasma IL-17A as a biomarker to guide patient selection for anti-inflammatory interventions .

  • Longitudinal monitoring of IL-17A could help identify optimal intervention windows.