IL 9 Rat

What is rat IL-9 and what are its essential biological characteristics?

Rat Interleukin-9 (IL-9) is a 20-30 kDa secreted glycoprotein belonging to the IL-7/IL-9 family of hematopoietic cytokines . It is primarily produced by activated T helper 2 (Th2) lymphocytes and functions as a regulatory cytokine in various immunological processes . The rat IL-9 cDNA encodes a 144 amino acid precursor protein with an 18 amino acid signal peptide that is cleaved to form the mature cysteine-rich protein with a molecular mass of approximately 14 kDa, though the native protein is heavily glycosylated . The gene identifier for rat IL-9 is 116558 with the gene symbol Il9, and protein aliases include ILN, Interleukin, Interleukin9, and interleukin-9 .

How does rat IL-9 compare to mouse and human IL-9?

While the search results do not provide extensive comparative data, they indicate similarities between rat and mouse IL-9. Like mouse IL-9, rat IL-9 appears to function in T-cell differentiation and immune regulation. Both species have IL-9 proteins that undergo post-translational modifications, particularly glycosylation, which affects their molecular weight and functional properties . Research indicates that findings on IL-9-secreting TH9 cells have antitumor properties in both mouse and human cancers, suggesting conserved functional roles across species .

What cell types produce IL-9 in rats?

The primary source of IL-9 in rats is activated T helper 2 (Th2) lymphocytes . More recently, a distinct subset of CD4+ T cells called TH9 cells has been identified as specialized producers of IL-9 . These cells differentiate from naive CD4 T cells when stimulated with transforming growth factor beta (TGF-β) and IL-4 . Additionally, research has shown that TGF-β can reprogram existing Th2 cells to secrete IL-9, effectively converting them to TH9 cells .

What techniques are available for detecting IL-9 in rat samples?

Several validated methods exist for detecting IL-9 in rat samples:

ELISA Assays: Rat IL-9 ELISA kits can quantitate IL-9 in rat serum, plasma, and cell culture supernatants . These assays recognize both natural and recombinant rat IL-9 with high specificity.

Flow Cytometry: Flow cytometric analysis using anti-rat IL-9 antibodies can detect intracellular IL-9 in rat cells. This typically requires cell fixation with paraformaldehyde and permeabilization with saponin . For optimal results, cells may need stimulation with agents such as PMA (phorbol 12-myristate 13-acetate) and calcium ionophore.

Reporter Systems: While not explicitly mentioned for rats, reporter systems like IL-9-GFP have been used in mouse models and could potentially be adapted for rat studies .

How should I prepare rat samples for optimal IL-9 detection?

For flow cytometry detection of intracellular IL-9, rat splenocytes should be treated with appropriate stimulants (e.g., 50 ng/mL PMA and 500 ng/mL Ca2+ ionophore for 24 hours), followed by fixation with paraformaldehyde and permeabilization with saponin before staining with anti-IL-9 antibodies . For ELISA assays, sample preparation will depend on the specific kit instructions, but generally involves collection of serum, plasma, or cell culture supernatants under sterile conditions .

What controls are essential when measuring IL-9 in rat experiments?

When conducting antibody-based detection methods such as flow cytometry, appropriate isotype controls are essential. For anti-rat IL-9 antibodies, a suitable control would be rat IgG1 isotype control (such as clone HRPN) . In neutralization experiments, including both the neutralizing antibody and appropriate isotype controls is critical for result interpretation . When using fluorescently labeled secondary antibodies, include controls with secondary antibody alone to account for non-specific binding .

What are the primary biological functions of IL-9 in rats?

IL-9 in rats serves several important immunoregulatory functions:

• Regulates multiple cell types involved in Th2-associated asthma responses, including B and T lymphocytes, mast cells, eosinophils, and epithelial cells

• Plays a critical role in the antitumor functions of TH9 cells, as demonstrated in adoptive transfer experiments

• Contributes to TH9 cell-mediated immune responses, which are essential for the efficacy of certain cancer immunotherapy treatments such as dendritic cell vaccination and anti-GITR therapy

How is IL-9 production regulated in rat immune cells?

IL-9 production in rats is regulated by a complex network of cytokines and signaling pathways. TGF-β plays a particularly important role, reprogramming Th2 cells to secrete IL-9 and driving the differentiation of TH9 cells . The combination of TGF-β and IL-4 stimulates the differentiation of naive CD4 T cells into IL-9-producing TH9 cells . Proinflammatory factors, including IL-1β and tumor necrosis factor alpha (TNF-α), enhance TH9 differentiation and subsequent IL-9 production . Additionally, recent evidence suggests that STING (Stimulator of Interferon Genes) signaling may influence T cell differentiation and potentially IL-9 production, though the exact mechanism needs further investigation .

What is the functional relationship between IL-9 and TH9 cells in rat models?

TH9 cells are specialized CD4+ T cells that produce high levels of IL-9 . These cells exert IL-9-mediated antitumor functions when adoptively transferred into tumor-bearing mice, suggesting IL-9 is the primary mediator of their anticancer activity . In experimental settings, TH9 cells stimulated with cyclic GMP-AMP synthase (cGAMP) before in vitro polarization showed enhanced antitumor efficacy when adoptively transferred into B16-OVA tumor-bearing mice . This indicates that manipulating TH9 cells can modulate IL-9 production and subsequent antitumor functions.

How is IL-9 involved in rat models of asthma and allergic inflammation?

IL-9 regulates multiple cell types involved in Th2-associated asthma responses, including B and T lymphocytes, mast cells, eosinophils, and epithelial cells . While the search results don't provide specific experimental details for rat asthma models, the involvement of IL-9 in regulating these cell types suggests its importance in allergic airway inflammation. Understanding IL-9's role in these models can provide insights into potential therapeutic targets for asthma and allergic conditions.

What is the role of IL-9 in rat cancer models?

IL-9, primarily through TH9 cells, demonstrates significant antitumor properties in experimental cancer models . When adoptively transferred into tumor-bearing mice, IL-9-secreting TH9 cells exert potent antitumor functions . The experimental protocol typically involves:

• Establishing tumors (e.g., B16-OVA melanoma) in mice

• Generating and potentially manipulating TH9 cells in vitro

• Adoptively transferring 2×10^6 effector TH9 cells intravenously

• Monitoring tumor growth by measuring tumor dimensions three times weekly

In lung metastasis models, 2.5×10^5 B16-OVA cells are injected intravenously, followed by TH9 cell transfer one day later, with lung tumor foci enumerated 14 days post-transfer .

How can IL-9-producing cells be used in adoptive transfer experiments?

The following protocol outlines a method for using IL-9-producing TH9 cells in adoptive transfer experiments:

• Generate effector TH9 cells from naive CD4 T cells (e.g., from OT-II transgenic mice) by stimulating with TGF-β and IL-4

• Optionally stimulate cells with cGAMP or other agents before in vitro polarization to enhance their function

• Inject 2×10^6 effector TH9 cells intravenously into tumor-bearing recipients (typically 5 days after subcutaneous tumor implantation)

• Monitor tumor growth by measuring dimensions three times weekly, euthanizing mice when tumors exceed 300 mm^2 or become ulcerated

For intravenous tumor models, inject tumor cells first, followed by TH9 cell transfer one day later, and evaluate outcomes after 14 days .

How can I establish IL-9 neutralization in rat experimental models?

While the search results don't provide rat-specific protocols, neutralizing antibodies against IL-9 can be used with appropriate isotype controls, such as rat IgG1 isotype control (clone HRPN, BioXCell) . When designing neutralization experiments, consider:

• Determining appropriate antibody dosage through dose-response studies

• Including proper isotype controls at equivalent concentrations

• Validating neutralization efficiency through functional assays

• Administering antibodies at time points relevant to the experimental question

The effectiveness of neutralization should be verified through downstream measurements of IL-9-dependent activities.

What factors should be considered when studying IL-9 in the context of STING signaling?

Recent research highlights the importance of STING (Stimulator of Interferon Genes) signaling in T cell differentiation and function . When studying IL-9 in relation to STING signaling, several factors should be considered:

• Use of appropriate genetic models, such as Sting1^-/- or STING V154M/WT mice, which could be adapted or compared to rat models

• Investigation of how STING agonists like cGAMP affect TH9 cell differentiation and IL-9 production

• Consideration of the interplay between STING, type I interferon signaling, and IL-9 production, potentially using Ifnar^-/- models

• Assessment of how STING signaling affects the antitumor functions of IL-9-producing cells in vivo

How should researchers interpret conflicting data on IL-9 functions?

When confronted with conflicting data on IL-9 functions, researchers should:

• Carefully evaluate the experimental systems used, as IL-9 function may be context-dependent

• Consider strain differences in rats, as genetic background can influence cytokine responses

• Examine the specific cell types and activation conditions used, as these can dramatically affect IL-9 production and function

• Assess the presence of other cytokines in the experimental system, as cytokine networks involve complex interactions

• Compare in vitro versus in vivo results, recognizing that cell culture findings may not always translate to whole animal models

• Verify antibody specificity and detection method sensitivity, as technical variations can lead to apparently conflicting results

What are the key properties of rat IL-9 for experimental planning?

Table 1: Physical and Biochemical Properties of Rat IL-9

What factors influence TH9 cell differentiation and IL-9 production?

Table 2: Regulators of TH9 Differentiation and IL-9 Production

How should I design experiments to study IL-9 functions in rat models?

Table 3: Experimental Approaches for Studying IL-9 in Rats

What are the promising new areas of rat IL-9 research?

Emerging areas in rat IL-9 research include:

• Further exploration of the role of STING signaling in controlling TH9 cell differentiation and IL-9 production

• Investigation of the mechanisms by which IL-9-producing cells mediate antitumor immunity and how this can be enhanced

• Development of more sophisticated genetic models to study IL-9 function in vivo

• Examination of IL-9's role in neuroinflammation and potential neuroimmune interactions

• Exploration of IL-9 as a therapeutic target or agent in various disease contexts

How might epigenetic regulation influence IL-9 expression in rats?

While the search results don't directly address epigenetic regulation of IL-9, this represents an important frontier in cytokine research. Investigators might consider:

• Examining histone modifications at the Il9 locus during TH9 cell differentiation

• Investigating DNA methylation patterns that correlate with IL-9 expression

• Studying the role of microRNAs in post-transcriptional regulation of IL-9

• Exploring how environmental factors might induce epigenetic changes affecting IL-9 production

• Comparing epigenetic signatures between different IL-9-producing cell populations

What translational potential exists for rat IL-9 research findings?

Findings from rat IL-9 research have several potential translational applications:

• Development of IL-9-based or IL-9-targeting therapeutics for cancer immunotherapy, given the potent antitumor properties of IL-9-producing TH9 cells

• Exploration of the essential role of IL-9 in the efficacy of dendritic cell vaccination and anti-GITR therapy, suggesting combination approaches for enhanced cancer treatment

• Investigation of IL-9 manipulation for allergic and inflammatory conditions, given its role in regulating cells involved in asthma responses

• Consideration of the TGF-β/IL-9 axis as a therapeutic target in various disease contexts