IL 9 Human

What is Interleukin-9 and how was it discovered?

Interleukin-9 (IL-9) was first described in the late 1980s as a T-cell and mast cell growth factor, initially termed P40 (based on molecular weight) or Mast cell growth-enhancing activity (MEA). The cloning and complete amino acid sequencing revealed IL-9 as structurally distinct from other T cell growth factors . It was subsequently renamed IL-9 based on its biological effects on both myeloid and lymphoid cells.

Human IL-9 is a 14 kDa peptide encoded by a gene located on chromosome 5, in a syntenic region 3.2 Mb telomeric from the IL5/IL13/IL4 loci . The gene encodes a 144 amino acid protein including the leader sequence.

Research methodology for IL-9 identification:

• Protein purification followed by amino acid sequencing

• cDNA cloning and expression analysis

• Functional assays to determine biological activity

• Chromosomal mapping to identify genomic location

Which cells produce IL-9 in humans and how is this production regulated?

The major source of IL-9 is T lymphocytes, but several subsets with differing regulatory mechanisms have been identified:

• Th2 cells: IL-9 was initially associated with the Th2 phenotype, as demonstrated in T cells isolated from Leishmania major-infected Balb/c mice, which generate Th2-biased immune responses .

• Th17 cells: Can secrete both IL-17A/F and IL-9. Human Th17 cells can co-express IL-17A and IL-9, though IL-23 (required for maintaining the IL-17-secreting phenotype) inhibits IL-9 production .

• Regulatory T cells (Tregs): Both natural Tregs (nTregs) and inducible Tregs (iTregs) that express Foxp3 can secrete IL-9, although evidence regarding human Treg IL-9 production remains conflicting .

• Th9 cells: Recently characterized subset dedicated to high IL-9 production when differentiated in the presence of TGF-β and IL-4 .

• Mast cells: Produce IL-9 in response to LPS and IL-1, with regulation involving NF-κB binding sites in the IL-9 promoter .

Key transcription factors regulating IL-9 expression include:

• PU.1: An ETS-family transcription factor that binds directly to the Il9 gene. PU.1-deficient T cells show greatly diminished IL-9 production when cultured with TGF-β and IL-4 .

• IRF4: Required for Th9 generation and binds the Il9 gene directly, possibly in concert with PU.1. IRF4 expression correlates with both human and mouse Th9 differentiation .

• GATA1: In mast cells, promotes IL-9 production and Il9 promoter activation in a p38 MAPK-dependent manner .

What is the structure and signaling mechanism of the human IL-9 receptor?

The IL-9 receptor consists of two subunits:

• IL-9 receptor alpha chain (IL-9Rα): A member of the hematopoietin receptor superfamily characterized by a WSXWS motif in the extracellular domain and Box1/Box2 motifs in the intracellular domain. The human IL-9 receptor gene contains 11 exons and encodes a 522-amino acid protein .

• Common gamma chain (γc): Shared with other cytokine receptors including IL-2, IL-4, and IL-7 .

IL-9 signaling pathway involves:

• Ligand binding induces Jak1 activation associated with the receptor

• Phosphorylation of a single tyrosine residue (Tyr407) in IL-9Rα, which is critical for IL-9-dependent responses

• Activation of STAT1, STAT3, and STAT5 transcription factors

• The Box1 intracellular domain (amino acids 338-422) containing a YLPQ motif is essential for IL-9-induced cell growth, STAT3 activation, and gene expression

• Additional activation of MAP kinase and Insulin Receptor Substrate-PI3 kinase pathways

Non-hematopoietic cells (e.g., airway epithelial cells, smooth muscle cells) express IL-9Rα but may lack the common gamma chain, suggesting an alternative receptor complex configuration in these cells .

How do researchers measure IL-9 expression and activity in experimental settings?

Standard methodological approaches for IL-9 research include:

• Gene expression analysis:

  • Quantitative RT-PCR for IL-9 mRNA

  • Chromatin immunoprecipitation (ChIP) to detect transcription factor binding

  • RNA-seq for global transcriptomic profiles

• Protein detection:

  • ELISA for quantification in supernatants and serum

  • Intracellular cytokine staining with flow cytometry

  • Western blotting for signaling pathway activation

  • Immunohistochemistry for tissue localization

• Functional assays:

  • T cell and mast cell proliferation assays

  • Reporter cell lines expressing IL-9 receptor components

  • Phospho-flow cytometry to detect STAT activation

  • Neutralizing antibodies to block IL-9 activity

• Genetic approaches:

  • CRISPR-Cas9 for gene editing of IL-9 or IL-9R

  • siRNA knockdown of transcription factors

  • Transgenic overexpression or knockout models

• Computational methods:

  • Analysis of ligand-receptor pairs for cell-cell communication networks as described in search result

  • Calculation of intercellular communication scores for ranking active ligand-receptor pairs

How do different T helper cell subsets differ in their IL-9 production capacity?

Comparative analysis of IL-9 production across T helper subsets reveals distinct patterns:

Research approaches for comparative analysis:

• Parallel differentiation under defined conditions:

  • Naïve CD4+ T cells cultured with specific cytokine combinations

  • Th2: IL-4 + anti-IFNγ

  • Th9: TGF-β + IL-4

  • Th17: TGF-β + IL-6 (± IL-23)

  • Treg: TGF-β + IL-2

• Time-course analysis to determine kinetics of IL-9 production

• Single-cell technologies to identify heterogeneity within populations

• Transcription factor manipulation to assess impact on IL-9 production

In human studies, Th17 cells have been shown to co-express IL-17A and IL-9 in long-term cultures, though IL-23 has inhibitory effects on IL-9 production .

What is the relationship between IL-9 and inflammatory diseases in humans?

IL-9 is associated with various inflammatory conditions in humans:

• Allergic inflammation and asthma:

  • IL-9 expression is increased in lungs of asthmatic patients

  • IL-9R expression is found in lungs of asthmatic individuals but not healthy controls

  • Seasonal ragweed pollen enhances IL-9 production from peripheral blood mononuclear cells of allergic patients

  • Blocking antibodies to IL-9 are being developed as therapy for atopic disease

• Genetic associations with disease:

  • SNPs in the IL9R gene (X chromosome) are protective for wheezing in boys but not girls

  • IL9R polymorphisms provide modest protection against allergen sensitization

  • IL9 polymorphisms show sex-restricted differences in:

    • Lung function

    • Allergen sensitization

    • IgE levels

    • RSV infection severity

  • An IL9R polymorphism (rs3093457) has been linked to rheumatoid arthritis

Methodological approaches for studying IL-9 in human disease:

• Genetic association studies to identify disease-relevant polymorphisms

• Analysis of IL-9 and IL-9R expression in patient samples

• Ex vivo stimulation of patient-derived cells

• Therapeutic trials with IL-9-targeting agents

How do IL-9 and IL-17 interact in inflammatory conditions?

IL-9 and IL-17 show intriguing relationships in inflammatory settings:

• Co-production by T cells:

  • Th17 cells can produce both IL-17A/F and IL-9

  • Human Th17 cells can co-express IL-17A and IL-9 in long-term cultures

  • IL-23 enhances IL-17 but suppresses IL-9 production, creating a regulatory balance

• Shared and distinct regulation:

  • TGF-β promotes both IL-9 and IL-17 production

  • IRF4 is required for both Th9 and Th17 development

  • RORγt is specific for Th17, while high PU.1 expression characterizes Th9 cells

• Pathological significance:

  • Both cytokines are implicated in autoimmunity, allergy, and infection

  • Th17 cells producing IL-9 may co-exist and interact with Th9 cells during these conditions

  • Each cytokine contributes distinct effector mechanisms to inflammatory responses

Research strategies for investigating IL-9/IL-17 interactions:

• Co-culture experiments with Th9 and Th17 cells

• In vivo models with selective cytokine blockade

• Single-cell analysis of patient samples to identify co-expression patterns

• Transcriptomic analysis of cells expressing both cytokines

How does IL-9 receptor signaling differ between hematopoietic and non-hematopoietic cells?

A fundamental difference exists in IL-9 receptor configuration across cell types:

This distinction presents a critical research question: How do non-hematopoietic cells that lack the common gamma chain respond to IL-9?

Recent research has focused on identifying a novel IL-9 receptor complex in non-hematopoietic cells. While structural cells express IL-9Rα, they lack expression of the γc chain, raising questions about signal transduction mechanisms .

Experimental approaches to address this question:

• Immunoprecipitation studies to identify novel IL-9R binding partners

• CRISPR-Cas9 screening to identify essential signaling components

• Comparative phospho-proteomic analysis between cell types

• Receptor mutagenesis to identify critical binding regions

What is the role of mast cells in regulating IL-9 production in human immune responses?

Mast cells play a dual role in IL-9 biology:

• As IL-9 producers:

  • Produce IL-9 in response to LPS and IL-1

  • Regulation involves NF-κB binding sites in the IL-9 promoter

  • GATA1 promotes IL-9 production in a p38 MAPK-dependent manner

• As regulators of T cell IL-9 production:

  • IL-33-primed mast cells foster Th2 cell responses

  • Allow IL-9-producing Th cells to emerge in an OX40L-dependent manner

  • Form a communication network with memory CD4+ T cells

Methodological framework for investigating mast cell-T cell interactions:

• Co-culture systems:

  • IL-33-primed mast cells cultured with CD4+ memory T cells

  • Analysis of cytokine production using intracellular staining or secretion assays

• Computational analysis of cellular communication:

  • Analysis of ligand-receptor pairs between cell types

  • Thresholding to identify "active" L-R pairs

  • Communication score computation to rank active L-R pairs

• Blocking studies:

  • Neutralizing antibodies against candidate mediators (e.g., OX40L)

  • siRNA knockdown of specific factors in mast cells

• In vivo validation:

  • Mouse models with mast cell deficiency

  • Adoptive transfer of wild-type or modified mast cells

Recent research demonstrated that IL-33-primed mast cells fostered Th2 cell responses and allowed IL-9-producing Th cells to emerge in an OX40L-dependent manner, highlighting a critical regulatory circuit in human allergic responses .

What are the current experimental models for studying human IL-9 biology?

Researchers utilize several complementary approaches to study human IL-9:

• Primary human cell cultures:

  • Peripheral blood mononuclear cells (PBMCs)

  • Purified T cell subsets differentiated under specific conditions

  • Cord blood-derived mast cells

  • Bronchial epithelial cells from healthy donors or patients

• Cell lines:

  • Human T cell lines expressing IL-9

  • Mast cell lines (e.g., HMC-1)

  • Reporter cell lines expressing human IL-9 receptor components

• Ex vivo analysis:

  • Bronchoalveolar lavage (BAL) fluid from asthma patients

  • Skin biopsies from atopic dermatitis patients

  • Synovial fluid from rheumatoid arthritis patients

• Humanized mouse models:

  • Mice reconstituted with human immune cells

  • Transgenic mice expressing human IL-9 or IL-9R

• Computational approaches:

  • Analysis of ligand-receptor pairs for cell-cell communication

  • Integration of multi-omics data to model IL-9 networks

How can researchers accurately measure IL-9 receptor expression and signaling in human samples?

Comprehensive assessment of IL-9 receptor biology requires multi-faceted approaches:

• Receptor expression analysis:

  • Flow cytometry with anti-IL-9Rα and anti-γc antibodies

  • Quantitative PCR for receptor subunit mRNA

  • Western blotting for protein expression

  • Immunohistochemistry for tissue localization

• Signaling pathway evaluation:

  • Phospho-flow cytometry to detect STAT activation

  • Western blotting for phosphorylated signaling molecules

  • Mass spectrometry for comprehensive phospho-proteomics

  • Time-course analysis to determine signaling kinetics

• Functional assessment:

  • Dose-response curves with recombinant IL-9

  • Inhibitor studies to block specific pathways

  • siRNA knockdown of receptor components

  • CRISPR-Cas9 gene editing to modify receptor structure

• Novel receptor complex identification:

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity ligation assays to detect protein interactions

  • FRET/BRET analysis for real-time interaction monitoring

Statistical approaches should include appropriate normalization strategies and account for the often non-linear nature of receptor signaling relationships.

What are the emerging therapeutic approaches targeting IL-9 in human diseases?

Several strategies are being developed to target the IL-9 pathway:

• Neutralizing antibodies:

  • Anti-IL-9 monoclonal antibodies for blocking circulating IL-9

  • Anti-IL-9R antibodies to prevent receptor engagement

  • Antibodies are being developed as therapy for atopic diseases

• Receptor antagonists:

  • Small molecule inhibitors of IL-9R signaling

  • Peptide-based antagonists that compete for receptor binding

• Pathway inhibitors:

  • JAK inhibitors that block downstream signaling

  • STAT inhibitors targeting IL-9-activated transcription factors

• Cell-based approaches:

  • Modulation of IL-9-producing cells (Th9, Th17)

  • Engineering regulatory T cells to suppress IL-9 responses

• Genetic approaches:

  • Targeting polymorphisms associated with IL-9/IL-9R dysregulation

  • Gene therapy to correct IL-9 pathway abnormalities

Current clinical development status focuses primarily on monoclonal antibodies for allergic conditions, with several candidates in early clinical trials.

What unresolved questions remain in human IL-9 biology?

Despite decades of research, several critical questions remain:

• Cell type-specific functions:

  • Relative contribution of different IL-9-producing cells in human disease

  • Tissue-specific effects of IL-9 in different organs

• Receptor biology:

  • Composition of the IL-9 receptor in non-hematopoietic cells that lack γc

  • Differences in signaling pathways between cell types

• Regulatory mechanisms:

  • Epigenetic regulation of IL-9 expression

  • Post-transcriptional control of IL-9 production

  • Stability of the IL-9-producing phenotype

• Disease relevance:

  • IL-9's role in understudied conditions (e.g., autoimmunity, cancer)

  • Biomarkers to identify IL-9-dependent disease processes

  • Predictors of response to IL-9-targeting therapies

• Developmental biology:

  • IL-9's role in human development and tissue homeostasis

  • Age-related changes in IL-9 responsiveness

Addressing these questions will require integrative approaches combining human studies, innovative experimental models, and advanced computational analyses.