IL4R Human

What is IL-4R and what are its key structural characteristics?

IL-4R (Interleukin-4 Receptor), also known as IL-4RA and CD124, is a transmembrane glycoprotein belonging to the class I receptor family. The human IL-4R has an extracellular domain that binds to IL-4, a transmembrane region, and an intracellular signaling domain. The receptor is highly expressed by activated T-cells and couples with the common γ chain to form the type I receptor for IL-4. IL-4R can also associate with IL-13 R alpha 1 to form the type II receptor complex, which allows for response to both IL-4 and IL-13 signaling .

What cellular functions is IL-4R involved in?

IL-4R plays critical roles in multiple immune processes including Th2 cell differentiation, immunoglobulin class switching, and alternative macrophage activation. The receptor is essential for mediating IL-4 signaling, which stimulates B-cell activation, T-cell proliferation, and CD4+ T-cell differentiation into Th2 cells. Beyond its immune functions, IL-4R has been implicated in allergic inflammation, tumor progression, and atherogenesis . Recent research has also revealed neuronal functions, including modulation of synaptic vesicle recruitment and neuronal network activity .

How does IL-4R expression differ between normal and pathological tissues?

Expression patterns of IL-4R show significant differences between normal and diseased tissues. For example, in brain tissue, IL-4R protein is typically undetectable in normal adult and pediatric specimens despite the presence of IL-4Rα mRNA. In contrast, 15 of 18 glioblastoma multiforme (GBM) tumors and various other brain tumor samples show moderate to intense positive staining for IL-4Rα. Similar differential expression is seen in astrocytomas of grades I, II, and III compared to normal brain tissue . This overexpression in tumor tissues makes IL-4R a potential target for cancer therapeutics.

What are the challenges in detecting IL-4R isoforms, and how can they be overcome?

Detection of IL-4R isoforms, particularly IL-4δ2, presents significant challenges for researchers. Current commercial ELISA assays typically cannot distinguish between full-length IL-4 and the IL-4δ2 isoform. Similarly, probes included in Affymetrix or Illumina microarray kits may target IL-4δ2 rather than full-length IL-4, leading to misleading data. The primary difficulty stems from the 100% homology between these isoforms, with the only difference being the 16 amino acids absent in IL-4δ2 .

To overcome these challenges, researchers should:

• Use isoform-specific primers for RT-PCR that span exon junctions

• Develop custom antibodies targeting the specific regions absent in IL-4δ2

• Employ BaseScope probes for in situ hybridization to detect specific exons, as demonstrated in the detection of exon 7 in IL-4Rα knockout models

• Combine multiple detection methods to confirm isoform identity

How can researchers effectively create and validate IL-4R knockout models?

Creating effective IL-4R knockout models requires careful planning and validation. Current approaches include:

• CRISPR/Cas9 gene editing: Successful knockout has been achieved using targeted deletions, such as the 10 bp deletion in exon 11 and 332 bp deletion in exon 11 reported in HEK-293T cell lines .

• Conditional knockout using Cre-loxP system: For tissue-specific deletion, as demonstrated in neuron-specific IL-4Rα knockout mice (il4ra fl/fl.Syn cre+) .

Validation methods should include:

• Genomic PCR to confirm deletion

• RNA analysis using probes specific to deleted exons

• Protein expression analysis using Western blot or immunostaining

• Functional assays to confirm loss of receptor activity

• Cell-type specific validation, such as combining BaseScope probes for cell markers (e.g., nefh) and IL-4Rα exons to confirm cell-specific deletion

What methodologies are most effective for studying IL-4R signaling in primary cells?

To effectively study IL-4R signaling in primary cells, researchers should consider:

• Cell isolation and culture techniques:

  • For neuronal studies: differentiation of human neurons from iPSC-derived neural progenitor cells with verification of network formation and electrophysiological activity

  • For immune cells: isolation of PBMCs with verification of IL-4R expression

• Signaling analysis methods:

  • Electrophysiological recordings to assess synaptic function in neuronal cells

  • Cumulative amplitude analysis to estimate readily releasable pool (RRP) and resting pool size in synaptic studies

  • Phosphorylation assays for downstream signaling molecules (e.g., JAK1, STAT6)

  • Transcriptomic analysis using RNAseq to identify regulated genes after IL-4 treatment

• Functional readouts:

  • For immune cells: proliferation assays, cytokine production, cell differentiation markers

  • For neuronal cells: synaptic function tests, dendrite morphology analysis, and behavioral assessments

How does IL-4R homodimerization affect downstream signaling events?

Homodimerization of human IL-4Rα chain, particularly its intracellular domain, induces Cɛ germline transcripts, accompanied by Jak1 phosphorylation and activation . This mechanism has been studied using chimeric receptor constructs, including EpoR/IL-4Rα and CD8α/IL-4Rα, which allow for controlled dimerization and analysis of the specific contribution of the IL-4Rα intracellular domain to signaling.

The signaling pathways activated through homodimerization differ from those activated by the conventional IL-4R heterodimeric complexes. Understanding these differences is crucial for developing targeted therapeutic approaches and explains some of the pleiotropic effects of IL-4 signaling in different cell types. Researchers investigating this phenomenon should consider:

• Using chimeric receptor systems to isolate the effects of receptor dimerization

• Analyzing phosphorylation of downstream signaling molecules like JAK1, STAT6, and other adaptors

• Comparing transcriptional profiles induced by homodimeric versus heterodimeric receptor signaling

What are the molecular mechanisms through which IL-4R modulates neuronal network activity?

IL-4R signaling in neurons modulates network activity through several mechanisms:

• Synaptic vesicle recruitment: IL-4Rα deficiency reduces the readily releasable pool (RRP) size (2,269.7 ± 284.9 vs. 4,025.6 ± 585.5 pA in controls) and decreases the replenishment rate of neurotransmitter vesicles from the resting vesicle pool (103.3 ± 12.7 vs. 175.9 ± 19.1 in controls) .

• Gene expression regulation: Human neurons differentiated from iPSC-derived neural progenitor cells show 908 differentially regulated genes upon IL-4 treatment, with 360 genes showing decreased expression and 548 genes significantly increased .

• Homeostatic regulation: IL-4/IL-4R signaling appears to play a role in maintaining homeostasis of the CNS through fine-tuning of synaptic transmission. Long-term effects of IL-4Rα deficiency in adult mice include increased neuronal network activity and behavioral deficits .

Importantly, these effects appear to be direct neuronal effects rather than indirect effects mediated through glial cells or inflammation, as demonstrated using neuron-specific IL-4Rα knockout models.

How do the type I and type II IL-4 receptor complexes differ in their signaling outcomes?

The type I IL-4 receptor complex consists of IL-4Rα associated with the common gamma chain, while the type II complex involves IL-4Rα associating with IL-13 R alpha 1 . These different configurations lead to distinct signaling outcomes:

Understanding these differences is critical for designing targeted therapeutics and interpreting experimental results when studying IL-4 signaling in different tissue contexts.

How does IL-4R expression in brain tumors inform potential targeted therapies?

IL-4R is significantly overexpressed in brain tumors compared to normal brain tissue. Studies have shown that 15 of 18 glioblastoma multiforme (GBM) tumors and 12 other brain tumor samples exhibit moderate to intense positive staining for IL-4Rα, while normal brain tissues show no detectable IL-4R protein despite expressing IL-4Rα mRNA . This differential expression makes IL-4R an attractive target for brain tumor therapies.

IL-4 cytotoxin, composed of circularly permutated IL-4 and a mutated form of Pseudomonas exotoxin [IL4(38–37)-PE38KDEL], has demonstrated selective cytotoxicity against IL-4R-expressing cells. Primary GBM explant cell cultures were 25–74 times more sensitive to IL-4 cytotoxin compared with normal human astrocytes or NT2 neuronal cell lines . This differential sensitivity provides a therapeutic window for targeted treatment.

Researchers developing IL-4R-targeted therapies should consider:

• Confirming IL-4R expression in patient samples using validated immunohistochemical methods

• Testing the specificity of IL-4R-targeted agents against both tumor and normal tissue

• Exploring combination approaches with standard treatments

• Investigating mechanisms of resistance to IL-4R-targeted therapies

What experimental approaches best demonstrate the functional significance of IL-4R in allergic and inflammatory conditions?

To effectively study IL-4R's role in allergic and inflammatory conditions, researchers should employ:

• Genetic models:

  • Conditional knockout mice targeting IL-4Rα in specific cell types (e.g., T cells, B cells, macrophages, epithelial cells)

  • Knock-in models with mutations in key signaling residues

• Pharmacological approaches:

  • IL-4R antagonists or blocking antibodies (such as Dupilumab biosimilars)

  • Selective inhibitors of downstream signaling pathways

• Functional assessments:

  • Airway hyperresponsiveness measurements

  • Analysis of inflammatory cell infiltration

  • Measurement of Th2 cytokine production

  • Evaluation of IgE class switching and production

  • Alternative macrophage activation markers

• Translational studies:

  • Ex vivo analysis of human samples from allergic patients

  • Correlation of IL-4R polymorphisms with disease phenotypes

  • Biomarker studies to predict response to IL-4R-targeted therapies

These approaches should be combined to provide comprehensive evidence of IL-4R's role in disease pathogenesis and the potential therapeutic benefit of targeting this pathway.

How can researchers distinguish between direct and indirect effects of IL-4R signaling in complex tissues?

Distinguishing direct from indirect effects of IL-4R signaling presents a significant challenge in complex tissues. Effective research strategies include:

• Cell-type specific knockout models:

  • Neuron-specific IL-4Rα knockout (il4ra fl/fl.Syn cre+) to isolate neuronal effects

  • Similar approaches for other cell types using appropriate Cre drivers

• Chimeric receptor systems:

  • EpoR/IL-4Rα and CD8α/IL-4Rα chimeras that allow specific activation of IL-4R signaling pathways in selected cell types

  • Inducible systems to control timing of receptor activation

• In vitro isolation:

  • Pure cell culture systems (e.g., iPSC-derived neurons) to study cell-autonomous effects

  • Co-culture systems to study specific intercellular interactions

• Temporal analysis:

  • Time-course experiments to distinguish primary from secondary effects

  • Rapid signaling events (seconds to minutes) versus transcriptional changes (hours)

• Molecular validation:

  • Combined BaseScope probes for cell-type markers and IL-4Rα to confirm cell-specific expression or deletion

  • Cell-specific transcriptomics (e.g., single-cell RNA-seq)

What are the functional implications of alternative IL-4R splice variants, and how can they be effectively studied?

Alternative IL-4R splice variants, such as IL-4δ2, present both challenges and opportunities for research. IL-4δ2 lacks 16 amino acids present in full-length IL-4 but maintains 100% homology in the remaining sequence. This isoform binds specifically to human PBMCs and tumor lines that express IL-4R and IL-13R .

Functional implications may include:

• Altered receptor binding kinetics

• Modified signal transduction pathways

• Different biological responses in various cell types

• Potential antagonistic effects on full-length IL-4 signaling

Effective study approaches include:

• Detection strategies:

  • Development of isoform-specific antibodies targeting the junction regions

  • Custom ELISA assays that can distinguish between isoforms

  • RT-PCR with primers spanning exon junctions specific to each variant

  • Careful selection of microarray probes that distinguish between isoforms

• Functional analysis:

  • Comparison of signaling pathways activated by different isoforms

  • Competition binding assays between isoforms

  • Isoform-specific expression constructs for controlled studies

  • CRISPR-mediated editing to selectively disrupt specific isoforms

• Clinical relevance:

  • Analysis of isoform expression ratios in disease states

  • Correlation of isoform expression with disease progression or treatment response

  • Development of isoform-specific therapeutic approaches

How does the readily releasable pool (RRP) reduction in IL-4Rα-deficient neurons impact broader neural circuit function?

The reduction in readily releasable pool (RRP) size (2,269.7 ± 284.9 vs. 4,025.6 ± 585.5 pA) and decreased replenishment rate (103.3 ± 12.7 vs. 175.9 ± 19.1) observed in IL-4Rα-deficient neurons has several implications for neural circuit function:

• Synaptic transmission effects:

  • Reduced neurotransmitter release during sustained activity

  • Altered short-term synaptic plasticity, particularly during high-frequency stimulation

  • Changed signal-to-noise ratio in information processing

• Network-level consequences:

  • Potential circuit hyperexcitability as a compensatory mechanism

  • Altered excitatory/inhibitory balance depending on which neuronal populations are affected

  • Modified network synchronization and oscillatory patterns

• Behavioral manifestations:

  • Potential cognitive deficits in learning and memory tasks

  • Changes in sensory processing or motor function

  • Altered susceptibility to seizures or other network-level pathologies

Research approaches to investigate these aspects should include:

• In vitro electrophysiology to characterize synaptic properties under various stimulation conditions

• In vivo circuit recordings to assess network dynamics

• Computational modeling to predict circuit-level effects

• Comprehensive behavioral testing batteries to identify subtle phenotypes

• Pharmacological manipulations to probe compensatory mechanisms

What methodological considerations are critical when developing IL-4R-targeted therapeutic approaches?

When developing IL-4R-targeted therapeutic approaches, researchers should consider:

• Target specificity and expression profiles:

  • Differential expression between target tissues and normal tissues (e.g., brain tumors vs. normal brain)

  • Expression levels needed for therapeutic efficacy

  • Potential off-target effects in tissues with low but functional IL-4R expression

• Mechanism of action optimization:

  • Receptor antagonism vs. targeting receptor-expressing cells

  • Partial vs. complete inhibition of signaling

  • Targeting type I vs. type II receptor complexes

  • Consideration of IL-4R isoforms and their functional significance

• Delivery systems and pharmacokinetics:

  • Tissue penetration (particularly important for CNS applications)

  • Half-life and dosing requirements

  • Immunogenicity of biologics (especially for modified proteins like IL4(38–37)-PE38KDEL)

• Efficacy assessment:

  • Appropriate in vitro models that recapitulate target tissue biology

  • Relevant in vivo models with confirmed IL-4R expression patterns

  • Pharmacodynamic markers to confirm target engagement

  • Functional outcomes relevant to the disease being targeted

• Combination approaches:

  • Synergistic effects with standard therapies

  • Potential for increased therapeutic window

  • Management of resistance mechanisms

These considerations should guide systematic development from preclinical studies through clinical translation to maximize the therapeutic potential of IL-4R targeting.