IL5RA Mouse

What is IL5RA and what is its functional significance in mouse models?

IL5RA, also known as interleukin 5 receptor alpha or CD125, is a specific subunit of a heterodimeric cytokine receptor in mice. It functions by pairing with a signal transducing beta subunit that is shared with IL-3 and GM-CSF receptors to form a functional IL-5 receptor complex. In mice, IL5RA is predominantly expressed on eosinophils and basophils, serving as a cell surface receptor that plays crucial roles in the survival, differentiation, and chemotaxis of eosinophils .

The IL5RA protein is a membrane-localized receptor with a canonical length of 420 amino acid residues and a mass of approximately 47.7 kDa, subject to post-translational modifications including glycosylation . As a member of the Type I cytokine receptor family, IL5RA functions as a marker for identifying eosinophils, basophils, and mast cells in research contexts . Recent studies have also revealed its importance in B1 cell development and maintenance, suggesting broader immunological functions beyond eosinophil regulation .

How is IL5RA expression regulated across different mouse tissues and cell types?

IL5RA expression in mice shows distinct tissue and cell-type specificity patterns:

• Hematopoietic tissues: Highest expression occurs in bone marrow (particularly in eosinophil progenitors) and specific immune cell subsets in peripheral blood and spleen

• Immune cells: Predominant expression on eosinophils and basophils, with additional expression on certain B cell populations, particularly B1 cells

• Brain tissue: Differential expression across brain regions according to Allen Brain Atlas data, with specific regional patterns

• Cell clusters: Single-cell RNA sequencing has identified specific B cell clusters (clusters 0 and 2) with enriched IL5RA expression in mice

Recent research using transgenic IL5ra reporter mouse models has enabled more precise tracking of IL5RA expression patterns in vivo . Interestingly, some studies have found that certain cell populations detectible with anti-IL5RA antibodies do not express the IL5ra reporter transgene, suggesting potential differences in protein isoform expression or detection sensitivity across methodologies .

What signaling pathways and cellular processes are regulated by IL5RA in mouse models?

IL5RA signaling in mice regulates multiple important cellular processes through several interconnected pathways:

• Eosinophil development and function: Controls terminal differentiation of eosinophils from progenitor cells, mediates survival signals, and regulates eosinophil activation

• B1 cell maintenance: Supports the development and function of B1 cells through regulation of specific gene sets

• Downstream gene regulation: Activates expression of target genes including Ass1, Anxa2, Ccnd1, and Zcwpw1 in B1 cells as demonstrated by qPCR studies

• JAK/STAT signaling: Primarily signals through JAK2 and STAT5 activation

• Tissue protection mechanisms: Recent studies indicate IL5RA-dependent eosinophils provide hepatoprotective functions during liver ischemia-reperfusion injury

• Alarmin response integration: Functions downstream of alarmin-initiated pathways, as IL-33 signaling triggers IL-5 production by ILC2s, which then acts through IL5RA

The complex signaling network associated with IL5RA positions it as a critical mediator between environmental signals and cellular responses in both homeostatic and inflammatory conditions.

What are the most effective methods for detecting IL5RA expression in mouse tissues?

Multiple complementary techniques can be employed to detect IL5RA expression in mouse tissues, each with specific advantages:

Flow Cytometry:

• Most widely used for quantitative single-cell analysis

• Requires validated anti-IL5RA (CD125) antibodies

• Allows for multiparameter analysis and sorting of IL5RA-positive cells

• Optimal for analyzing expression across heterogeneous cell populations

PCR-Based Detection:

• Real-time quantitative PCR with validated primers for mouse Il5ra

• PrimePCR assays are available specifically designed following MIQE guidelines

• Allows precise quantification of transcript levels across experimental conditions

• Can detect multiple transcript variants

Transgenic Reporter Systems:

• IL5RA reporter mice express fluorescent proteins under Il5ra promoter control

• Enable direct visualization and isolation of IL5RA-expressing cells

• Particularly valuable for developmental studies and lineage tracing

• Can reveal regulatory elements controlling Il5ra expression

Single-Cell RNA Sequencing:

• Provides comprehensive transcriptional profiling at single-cell resolution

• Has identified previously unknown IL5RA-expressing cell populations

• Enables correlation of IL5RA expression with global gene expression patterns

• Recent studies used scRNA-seq to identify IL5RA enrichment in specific B cell clusters

When selecting methods, researchers should consider that different detection approaches may yield slightly different results. For example, studies have observed populations detectable with anti-IL5RA antibodies that do not express IL5ra reporter transgenes , suggesting the importance of validating findings across multiple detection platforms.

How can researchers effectively evaluate IL5RA functional activity in mouse models?

Assessing IL5RA functionality requires approaches that evaluate receptor-mediated signaling and biological outcomes:

Phospho-flow Cytometry:

• Measures phosphorylation of downstream molecules (JAK2, STAT5)

• Provides single-cell resolution of pathway activation

• Can be performed ex vivo after IL-5 stimulation

• Directly links receptor expression to signaling activity

Genetic Approaches:

• Compare phenotypes between wild-type and Il5ra-deficient mice

• Use conditional knockout models to assess tissue-specific functions

• Employ IL5RA reporter mice to track receptor-expressing cells

• Recent studies have shown that IL5RA-deficient mice exhibit exacerbated liver injury, demonstrating a protective role for IL5RA signaling

Functional Readouts:

• Measure eosinophil numbers in blood and tissues

• Assess IL-5-responsive gene expression (Ass1, Anxa2, Ccnd1, Zcwpw1)

• Evaluate B1 cell frequencies and function

• Compare tissue pathology in disease models (e.g., liver IR injury models have revealed protective functions of IL5RA-expressing eosinophils)

Intervention Studies:

• Anti-IL-5 or anti-IL5RA neutralizing antibodies to block signaling

• Recombinant IL-5 administration to enhance signaling

• Comparison of effects across different genetic backgrounds

• Studies have shown that recombinant IL-13 administration can reduce hepatic IR injury, suggesting downstream effects of IL5RA signaling pathways

A comprehensive assessment of IL5RA functionality should combine multiple approaches to establish both the signaling capacity of the receptor and its biological consequences in relevant cell populations and disease contexts.

What considerations are important when selecting antibodies for IL5RA detection in mouse research?

Selecting appropriate anti-IL5RA antibodies requires careful consideration of several factors:

Antibody Validation:

• Use IL5ra knockout mice as negative controls

• Compare multiple antibody clones in parallel

• Validate with orthogonal methods (e.g., reporter mice, mRNA expression)

• Test across relevant tissue types and experimental conditions

Format Considerations:

• Flow cytometry applications require antibodies recognizing extracellular domains

• Western blot applications typically require antibodies against denatured epitopes

• Functional studies need antibodies that either block or don't interfere with IL-5 binding

• Multiple conjugates (FITC, biotin, HRP, Alexa dyes) are available for different applications

Clone Selection:

• Different clones may have varying abilities to detect post-translationally modified IL5RA

• Some clones may recognize specific isoforms of IL5RA (up to 5 different isoforms have been reported)

• Epitope accessibility can differ in various fixation and permeabilization protocols

• Consider species cross-reactivity if comparing mouse and human systems

Application-Specific Products:

• Flow cytometry: Pre-conjugated antibodies with optimized fluorophores

• Immunohistochemistry: Antibodies validated for paraffin-embedded tissues

• Western blotting: Antibodies optimized for denatured protein detection

• Functional blocking: Antibodies that interfere with IL-5 binding

Research has revealed that some IL5RA-expressing populations may be detectable with certain monoclonal antibodies but not with IL5RA reporter transgenes , suggesting heterogeneity in epitope expression or differences in detection sensitivity. Researchers should carefully validate antibodies for their specific application and experimental system.

What transgenic and knockout IL5RA mouse models are available and how are they best utilized?

Several engineered mouse models have been developed to study IL5RA biology, each with specific research applications:

IL5RA Knockout (Il5ra−/−) Mice:

• Complete deletion of functional IL5RA expression

• Show impaired eosinophil development and function

• Exhibit increased susceptibility to certain tissue injuries

• Studies have demonstrated exacerbated liver ischemia-reperfusion injury in these mice

IL5RA Reporter Mice:

• Express fluorescent proteins under Il5ra promoter control

• Allow direct visualization and tracking of IL5RA-expressing cells

• Enable isolation of IL5RA-expressing populations by FACS

• Recent studies report development of new IL5RA reporter mouse strains that have expanded research capabilities

Conditional IL5RA Models:

• Allow cell-type specific deletion when crossed with appropriate Cre lines

• Examples include eosinophil-specific knockout models

• Particularly valuable for distinguishing direct from indirect effects

• Can be combined with inducible systems for temporal control

Combination Models:

• IL5RA models can be crossed with other relevant gene knockouts

• Studies have utilized mice with eosinophil-specific deletion of IL-4/IL-13 (IL-4/IL-13ΔEOS) to examine downstream effector mechanisms

• Combinations with ST2−/− or IL-33−/− models have revealed upstream regulators

• ILC2-deficient models (Nmur1iCre-eGFP Id2fl/fl) have demonstrated the role of ILC2s in maintaining IL5RA+ B1 cells

Each model provides distinct advantages for investigating specific aspects of IL5RA biology. Selection should be based on the particular research question, with consideration of potential developmental effects, compensation mechanisms, and genetic background influences.

How does the absence of IL5RA affect immune responses in mouse disease models?

IL5RA deficiency leads to several distinctive phenotypes across different disease models:

Eosinophil-Related Phenotypes:

• Profound reduction in eosinophil numbers in blood, bone marrow, and tissues

• Impaired eosinophil recruitment to inflammatory sites

• Reduced eosinophil development from progenitors

• Altered tissue distribution patterns

Tissue Injury Models:

• Exacerbated liver ischemia-reperfusion injury

• Increased serum ALT and AST levels indicating greater hepatocyte damage

• Worsened histological evidence of tissue necrosis

• Greater neutrophil accumulation in injured tissues

B Cell Abnormalities:

• Reduced numbers of IL5RA+ B1 cells

• Downregulation of IL-5 target genes (Ass1, Anxa2, Ccnd1, Zcwpw1) in B cells

• Altered antibody production profiles

• These findings suggest IL5RA signaling is important for B1 cell maintenance

Type 2 Immunity:

• Modified responses to parasitic infections

• Altered allergic inflammation

• Changes in ILC2-dependent immune pathways

• Modified mucosal immune responses

The diverse phenotypes observed in IL5RA-deficient mice highlight the complex roles of this receptor across multiple immune processes and disease contexts. These findings have expanded our understanding of IL5RA beyond its canonical role in eosinophil development to include tissue protection and B cell regulation.

What methodological challenges arise when working with IL5RA reporter mice?

IL5RA reporter mice present unique methodological considerations that researchers should address:

Reporter Validation:

• Essential to confirm correlation between reporter expression and endogenous IL5RA protein

• Validate across multiple tissues and cell types

• Test under different activation conditions

• Compare with anti-IL5RA antibody staining

Technical Optimization:

• Determine optimal flow cytometry parameters for reporter detection

• Establish imaging protocols that preserve reporter fluorescence

• Consider tissue autofluorescence and implement appropriate controls

• Optimize fixation methods that maintain reporter signal

Data Interpretation:

• Distinguish between current and historical expression (reporter protein may persist after transcription ceases)

• Consider threshold detection differences between reporter and endogenous protein

• Interpret quantitative data in the context of reporter sensitivity

• Be cautious about extrapolating from reporter brightness to expression level

Experimental Design:

• Include appropriate wild-type controls lacking the reporter

• Consider using heterozygous reporter mice if homozygosity affects function

• Account for potential developmental effects of the reporter construct

• Use reporter expression in conjunction with other markers for comprehensive analysis

Addressing these challenges requires careful experimental design and appropriate controls to maximize the utility of IL5RA reporter mice as research tools.

How does IL5RA signaling interact with other cytokine pathways in inflammatory responses?

IL5RA signaling operates within complex cytokine networks with several key interactions:

IL-33/ST2 Axis:

• Research demonstrates that IL-33 signaling through ST2 induces IL-5 production by ILC2s

• IL-33−/− and ST2−/− mice show impaired eosinophil accumulation in tissues

• This establishes a sequential pathway: IL-33 → ILC2 activation → IL-5 production → IL5RA signaling

• In liver ischemia-reperfusion models, this pathway is critical for eosinophil-mediated tissue protection

Type 2 Cytokine Network:

• IL5RA signaling functions within the broader Type 2 cytokine network (IL-4, IL-13, TSLP)

• IL-13 administration to wild-type mice reduces hepatic IR injury

• Mice with eosinophil-specific deletion of IL-4/IL-13 show increased tissue injury

• This suggests IL5RA-dependent eosinophils mediate protection partly through IL-4/IL-13 production

ILC2 Dependencies:

• Group 2 innate lymphoid cells (ILC2s) are a critical non-redundant source of IL-5

• ILC2-deficient mice show reduced IL5RA+ B1 cells and downregulation of IL-5 target genes in B1 cells

• This positions ILC2s as essential upstream regulators of IL5RA-dependent processes

• Alarmin-responsive ILC2s link tissue damage to IL5RA signaling through IL-5 production

Shared Receptor Components:

• IL5RA uses a common beta chain shared with IL-3 and GM-CSF receptors

• This creates potential for competitive binding and coordinated regulation

• Understanding this shared signaling machinery helps explain redundancies and specificities

• The beta subunit is required for the biological activities of IL-5

Coordinated Transcriptional Responses:

• IL5RA signaling induces specific gene expression programs

• These may overlap with or diverge from programs induced by other cytokines

• qPCR analysis has identified specific IL-5 target genes downregulated in IL-5-deficient conditions

• This transcriptional integration determines the ultimate cellular responses

Understanding these interactions requires sophisticated experimental approaches, including conditional deletion models, temporal control of cytokine availability, and systems biology approaches to model network behavior.

What is the role of IL5RA in B cell development and function?

Research has revealed significant roles for IL5RA in B cell biology beyond its canonical function in eosinophils:

B1 Cell Regulation:

• IL5RA expression is enriched in specific B cell subsets, particularly B1 cells

• Single-cell RNA sequencing has identified specific B cell clusters (clusters 0 and 2) with enriched IL5RA expression

• IL5RA+ B1 cells are significantly reduced in ILC2-deficient mice

• This indicates ILC2-derived IL-5 is essential for maintaining IL5RA+ B1 cells

IL-5 Target Gene Expression:

• Multiple IL-5-dependent genes are downregulated in B1 cells from ILC2-deficient mice

• These genes (Ass1, Anxa2, Ccnd1, Zcwpw1) are not expressed in B2 cells

• qPCR confirmation shows reduced expression of these genes in sort-purified B1 cells from IL-5-deficient mice

• This reveals a specific transcriptional program activated by IL5RA signaling in B1 cells

ILC2 Dependency:

• The reduction in IL5RA+ B1 cells and IL-5 target genes in B1 cells from ILC2-deficient mice suggests ILC2s as an essential source of IL-5

• This establishes an ILC2-IL-5-IL5RA-B1 cell axis

• The axis may be critical for maintaining certain B1 cell populations and their functions

• This network connects innate lymphoid cell activation to B1 cell regulation

Signaling Mechanisms:

• IL5RA in B cells is thought to signal through similar pathways as in eosinophils

• The receptor may interact with syndecan binding protein (syntenin), which is required for IL5-mediated activation of the transcription factor SOX4

• Several alternatively spliced transcript variants of IL5RA have been reported , which may have distinct functions in B cells

• These signaling mechanisms link IL5RA to specific B cell transcriptional programs

The specific role of IL5RA in B cells represents an important area for further research, particularly in understanding how the IL-5/IL5RA axis connects different branches of the immune system and regulates B cell subpopulations with distinct functions.

How do IL5RA-expressing eosinophils contribute to tissue protection in injury models?

Recent research has uncovered unexpected tissue-protective functions of IL5RA-expressing eosinophils:

Hepatoprotective Function:

• Eosinophils demonstrate critical hepatoprotective function during liver ischemia-reperfusion (IR) injury

• IL5RA-deficient mice develop exacerbated hepatic IR injury with increased serum ALT and AST levels

• Histological analysis confirms greater hepatocyte necrosis in these models

• This provides compelling evidence for a critical hepatoprotective function of eosinophils

IL-33/ST2 Pathway Involvement:

• ST2−/− and IL-33−/− mice show impaired eosinophil accumulation in the liver during injury

• These mice develop more severe hepatic IR injury similar to IL5RA-deficient mice

• This establishes the IL-33/ST2 axis as an upstream regulator of eosinophil recruitment

• The pathway connects tissue damage (IL-33 release) to protective eosinophil responses

Type 2 Cytokine Mediation:

• IL-13 administration to wild-type mice reduces hepatic IR injury

• Mice with eosinophil-specific deletion of IL-4/IL-13 show increased hepatic IR injury

• This indicates eosinophil-derived IL-4/IL-13 contribute to tissue protection

• IL5RA signaling may thus regulate tissue protection partly through modulating eosinophil cytokine production

Neutrophil Regulation:

• IL5RA-deficient mice show increased neutrophil accumulation in injured tissues

• This suggests eosinophils may limit neutrophil-mediated tissue damage

• The mechanism may involve direct inhibition or competitive recruitment

• Neutrophil-eosinophil cross-regulation represents an important area for further study

These findings challenge traditional views of eosinophils as primarily pathogenic cells and highlight the context-dependent nature of IL5RA signaling in tissue homeostasis and injury. The protective role of eosinophils has important implications for therapeutic approaches targeting the IL-5/IL5RA pathway in various disease contexts.

How can researchers address variability in IL5RA detection across different experimental conditions?

Experimental variability in IL5RA detection requires systematic approaches to ensure reliable results:

Standardized Protocols:

• Develop consistent protocols for sample preparation, antibody staining, and analysis

• Document detailed methods including buffer compositions, incubation times, and temperatures

• Maintain consistent instrument settings for flow cytometry and imaging

• Use standardized positive and negative controls across experiments

Detection Method Comparison:

• Compare results from multiple detection methods (antibodies, reporter mice, PCR)

• Research has shown that some cell populations detectible with anti-IL5RA antibodies do not express IL5ra reporter transgenes

• These discrepancies may reflect biological differences or technical limitations

• Use multiple complementary approaches for comprehensive analysis

Antibody Validation:

• Validate antibodies using IL5RA knockout tissues as negative controls

• Compare multiple antibody clones and conjugates

• Test under different fixation and permeabilization conditions

• Consider the detection of specific IL5RA isoforms (up to 5 different isoforms have been reported)

PCR Considerations:

• Use validated primer sets designed following MIQE guidelines

• Consider primer design to detect all relevant transcript variants

• Include appropriate housekeeping genes for normalization

• Implement standard curves to ensure quantitative accuracy

Experimental Design:

• Include biological and technical replicates

• Blind analysis when possible to reduce observer bias

• Use appropriate statistical methods for analysis

• Power calculations to determine adequate sample sizes

By systematically addressing these factors, researchers can improve the reliability and reproducibility of IL5RA detection across different experimental conditions and research settings.

What approaches can help resolve differences between IL5RA protein and gene expression data?

Discrepancies between protein and transcript measurements are common in IL5RA research and require specific resolution strategies:

Temporal Considerations:

• Protein expression typically lags behind transcript expression

• Design time-course experiments to capture both mRNA and protein dynamics

• Consider protein half-life and mRNA stability

• Account for potential delays between transcription and translation

Isoform Analysis:

• Multiple IL5RA transcript variants and protein isoforms exist

• Ensure detection methods capture all relevant isoforms

• Use isoform-specific primers or antibodies when appropriate

• Consider alternative splicing regulation in different cell types

Post-transcriptional Regulation:

• Investigate microRNA-mediated regulation of IL5RA translation

• Assess RNA-binding proteins that may affect translation efficiency

• Consider nonsense-mediated decay of certain transcript variants

• Evaluate translational efficiency in different cellular contexts

Post-translational Modifications:

• IL5RA undergoes glycosylation and potentially other modifications

• These modifications may affect antibody binding and protein function

• Consider using methods to detect specific modifications

• Evaluate modification patterns across different cell types

Integrated Analysis Approaches:

• Combine RNA-seq with proteomics data

• Use single-cell multi-omics where feasible

• Apply computational methods to integrate data types

• Develop models that account for regulatory relationships

By systematically investigating these factors, researchers can better understand the relationship between IL5RA transcript and protein levels, improving interpretation of experimental results and resolving apparent discrepancies between different measurement approaches.

How can researchers distinguish between direct and indirect effects of IL5RA signaling in complex disease models?

Determining causality in complex systems requires specialized experimental approaches:

Cell-Specific Conditional Knockout Models:

• Use IL5RA conditional knockout mice crossed with cell-type specific Cre lines

• Compare phenotypes between global and conditional knockouts

• This approach has been used with eosinophil-specific deletion of IL-4/IL-13 to examine downstream mediators

• Target multiple cell types individually to dissect complex phenotypes

Bone Marrow Chimeras:

• Create mixed bone marrow chimeras with wild-type and IL5RA-deficient cells

• Allows identification of cell-intrinsic versus environmental effects

• Can reveal non-hematopoietic contributions to phenotypes

• Enables assessment of competitive fitness of IL5RA-sufficient versus deficient populations

Ex Vivo and In Vitro Validation:

• Isolate specific cell populations to test direct IL-5 responsiveness

• Compare responses between cells from wild-type and IL5RA-deficient mice

• Use pharmacological inhibitors with appropriate controls

• Recent studies have used sort-purified cells for qPCR confirmation of IL-5 target genes

Temporal Intervention Studies:

• Use neutralizing antibodies at different disease stages

• Compare prophylactic versus therapeutic intervention

• Studies have shown protective effects of recombinant IL-13 administration in liver injury models

• Monitor disease kinetics to identify primary versus secondary effects

Omics Approaches:

• Apply single-cell transcriptomics to identify direct IL-5 responsive cells

• Recent research has used single-cell RNA sequencing to identify IL5RA-enriched B cell clusters

• Use computational approaches to infer causal relationships

• Integrate multiple data types to build comprehensive models

By combining these approaches, researchers can build strong cases for direct versus indirect effects of IL5RA signaling and develop more precise models of how IL5RA contributes to complex disease phenotypes and protective immune responses.