Recombinant Mouse Interleukin-11 protein (Il11) (Active)

What is mouse IL11 and how does it function in biological systems?

Mouse IL11 is a member of the IL6 family of cytokines consisting of 178 amino acids (position 22-199). It functions as a signaling molecule that binds to its receptor formed by IL6ST and either IL11RA1 or IL11RA2, activating a downstream cascade that promotes cell proliferation . At the molecular level, this signaling pathway leads to the activation of intracellular protein kinases and the phosphorylation of STAT3 . Two distinct signaling mechanisms exist: "classic signaling" occurs through interaction with membrane-bound IL11RA and IL6ST, while "trans-signaling" happens when IL11 binds to soluble IL11RA and then to IL6ST . Physiologically, IL11 stimulates the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells, induces megakaryocyte maturation leading to increased platelet production, and promotes hepatocyte proliferation in response to liver damage .

How has our understanding of IL11 function evolved over time?

What are the critical factors to consider when designing experiments with rmIL11?

When designing experiments with rmIL11, researchers must consider several critical factors to ensure valid and interpretable results:

• Species matching: Use rmIL11 for mouse experiments rather than rhIL11 to avoid partial agonist effects that could lead to misinterpretation of results . The species-specific recombinant protein ensures proper receptor engagement and downstream signaling.

• Dosing considerations: Research indicates that a dose of 200 μg/kg/day is effective for studying physiological effects in mice . This dosage has been shown to successfully induce measurable biological responses such as CD34+/VEGFR2+ cell mobilization and cardiac effects.

• Administration route and timing: Consider whether intraperitoneal injection (as used in cardiac studies) or continuous infusion via osmotic pumps (as used in vascular studies) is most appropriate for your experimental question. The timing of measurements is crucial as some effects peak at specific timepoints (e.g., CD34+/VEGFR2+ cell mobilization peaked at 72 hours post-administration) .

• Appropriate controls: Include both vehicle controls (PBS) and, when studying tissue-specific effects, consider tissue-specific receptor knockout models to confirm direct effects of IL11 .

• Comprehensive readouts: Incorporate multiple analysis methods (e.g., flow cytometry, histology, functional assays, transcriptomic analyses) to fully characterize the multifaceted effects of rmIL11 .

What methods are used to evaluate rmIL11 activity in experimental settings?

Multiple complementary methods can be employed to evaluate rmIL11 activity:

• Flow cytometry analysis: To quantify cellular responses such as mobilization of CD34+/VEGFR2+ mononuclear cells in peripheral blood .

• Physiological measurements: For cardiovascular studies, echocardiography provides in vivo assessment of cardiac function, while cardiomyocyte contractility assays can evaluate effects at the cellular level .

• Molecular signaling analysis: Western blotting (immunoblotting) to detect activation of downstream signaling molecules such as phosphorylated STAT3 .

• Transcriptomic profiling: Both bulk RNA-seq for tissue-level changes and single-nucleus RNA-seq (snRNA-seq) for cell-type specific responses provide comprehensive insights into transcriptional effects .

• Histomorphometry: To measure structural changes such as collateral vessel remodeling, analyzing parameters like luminal diameter in tissue sections .

• Functional scoring systems: For models of ischemia, hindlimb use and appearance scores can quantify functional recovery .

• ATAC-seq: To identify changes in chromatin accessibility that might influence gene expression patterns following IL11 treatment .

Why is the species specificity of IL11 critical for experimental interpretation?

The species specificity of IL11 represents one of the most crucial considerations in experimental design, as failure to account for this phenomenon has led to significant misconceptions in the field . This specificity is critical for several reasons:

How do findings from mouse IL11 studies translate to human biology?

Translating findings from mouse IL11 studies to human biology requires careful consideration of several factors:

• Direct translation challenges: Due to species specificity issues, effects observed with rmIL11 in mice may not directly translate to effects of rhIL11 in humans. Crystal structure studies are needed to further understand the complex receptor-ligand interactions across species .

• Shared pathways with differences: Both mouse and human IL11 activate similar downstream pathways involving STAT3 phosphorylation, but the tissue-specific consequences may differ . For example, while rmIL11 causes cardiac stress in mice, the exact profile of cardiac effects in humans needs careful evaluation.

• Clinical observations as validation: The cardiac side effects observed in patients receiving rhIL11 therapy align with experimental findings showing direct cardiomyocyte toxicities in mice, suggesting some conservation of pathological mechanisms across species .

• Receptor distribution considerations: Differences in IL11 receptor expression patterns between mice and humans may affect tissue-specific responses to IL11 stimulation.

• Compensatory mechanisms: The finding that IL11 is redundant for hematopoiesis despite its use as a therapeutic for thrombocytopenia suggests complex compensatory mechanisms may exist in both species but with different efficiencies .

What are the cardiovascular effects of rmIL11 and how are they measured?

Recombinant mouse IL11 exerts significant effects on the cardiovascular system, primarily characterized by direct cardiomyocyte toxicity leading to acute heart failure . These effects can be measured through several approaches:

• Cardiac function assessment: Echocardiography provides in vivo measurements of cardiac performance in mice treated with rmIL11, revealing functional impairments consistent with heart failure .

• Cardiomyocyte-specific responses: Single nucleus RNA-sequencing (snRNA-seq) of hearts from rmIL11-treated mice reveals cardiomyocyte stress signatures. After rmIL11 injection, cardiomyocytes predominantly segregate into a stress state (state 0) characterized by expression of cardiac stress markers including Ankrd1, Ankrd23, Xirp2, and Nppb .

• Molecular pathway analysis: KEGG pathway analysis of cardiomyocyte-specific differentially expressed genes following rmIL11 treatment shows significant enrichment of ribosomal pathways (93 of 130 genes upregulated, fold enrichment: 4.5, FDR: 2.3e-46), suggesting effects on protein translation and potentially a pro-hypertrophic response in stressed cardiomyocytes .

• Genetic validation: Cardiomyocyte-specific Il11ra1 knockout mouse models, generated either through AAV9-mediated Tnnt2-restricted Cre (vCMKO) or Myh6-Cre (m6CMKO), provide tools to confirm the direct effects of IL11 specifically on cardiomyocytes .

• Contractility measurements: Direct assessment of cardiomyocyte contractility in vitro complements in vivo functional studies to evaluate IL11's effects at the cellular level .

How does rmIL11 affect vascular remodeling and angiogenesis?

Recombinant IL11 has significant effects on vascular biology, though the literature reveals complex and sometimes context-dependent outcomes:

• Mobilization of proangiogenic cells: Treatment with rhIL11 (at 200 μg/kg/day) leads to in vivo mobilization of CD34+/VEGFR2+ mononuclear cells, with peak mobilization occurring at 72 hours post-administration. This represents a 20-fold increase in circulating CD34+/VEGFR2+ cells compared to PBS-treated controls .

• Collateral vessel remodeling: In a hindlimb ischemia model, rhIL11 treatment results in significant collateral vessel growth. Histomorphometry analyses show a 1.5-fold increase in collateral vessel luminal diameter by day 8 and a 3-fold increase by day 21 after femoral artery ligation compared to controls .

• Improved perfusion: Mice pre-treated with rhIL11 for 72 hours prior to femoral artery ligation demonstrate a 3-fold increase in plantar vessel perfusion, leading to faster blood flow recovery .

• Functional improvements: rhIL11-treated mice exhibit better hindlimb appearance and use scores in ischemia models, indicating functional benefits of the vascular remodeling process .

• Limited effects on angiogenesis: Despite promoting collateral vessel growth, no significant difference in angiogenesis (measured by isolectin B4+ cell analysis) was observed 21 days after femoral artery ligation, suggesting that rmIL11's effects may be specific to arteriogenesis rather than capillary formation .

What effects does rmIL11 have on hepatic and hematopoietic systems?

rmIL11 exhibits significant effects on both hepatic and hematopoietic systems:

Hepatic effects:

• rmIL11 promotes hepatocyte proliferation specifically in response to liver damage, suggesting a role in liver regeneration .

• The literature contains contradictory findings regarding IL11's role in liver disease. While earlier studies using rhIL11 in mice suggested protective effects against liver damage, more recent work indicates that endogenous mouse Il11 might actually contribute to hepatotoxicity .

• Mechanistically, rhIL11 dose-dependently inhibits endogenous mouse Il11-dependent hepatotoxicity, explaining why earlier studies using rhIL11 observed protective effects that were actually due to inhibition of native mouse Il11 signaling .

Hematopoietic effects:

• rmIL11 stimulates the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells .

• It induces megakaryocyte maturation resulting in increased platelet production .

• Despite its historical development as a platelet-boosting agent, more recent findings suggest IL11 is largely redundant for normal hematopoiesis .

• Administration of rhIL11 in mice results in a 2.8-fold increase in circulating platelets and a 2.5-fold increase in monocytes, without significant changes in red blood cells, total white blood cells, lymphocytes, or granulocytes .

How do we reconcile conflicting reports about IL11's protective versus pathological effects?

The apparent contradictions in IL11 literature can be largely reconciled by understanding several key factors:

What are the key experimental pitfalls to avoid when studying IL11 biology?

Based on historical misconceptions and recent clarifications in the IL11 field, researchers should be vigilant about the following experimental pitfalls:

• Using cross-species recombinant proteins without validation: The most critical pitfall is using rhIL11 in mouse experiments without acknowledging its partial agonist/antagonist effects. Always use species-matched recombinant proteins (rmIL11 for mouse studies, rhIL11 for human studies) .

• Misinterpreting gain-of-function vs. loss-of-function: When using recombinant IL11 proteins, carefully determine whether the experimental design truly represents a gain-of-function or loss-of-function scenario based on species matching and receptor engagement .

• Inadequate controls: Proper controls should include vehicle (e.g., PBS) administration and, when possible, receptor knockout models to confirm direct IL11 effects .

• Overlooking temporal dynamics: IL11 effects may vary significantly over time. For example, CD34+/VEGFR2+ cell mobilization peaks at 72 hours after rhIL11 administration , while other effects may occur earlier or later.

• Failing to assess cell type-specific responses: Bulk tissue analyses may obscure cell-type specific effects. Single-cell or single-nucleus approaches reveal that IL11 induces distinct responses in different cell populations, such as the stress signature observed specifically in cardiomyocytes .

• Not considering context-dependency: IL11 effects may differ substantially between healthy tissues and disease states. The experimental context (e.g., baseline condition, disease model, concurrent treatments) should be carefully considered when interpreting results.

How can genetically modified mouse models advance our understanding of IL11 biology?

Genetically modified mouse models provide powerful tools for dissecting IL11 biology with precision:

• Cell type-specific receptor knockouts: Cardiomyocyte-specific Il11ra1 knockout models using either AAV9-mediated and Tnnt2-restricted Cre (vCMKO) or Myh6-Cre (m6CMKO) have been developed to study IL11's direct effects on cardiomyocytes . Similar approaches could be applied to other cell types of interest, such as hepatocytes, fibroblasts, or hematopoietic stem cells, to delineate tissue-specific IL11 functions.

• Inducible expression systems: Transgenic mice with inducible expression of rmIL11 could help characterize the temporal dynamics of IL11's effects and avoid developmental confounders.

• Reporter mice: Developing IL11 or IL11RA reporter mice would facilitate visualization of expression patterns across tissues and in response to various stimuli or disease states.

• Humanized receptor models: Creating mice with humanized IL11 receptors would enable more translational studies using rhIL11 without the confounding partial agonist effects observed when using rhIL11 in wild-type mice.

• Signaling pathway mutants: Mice with mutations in specific IL11 downstream signaling components could help dissect which pathways mediate different IL11 functions.

What emerging technologies are advancing IL11 research?

Several cutting-edge technologies are enhancing our understanding of IL11 biology:

• Single-cell and single-nucleus RNA sequencing: This technology has revealed cell type-specific responses to rmIL11, including a distinct stress signature in cardiomyocytes . Further applications could identify additional cell populations responsive to IL11 and characterize heterogeneity in these responses.

• ATAC-seq for chromatin accessibility: Analysis of chromatin accessibility changes following IL11 treatment provides insights into transcriptional regulation mechanisms .

• Spatial transcriptomics: Emerging spatial profiling methods could map IL11 signaling effects within tissue microenvironments, revealing localized responses not detectable in bulk analyses.

• Advanced protein structural analyses: Crystal structure studies of species-specific IL11-receptor interactions would help explain the complex pharmacology observed across species .

• In vivo imaging of signaling dynamics: Development of biosensors for real-time visualization of IL11 signaling in live animals could provide unprecedented insights into the temporal and spatial dynamics of pathway activation.

• Organoid models: Three-dimensional organoid cultures derived from various tissues could serve as platforms for studying IL11 effects in more physiologically relevant contexts than traditional 2D cell culture.