ELANE Mouse

What is the ELANE gene and what is its significance in mouse models?

ELANE (Elastase, Neutrophil Expressed) encodes neutrophil elastase, a serine protease expressed in neutrophil azurophil granules. This protein plays a crucial role in innate immunity and neutrophil function. In research contexts, ELANE has significant importance as mutations in this gene are associated with severe congenital neutropenia (SCN) in humans. Mouse models with ELANE mutations are developed to study the molecular mechanisms underlying neutropenia and to explore potential therapeutic approaches. These models allow researchers to investigate the effects of specific ELANE mutations found in human patients with neutropenia in a controlled experimental environment. ELANE has also recently been found to exhibit selective anticancer activity, making it a gene of interest in both immunology and oncology research .

How are ELANE mouse models generated?

ELANE mouse models are typically generated through targeted mutation approaches using homologous recombination in embryonic stem (ES) cells. The process involves several key steps:

• Design of a targeting vector containing the desired mutation in the Elane gene

• Introduction of this vector into embryonic stem cells where homologous recombination occurs

• Selection of ES cell clones with the properly integrated mutation

• Microinjection of these ES cells into blastocysts

• Implantation into pseudopregnant foster females

• Breeding of chimeric offspring to establish germline transmission

For example, the G193X Elane mouse model was created by introducing a mutation that produces a stop codon at amino acid G193, deleting the carboxyl-terminal 27 amino acids of the mature neutrophil elastase protein. This mutation reproduces the G192pter ELANE mutation found in some SCN patients . The successful ES cell clones were identified and used to generate chimeric mice, with subsequent breeding to establish stable transgenic lines. Genotyping of these mice typically involves PCR using specific primers targeting the Elane locus, followed by restriction enzyme digestion to distinguish between wild-type and mutant alleles .

What are the key phenotypic characteristics of ELANE mutant mice?

Interestingly, despite expectations based on human SCN, many ELANE mutant mice do not exhibit overt neutropenia under basal conditions. For instance, the G193X Elane mice show normal growth, development, and fertility, and are grossly indistinguishable from wild-type littermates . This suggests complex species-specific differences in neutrophil development regulation.

Key characteristics observed in various ELANE mouse models include:

• Normal basal granulopoiesis in many models, contrasting with human SCN phenotypes

• Increased sensitivity of granulocytic precursors to chemical induction of ER stress

• Rapid degradation of mutant NE protein in some models (e.g., G193X Elane mice)

• Normal stress granulopoiesis following myeloablative therapy

• Molecular evidence of unfolded protein response (UPR) activation in some models

The G193X Elane mice, while phenotypically normal, show complete absence of detectable NE protein despite normal mRNA expression, indicating post-translational degradation of the truncated protein . This rapid degradation may explain the lack of neutropenia in these mice, as the misfolded protein is efficiently removed before triggering substantial ER stress.

How does the unfolded protein response relate to ELANE mutations in neutropenia?

The unfolded protein response (UPR) has emerged as a key mechanism in the pathogenesis of ELANE-associated neutropenia. Research suggests a model where ELANE mutations result in NE protein misfolding, which induces endoplasmic reticulum (ER) stress, activates the UPR, and ultimately blocks granulocytic differentiation .

In G193X Elane mice, though basal UPR activation is not significant, granulocytic precursors show increased sensitivity to chemical induction of ER stress . This suggests that while the mutant protein alone may not trigger sufficient ER stress to impair granulopoiesis, it may predispose cells to ER stress when faced with additional challenges.

The UPR involves three main sensors: PERK, IRE1, and ATF6. Experiments with G193X Elane mice found that inactivation of PERK (protein kinase RNA-like ER kinase), one of the major sensors of ER stress, either alone or in combination with the G193X Elane mutation, did not significantly affect granulopoiesis . This indicates that either the PERK branch of the UPR is not critical for ELANE mutation-associated neutropenia, or that compensatory mechanisms exist in mice that are absent in humans.

Why do ELANE mouse models often fail to recapitulate human neutropenia phenotypes?

The discrepancy between human ELANE mutations causing severe neutropenia and mouse models showing normal neutrophil counts represents an important research puzzle. Several hypotheses explain this species difference:

• Protein sequence divergence: Human and mouse NE share only 76% amino acid identity, which may affect how mutations impact protein folding and function .

• Differential protein quality control: Mice may have more efficient systems for degrading misfolded proteins, preventing accumulation of toxic intermediates.

• Species-specific granulopoiesis regulation: The mechanisms controlling neutrophil development may have species-specific features that make mice more resistant to perturbations in NE function.

• Compensatory pathways: Mice may activate compensatory mechanisms that humans lack or cannot sufficiently upregulate.

For example, the V72M Elane mouse model, which reproduces a mutation found in SCN patients, showed normal granulopoiesis despite valine at amino acid 72 being conserved between species. This suggests that this specific mutation may induce different structural changes in mouse NE compared to human NE . Similarly, the G193X Elane mice produce a truncated NE protein that is rapidly degraded, potentially preventing the protein from accumulating and causing ER stress .

These differences highlight the importance of considering species-specific factors when using mouse models to study human diseases and may explain why different ELANE mutations have varying phenotypic consequences in mice versus humans.

What is the role of ELANE in selective cancer cell killing?

Recent research has revealed that ELANE possesses remarkable cancer-selective cytotoxic properties. Cui et al. demonstrated that ELANE can selectively kill a wide range of cancer cells while sparing proximal non-cancer cells and significantly attenuate tumorigenesis . This selective killing spans 35 different human or murine cancer cell lines across 11 tumor types, making ELANE a potential broad-spectrum anticancer agent .

The mechanism of ELANE's selective toxicity involves:

• Liberation of the CD95 death domain (DD) that interacts with histone H1 isoforms

• Triggering of an abscopal effect mediated by CD8+ T cells that prevents distant metastasis

Several factors contribute to this cancer-selective property:

• Higher levels of CD95 in cancer cells compared to normal cells

• Increased expression of histone H1 isoforms in malignant cells

• The interaction between CD95-DD and histone H1 isoforms occurs predominantly in cancer cells

This discovery presents a promising direction for developing selective anticancer therapies based on the naturally evolved specificity of neutrophil elastase against genetically diverse targets. Mouse models with controlled ELANE expression could be valuable tools for further investigating these anticancer mechanisms and developing therapeutic approaches.

What techniques are used to verify ELANE expression and function in mouse models?

Researchers employ multiple complementary techniques to verify ELANE expression and function in mouse models:

1. RNA Expression Analysis:

• RNA isolation from bone marrow using TRIzol reagent

• Reverse transcription and PCR with gene-specific primers

• Restriction enzyme digestion of PCR products to distinguish wild-type from mutant transcripts

2. Protein Expression Analysis:

• Western blotting using specific anti-NE antibodies

• Treatment with N-glycosidase F (PNGase F) to remove N-linked oligosaccharides for better detection

• Generation of custom antibodies (e.g., rabbit anti-mouse NE antibody) targeting specific regions of the NE protein

3. Enzymatic Activity Assays:

• Measurement of elastase activity using chromogenic, neutrophil elastase-specific substrates such as N-methoxysuccinyl Ala-Ala-Pro-Val p-nitroanilide

• Normalization of elastolytic activity per microgram of protein

4. Functional Assays:

• Analysis of granulopoiesis under basal and stress conditions

• Evaluation of ER stress response in isolated granulocytic precursors

• Assessment of neutrophil function and bacterial killing capacity

For example, in G193X Elane mice, researchers confirmed normal mRNA expression but found no detectable NE protein, suggesting post-translational degradation of the mutant protein . This combination of techniques provides a comprehensive assessment of ELANE expression and function at multiple levels, crucial for accurately characterizing mouse models.

How can researchers isolate and culture neutrophil precursors from ELANE mouse models?

Isolation and culture of neutrophil precursors from ELANE mouse models are essential for studying granulopoiesis and the effects of ELANE mutations. The following methodological approach is typically used:

Isolation of Hematopoietic Progenitors:

• Harvest bone marrow cells from femurs and tibias of mice

• Isolate c-Kit+lineage- hematopoietic progenitors using magnetic cell separation or fluorescence-activated cell sorting (FACS)

• For fetal liver-derived progenitors, harvest fetal livers at 12-14 days post-conception

Culture Conditions for Granulocytic Differentiation:

• Culture isolated progenitors in medium containing:

  • Kit ligand (stem cell factor)

  • Granulocyte-colony stimulating factor (G-CSF)

  • Appropriate base medium (e.g., RPMI 1640) supplemented with 10-20% fetal bovine serum

• Maintain cultures for 3-7 days to obtain populations enriched for granulocytic precursors

Analysis of Differentiation:

• Monitor cell morphology by Wright-Giemsa staining

• Assess surface marker expression (e.g., Gr-1, Mac-1) by flow cytometry

• Evaluate cell proliferation and viability using appropriate assays

Induction of ER Stress:

• Treat cultured cells with ER stress-inducing agents such as tunicamycin or thapsigargin

• Assess activation of UPR pathways through expression of markers like BiP, CHOP, and XBP1 splicing

Using this approach, researchers have demonstrated that granulocytic precursors from G193X Elane mice show increased sensitivity to chemical induction of ER stress, even though basal UPR activation is not significant . This methodology allows for detailed analysis of cell-autonomous effects of ELANE mutations on neutrophil development and response to stress.

What are the optimal genetic backgrounds for ELANE mouse studies?

The choice of genetic background can significantly influence the phenotypic expression of ELANE mutations in mouse models. Based on research practices, several genetic backgrounds have been used for ELANE mouse studies, each with advantages and considerations:

Common Genetic Backgrounds:

• Inbred 129/SvJ background: Provides genetic homogeneity and reproducibility

• Inbred C57Bl/6 background: Widely used standard background, obtained by backcrossing for 6+ generations

• Outbred 129/SvJ × C57Bl/6 hybrid background: Offers genetic diversity that may better reflect human population variability

Considerations for Background Selection:

• Genetic background can influence penetrance and expressivity of mutations

• Strain-specific modifiers may compensate for or exacerbate ELANE mutation effects

• Background strain characteristics (e.g., hematopoietic profile, immune system features) should align with research questions

Experimental Approach for Background Effect Assessment:

• Generate and characterize the same ELANE mutation on multiple backgrounds

• Compare phenotypes across backgrounds to identify strain-dependent effects

• Consider using F2 intercrosses or recombinant inbred strains to map modifier loci

In studies with the G193X Elane mouse model, researchers analyzed mice on three different genetic backgrounds (inbred 129/SvJ, outbred 129/SvJ × C57Bl/6, and inbred C57Bl/6) to assess potential background effects on phenotype . This approach allows for detection of genetic modifiers that may influence the manifestation of ELANE mutations.

For transplantation studies, immunodeficient recipients such as NOD,B6.SCID Il2rγ−/−KitW41/W41 (NBSGW) mice may be preferred as they support multilineage engraftment of human hematopoietic cells without irradiation .

How should researchers interpret discrepancies between different ELANE mouse models?

Interpreting discrepancies between different ELANE mouse models requires a systematic approach that considers multiple factors:

Methodological Considerations:

• Mutation-specific effects: Different ELANE mutations may affect protein structure, stability, and function in distinct ways. For example, the G193X mutation causes truncation and rapid degradation of the NE protein, while other mutations might result in stable but misfolded proteins .

• Model generation techniques: The method used to introduce mutations (e.g., knock-in, transgenic overexpression, CRISPR/Cas9) can influence phenotype severity and specificity.

• Experimental conditions: Basal versus stress conditions may reveal phenotypes not apparent under standard housing conditions. G193X Elane mice show normal basal granulopoiesis but increased sensitivity to ER stress inducers .

Analytical Framework:

• Compare multiple models side-by-side using standardized assays

• Assess phenotypes at multiple levels:

  • Molecular (protein expression, activity)

  • Cellular (neutrophil development, function)

  • Organismal (susceptibility to infection, response to stress)

• Consider species-specific differences in neutrophil biology

Integration with Human Data:

• Correlate findings in mouse models with observations in patients carrying the corresponding ELANE mutations

• Use patient-derived cells (e.g., induced pluripotent stem cells) to validate mechanisms observed in mouse models

The comparison of different models can provide valuable insights. For instance, while the V72M Elane mice showed normal granulopoiesis despite this mutation causing SCN in humans, the G193X Elane mice revealed a potential mechanism - rapid degradation of mutant protein - that might explain the lack of phenotype in some mouse models . These apparent discrepancies can thus drive deeper mechanistic understanding when systematically analyzed.

What are the current limitations of ELANE mouse models for studying human neutropenia?

ELANE mouse models face several important limitations when used to study human neutropenia:

Phenotypic Divergence:

• Most ELANE mutant mouse models fail to develop neutropenia despite carrying mutations homologous to those causing SCN in humans

• Species-specific differences in neutrophil development regulation may account for this divergence

Molecular Differences:

• Only 76% amino acid identity between human and mouse NE proteins

• Structural differences may alter how mutations affect protein folding and function

• Species-specific differences in protein quality control mechanisms

Compensatory Mechanisms:

• Mice may have redundant pathways or compensatory mechanisms absent in humans

• The UPR may function differently between species, with varying consequences for granulopoiesis

Experimental Challenges:

• Limited access to primary human samples for direct comparison

• Difficulties in studying cell-autonomous effects in complex in vivo systems

• Limitations in accurately modeling human disease kinetics and progression

Future Directions to Address Limitations:

• Development of humanized models (e.g., mice expressing human ELANE)

• Combined approaches using mouse models and patient-derived iPSCs

• Systems biology approaches to identify species-specific differences in neutrophil development networks

• Study of ELANE knockout models compared to mutation models to distinguish loss-of-function from gain-of-toxic-function mechanisms

A promising approach has been the development of CRISPR/Cas9-mediated ELANE knockout models, which enable neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells from SCN patients . These findings suggest that complete absence of ELANE may be less detrimental than expression of mutant forms, highlighting the likely "gain-of-toxic-function" nature of ELANE mutations in SCN.

What novel applications exist for ELANE mouse models beyond neutropenia research?

ELANE mouse models have emerging applications beyond neutropenia research, opening new avenues for investigation:

Cancer Research:

• ELANE has demonstrated selective cytotoxicity against multiple cancer cell lines while sparing normal cells

• ELANE mouse models could serve as platforms to study mechanisms of cancer-specific cell death

• Potential for developing targeted cancer therapeutics based on ELANE's selectivity for cancer cells

• Opportunity to investigate the abscopal effect mediated by CD8+ T cells that prevents distant metastasis

Inflammatory Disorders:

• ELANE plays roles in several inflammatory conditions beyond neutropenia

• Mouse models can help study neutrophil elastase's contribution to tissue damage in inflammatory diseases

• Potential applications in studying auto-inflammatory syndromes as indicated by ClinVar annotations

Protein Folding and Quality Control:

• ELANE mutations provide a model system for studying protein misfolding and ER stress responses

• Insights may apply to other conditions involving protein misfolding (neurodegenerative diseases, etc.)

• Opportunity to test therapeutic approaches targeting protein quality control mechanisms

Innate Immunity:

• ELANE's role in neutrophil function makes these models valuable for studying innate immune responses

• Models can be used to investigate neutrophil's role in eliminating genetically diverse pathogens

• Potential applications in studying host-pathogen interactions

Therapeutic Development:

• Testing of treatments that modulate UPR or enhance protein folding

• Evaluation of gene editing approaches (like CRISPR/Cas9) for correcting ELANE mutations

• Development of small molecules that might stabilize specific mutant forms of ELANE

Recent studies showing ELANE's selective anticancer activity represent a particularly promising direction. Cui et al. demonstrated that ELANE can kill 35 different human or murine cancer cells across 11 tumor types while sparing healthy cells . This broad-spectrum anticancer activity operates through mechanisms involving CD95 death domain liberation and interaction with histone H1 isoforms, suggesting therapeutic applications beyond neutropenia treatment.