CD5L Mouse

What is CD5L and what are its primary functions in mice?

CD5L is a secreted glycoprotein predominantly produced by tissue macrophages that functions as a pattern recognition receptor (PRR) capable of recognizing various microbial pathogens and endogenous harmful substances . In mice, CD5L performs several critical immunomodulatory functions:

• Enhances neutrophil recruitment and activation by increasing circulating levels of CXCL1

• Promotes neutrophil phagocytosis, contributing to bacterial clearance

• Regulates inflammatory responses, particularly in sepsis and infection models

• Modulates Th17 cell pathogenicity and stability

• Influences lipid biosynthesis

• Suppresses apoptosis in various cell types including T cells and natural killer T cells

CD5L is highly conserved across mammalian species and circulates at high levels in the blood of wild-type mice . It can function both as a soluble factor and through direct cellular interactions, making it a versatile immunoregulatory molecule.

How are CD5L knockout mice generated and validated?

CD5L knockout (CD5L-/-) mice can be generated through targeted gene disruption technologies such as CRISPR/Cas9. The validation of successful knockout involves multiple approaches:

• Genetic validation: Confirming the insertion of frameshift mutations and stop codons in the CD5L gene

• Protein expression analysis: Using ELISA to verify the absence of CD5L in blood samples (compared to high levels in WT mice)

• Immunofluorescence: Confirming the absence of CD5L in primary producer cells like peritoneal F4/80+ macrophages

• Functional validation: Assessing phenotypic differences in response to inflammatory challenges

The knockout confirmation is critical as the resulting mice appear phenotypically normal under standard conditions, with no significant differences in leukocyte numbers or frequency of cell subsets in the spleen, thymus, peripheral blood, and peritoneal cavity compared to wild-type counterparts .

What baseline phenotypic differences exist between wild-type and CD5L-/- mice?

In unchallenged conditions, CD5L-/- mice show remarkably few baseline differences from wild-type mice:

How does CD5L deficiency affect outcomes in sepsis models?

CD5L deficiency significantly impacts survival and bacterial control in sepsis models:

In the cecal ligation and puncture (CLP) polymicrobial sepsis model, CD5L-/- mice show:

• Markedly increased mortality (>60% mortality vs. 100% survival in WT) in medium-grade CLP

• Greater weight loss indicating worse general condition

• Severely impaired bacterial clearance (10,000-fold higher CFU counts in blood compared to WT mice)

• Higher bacterial burden in organs including lungs, liver, and kidneys

• Delayed and reduced neutrophil recruitment to the infection site

• Altered cytokine profiles in peritoneal fluids and blood

• Higher serum AST values (72h post-CLP) suggesting greater organ damage

These outcomes demonstrate that CD5L plays a protective role in sepsis by enhancing neutrophil recruitment and bacterial clearance. The inability of CD5L-/- mice to control bacterial spread leads to systemic inflammation and increased mortality, even in attenuated sepsis models.

What is the effect of CD5L in pulmonary fibrosis models?

Interestingly, CD5L deficiency has a protective effect in pulmonary fibrosis models, in contrast to its protective role in sepsis:

This highlights the context-dependent role of CD5L, which may be protective in acute infectious challenges (like sepsis) but potentially detrimental in chronic inflammatory conditions like pulmonary fibrosis.

How does CD5L influence autoimmune neuroinflammation models?

CD5L plays a significant role in regulating Th17 cell pathogenicity in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis:

• CD5L-/- mice develop more severe and persistent EAE compared to WT mice (which begin recovery 12 days post-immunization)

• Higher frequencies of IL-17-producing CD4+ T cells and fewer IFNγ-producing cells are found in the CNS of CD5L-/- mice

• CD5L deficiency does not affect initial Th17 differentiation but influences their stability and effector functions

• CD5L-/- Th17 cells produce less IL-10 (an anti-inflammatory cytokine)

• Upon restimulation, CD5L-/- Th17 cells show greater IL-17 production and IL-23R expression

• CD5L-/- effector memory cells have higher frequencies of IL-17+ and lower frequencies of IL-10+ cells

These findings indicate that CD5L acts as a regulatory switch that restrains Th17 cell pathogenicity, potentially by modulating lipid biosynthesis. In the absence of CD5L, Th17 cells maintain a more inflammatory phenotype, contributing to exacerbated autoimmune neuroinflammation.

How does CD5L interact with immunoglobulins in mice?

CD5L interacts with IgM through a specific molecular mechanism:

• CD5L binds to the joining chain (J chain) in a Ca2+-dependent manner

• This interaction is further stabilized by a disulfide bond linkage between CD5L and IgM

• Surface plasmon resonance (SPR) measurements reveal that mouse CD5L binds to mouse Fcμ–J with a Kd of 36.1 ± 10.3 nM

• Cat CD5L shows higher affinity for IgM (Kd of 14.4 ± 8.4 nM), suggesting evolutionary conservation with species-specific affinity differences

• The interaction requires calcium ions, as binding does not occur in the absence of Ca2+

• Cryo-electron microscopy reveals that CD5L binds to the gap of the IgM pentamer, with structural details showing:

  • SRCR3 domain of CD5L interacts with the J chain

  • A disulfide bond forms between Cys191 in SRCR2 of CD5L and Cys414 in the Cμ3 domain of IgM

This molecular interaction may contribute to CD5L's immunomodulatory functions by affecting IgM-mediated immune responses.

What signaling pathways does CD5L regulate in innate immune responses?

CD5L regulates multiple signaling pathways in innate immune responses:

In the context of sepsis and infection, CD5L influences:

• Neutrophil recruitment and activation pathways via CXCL1 modulation

• Phagocytosis and bacterial clearance mechanisms

• Control of pro-inflammatory cytokine production

Transcriptomic analysis of peritoneal cells from CD5L-/- mice subjected to CLP reveals:

• 507 genes upregulated and 133 genes downregulated compared to WT mice

• Enhanced pro-inflammatory transcriptional profiles in CD5L-/- peritoneal cells

• Gene set enrichment analysis identified significant changes in multiple biological pathways

These findings suggest that CD5L acts as a regulator of innate immune responses, helping to balance pro-inflammatory and anti-inflammatory signals during infection and sepsis.

How does CD5L affect T cell function and inflammatory profiles?

CD5L significantly impacts T cell function through several mechanisms:

• Regulates lipid biosynthesis in Th17 cells, affecting their pathogenicity

• Does not affect initial Th17 differentiation or expression of signature genes (IL-17, IL-17F, IL-21, IL-23R, RORc, RORα)

• Modulates effector/memory Th17 cells after differentiation:

  • CD5L-/- Th17 cells show more stable IL-17 production upon restimulation

  • CD5L-/- Th17 cells express higher levels of IL-23R

  • CD5L-/- Th17 cells produce less IL-10, an anti-inflammatory cytokine

• Influences the stability of the Th17 cell lineage over time

• May regulate Th17 cell plasticity, as WT Th17 cells acquire IFNγ expression in vivo while CD5L-/- Th17 cells produce little IFNγ

These effects on T cell function reveal CD5L as a major regulatory switch that restrains Th17 cell pathogenicity, potentially by modulating cellular metabolism through effects on lipid biosynthesis.

What are the optimal methods for studying CD5L function in mouse models?

Several complementary approaches are recommended for studying CD5L function:

• Genetic models:

  • CD5L knockout mice (complete deletion)

  • Conditional knockout models (tissue-specific deletion)

  • Overexpression models

• Therapeutic administration approaches:

  • Recombinant CD5L protein administration (intravenous delivery at 2.5-5.0 mg/kg has been validated as safe)

  • Timing studies (preventive vs. therapeutic administration)

• Disease models for CD5L function assessment:

  • Cecal ligation and puncture (CLP) for polymicrobial sepsis (mild, medium, and high grade)

  • LPS-induced endotoxic shock

  • Bleomycin-induced pulmonary fibrosis

  • Experimental autoimmune encephalomyelitis (EAE)

• Analytical methods:

  • Flow cytometry for immune cell phenotyping

  • Bacterial colony-forming unit (CFU) counts for infection models

  • Multiplex cytokine analysis of biological fluids

  • RNA-sequencing and transcriptomic analysis

  • Histopathological scoring of tissues

  • Biochemical markers of organ damage (AST, creatinine)

These methodological approaches should be selected based on the specific aspect of CD5L function being investigated.

How can recombinant CD5L be used therapeutically in mouse models?

Recombinant CD5L (rCD5L) has shown promising therapeutic potential in mouse models:

Administration protocol:

• Dose: 2.5-5.0 mg/kg has been validated as safe and effective

• Route: Intravenous administration is most common

• Timing: Dependent on the model (preventive or therapeutic)

Safety profile:

• No adverse effects observed in naïve WT mice

• AST and creatinine levels remain normal

• No histopathological alterations in lungs, liver, or kidneys

Therapeutic effects in sepsis models:

• Significantly improves survival in high-grade CLP-induced sepsis in WT mice

• Lowers endotoxin and damage-associated molecular pattern (DAMP) levels

• Protects WT mice from LPS-induced endotoxic shock

• Enhances neutrophil recruitment and bacterial clearance

These findings suggest that rCD5L could have therapeutic potential for human sepsis treatment, though extensive translational studies would be required to validate this approach for clinical applications .

What considerations are important when interpreting CD5L knockout phenotypes?

When interpreting phenotypes of CD5L knockout mice, several important considerations should be taken into account:

• Context-dependent effects:

  • CD5L deficiency is detrimental in infection/sepsis models

  • CD5L deficiency is protective in pulmonary fibrosis

  • CD5L deficiency exacerbates autoimmune neuroinflammation

• Temporal dynamics:

  • Early vs. late effects may differ significantly

  • Acute vs. chronic disease models show opposite effects

• Cell type-specific considerations:

  • Effects on neutrophils (recruitment, function)

  • Effects on macrophages (polarization, apoptosis)

  • Effects on T cells (Th17 stability and pathogenicity)

• Molecular compensation:

  • Potential compensatory mechanisms in chronic knockout models

  • Acute depletion (via neutralizing antibodies) vs. genetic knockout

• Strain-specific effects:

  • Most studies use C57BL/6 background

  • Other genetic backgrounds may show different phenotypes

• Experimental variability factors:

  • Severity of disease induction (e.g., CLP grade, LPS dose)

  • Environmental factors (microbiome, housing conditions)

  • Age and sex of the animals

Careful consideration of these factors is essential for accurate interpretation of CD5L knockout phenotypes and for designing experiments that can distinguish direct from indirect effects of CD5L deficiency.

How might CD5L function differ between mouse models and human disease?

Understanding translational aspects of CD5L research requires careful consideration of species differences:

While the basic molecular structure and function appear conserved, species-specific differences in regulation, expression patterns, and interacting partners must be considered when translating findings from mouse models to human disease contexts . Future studies should directly compare mouse and human CD5L in parallel experimental systems to identify both conserved and divergent functions.

What are the molecular mechanisms by which CD5L regulates lipid metabolism in immune cells?

This advanced research question addresses a fundamental mechanism of CD5L function:

CD5L has been shown to regulate lipid biosynthesis in Th17 cells, which influences their pathogenicity . The specific mechanisms include:

• Modulation of polyunsaturated fatty acid (PUFA) metabolism

• Potential effects on cholesterol biosynthesis

• Regulation of lipid droplet formation in immune cells

• Impact on membrane lipid composition affecting signaling

• Potential interaction with transcriptional regulators of lipid metabolism

Further research is needed to fully elucidate:

• The direct versus indirect effects of CD5L on lipid metabolic enzymes

• Cell type-specific metabolic effects (T cells vs. macrophages)

• The relationship between CD5L's metabolic effects and its immunomodulatory functions

• How CD5L-mediated changes in lipid metabolism alter cellular responses to inflammatory stimuli

This represents an important frontier in understanding how immune regulation is linked to cellular metabolism.

How does CD5L interact with other pattern recognition receptors in coordinating immune responses?

CD5L functions as a pattern recognition receptor (PRR) that recognizes both microbial pathogens and endogenous harmful substances . Its interaction with other PRRs represents an important area for advanced research:

Potential interactions include:

• Cooperation or competition with Toll-like receptors (TLRs) in recognizing pathogen-associated molecular patterns

• Integration with NOD-like receptors (NLRs) in inflammasome regulation

• Coordination with C-type lectin receptors in fungal detection

• Interaction with scavenger receptors in clearance of cellular debris

Research approaches to investigate these interactions could include:

• Double knockout models (CD5L with other PRRs)

• Proteomics to identify physical interactions between CD5L and other PRRs

• Cell-specific conditional deletions to address tissue-specific PRR cooperation

• Pathway analysis to identify signaling convergence or divergence

Understanding these interactions would provide insight into how the innate immune system coordinates complex responses to diverse challenges through the integration of multiple pattern recognition systems.

What are the most significant unresolved questions in CD5L mouse research?

Despite significant advances, several critical questions remain unresolved in CD5L research:

• The precise molecular mechanism by which CD5L enhances neutrophil recruitment and phagocytosis in sepsis models

• How CD5L regulates macrophage polarization in different tissue contexts

• The structural basis for CD5L's ability to recognize diverse pathogens and endogenous ligands

• The relationship between CD5L's calcium-dependent interactions and its immunomodulatory functions

• Mechanisms underlying the context-dependent protective versus pathological roles of CD5L

• The potential for CD5L-based therapeutics in human disease, particularly sepsis

These questions represent important areas for future investigation that could significantly advance our understanding of immune regulation and potentially lead to novel therapeutic approaches.

What emerging technologies might advance CD5L research in mouse models?

Several cutting-edge technologies hold promise for advancing CD5L research:

• Single-cell RNA sequencing: To define cell-specific responses to CD5L and identify novel cellular targets

• CRISPR-based screening: To identify genes that interact functionally with CD5L

• Spatial transcriptomics: To map CD5L expression and effects in tissue contexts

• Intravital imaging: To visualize CD5L-dependent cellular interactions in real-time

• Cryo-electron microscopy: To further resolve CD5L protein complexes with binding partners

• Metabolomics: To comprehensively characterize CD5L's effects on cellular metabolism

• Humanized mouse models: To better translate findings toward human applications