CD14 Mouse

What is CD14 and what role does it play in mouse immune responses?

CD14 is a pattern recognition receptor primarily expressed on macrophages and monocytes that serves as a co-receptor for Toll-like receptors, particularly in the recognition of bacterial components such as lipopolysaccharide (LPS). In mice, CD14 exists in both membrane-bound (mCD14) and soluble (sCD14) forms. It mediates innate immune responses by facilitating the detection of pathogen-associated molecular patterns (PAMPs) and subsequent cellular activation .

How does CD14 expression differ across mouse tissues and cell types?

CD14 is predominantly expressed on cells of myeloid lineage, with the highest expression observed on liver macrophages (Kupffer cells), peritoneal macrophages, and circulating monocytes. During infection or inflammation, CD14 expression is significantly upregulated in affected tissues.

For instance, research has shown that Schistosoma mansoni infection causes approximately 30-fold and 100-fold increases in liver CD14 expression at 7 and 12 weeks post-infection, respectively . In specific models:

• Macrophage cell lines like RAW264.7 and J774A.1 express detectable levels of CD14

• CD14 is present at lower levels on microglial cells in the brain

• Hepatic stellate cells and sinusoidal endothelial cells express CD14 during inflammatory conditions

• Splenic, lung, and kidney tissues express varying levels of CD14, with expression increasing during infection

What are the physiological levels of soluble CD14 (sCD14) in mouse biological fluids?

According to quantitative assessments using ELISA, the normal ranges of sCD14 in mouse biological fluids are:

These values represent baseline conditions in healthy mice . During bacterial infections, especially meningitis, sCD14 levels increase dramatically in cerebrospinal fluid while showing only minor or transient increases in serum .

How are CD14 knockout mice generated and validated?

CD14 knockout (CD14 KO or CD14^-/-) mice are typically generated through targeted disruption of the CD14 gene using homologous recombination in embryonic stem cells. The general process involves:

• Creating a targeting vector that replaces critical exons of the CD14 gene with a neomycin resistance cassette

• Transfecting embryonic stem cells and selecting for cells with successful incorporation

• Injecting modified stem cells into blastocysts to create chimeric mice

• Breeding chimeric mice to generate heterozygous and then homozygous CD14-deficient mice

Validation of CD14 knockout is performed through:

• Genotyping using PCR to confirm genomic deletion

• Western blot analysis to verify absence of CD14 protein

• Flow cytometry to confirm lack of CD14 surface expression on monocytes/macrophages

• Functional assays demonstrating altered responses to LPS stimulation

Commercial CD14 knockout mice are available from repositories like Jackson Laboratory, as mentioned in search result .

What phenotypic differences are observed in CD14 knockout mice compared to wild-type mice?

CD14 knockout mice display several notable phenotypic differences compared to wild-type counterparts:

Inflammatory responses:

• Reduced pulmonary inflammation during Mycobacterium tuberculosis infection, resulting in improved survival rates (57% of wild-type mice succumbed while all CD14 KO mice survived)

• Lower levels of inflammatory markers (ALT, IL-6) and decreased liver necrosis after hepatic ischemia-reperfusion injury

• Decreased lung tissue damage during Achromobacter xylosoxidans infection, despite similar bacterial loads

Cell recruitment and activation:

• Reduced leukocyte recruitment to sites of inflammation (approximately 2-fold reduction in total lung leukocyte counts during M. tuberculosis infection)

• Altered expression of inflammatory cytokines with decreased production of pro-inflammatory mediators

Immune cell polarization:

• Increased alternative activation (M2) of macrophages during Schistosoma mansoni infection

• Enhanced expression of alternative activation markers including Ym1, RELMα, and Arg1 (2-3 fold increases) with corresponding decrease in classical activation marker Nos2

• Greater numbers of CD4^+Foxp3^+IL-10^+ regulatory T cells during parasite infection

Tissue pathology:

• Smaller liver granulomas during Schistosoma infection with increased collagen deposition

• Reduced lung inflammatory scores (9.5 ± 0.7 in CD14 KO vs. 13.9 ± 0.4 in wild-type)

Importantly, these phenotypic differences are most pronounced during infection or inflammatory challenges rather than in steady-state conditions .

How can CD14 shRNA transgenic mice be generated and what are their applications?

CD14 shRNA transgenic mice can be generated through pronuclear microinjection as demonstrated in search result . The methodology involves:

• Designing effective shRNA sequences targeting CD14 (e.g., shRNA-674 targeting bovine CD14, which showed conservation between species)

• Creating a construct containing:

  • The shRNA sequence under a suitable promoter (often U6 or H1)

  • A reporter gene (such as EGFP) for tracking expression

• Purifying the construct DNA for microinjection

• Performing pronuclear microinjection into fertilized mouse oocytes

• Implanting injected embryos into pseudopregnant females

• Screening offspring for transgene integration using PCR

• Breeding founder animals to establish transgenic lines

In the study cited, 37 founder pups were obtained through pronuclear microinjection, of which 3 were positive for the transgene. In the F1 generation, 11 of 33 mice (33%) were transgene-positive .

Applications of CD14 shRNA transgenic mice include:

• Studying the effects of partial CD14 knockdown versus complete knockout

• Investigating the role of CD14 in various infection models

• Evaluating the impact of CD14 downregulation on TLR4 signaling pathway

• Creating models for potential therapeutic interventions targeting CD14

• Using as a proof-of-concept before developing larger animal models

What are the most effective antibodies and protocols for detecting mouse CD14 by Western blot?

For effective detection of mouse CD14 by Western blot, researchers typically use:

Recommended antibodies:

• Goat Anti-Mouse CD14 Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF982) shows high specificity with approximately 10% cross-reactivity with recombinant human CD14

Optimized protocol:

• Sample preparation:

  • Lyse cells in appropriate buffer (RIPA buffer with protease inhibitors)

  • For tissue samples, homogenize in lysis buffer (20-50μg total protein recommended)

• SDS-PAGE conditions:

  • Run under reducing conditions using Immunoblot Buffer Group 1

  • Use 10-12% gels for optimal separation

• Transfer and blocking:

  • Transfer to PVDF membrane

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

• Primary antibody incubation:

  • Dilute Goat Anti-Mouse CD14 Antibody to 0.5 μg/mL in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

• Detection:

  • Use HRP-conjugated Anti-Goat IgG Secondary Antibody (e.g., R&D Systems, Catalog # HAF017)

  • Develop using enhanced chemiluminescence

Expected results: CD14 appears as a band of approximately 50-55 kDa in mouse macrophage cell lines like J774A.1 . Note that glycosylation patterns may cause slight variations in apparent molecular weight.

What methods are available for quantifying CD14 expression in mouse tissue samples?

Multiple complementary approaches are available for quantifying CD14 expression in mouse tissues:

1. Quantitative RT-PCR (qRT-PCR):

• Extract RNA using commercial kits (e.g., Purelink RNA MiniKit)

• Synthesize cDNA using reverse transcription kits (e.g., High Capacity cDNA Reverse Transcription kit)

• Perform qPCR using TaqMan system with mouse CD14-specific primers

  • Recommended primer: Mm00438094_g1 (Life Technologies)

  • Normalize to housekeeping genes like GAPDH (primer: Mm99999915_g1)

• Calculate relative expression using the 2^-ΔΔCT method

2. ELISA-based quantification:

• Commercial kits like Mouse CD14 Quantikine ELISA (R&D Systems)

  • Sensitivity: Mean minimum detectable dose 2.27 pg/mL (range: 0.66-6.15 pg/mL)

  • Calibrated against NS0-derived recombinant mouse CD14

• Appropriate for serum, plasma, and cell culture supernatants

3. Immunohistochemistry (IHC):

• Fix tissue in 10% buffered formaldehyde and prepare paraffin sections

• Dewax sections and block endogenous peroxidase activity

• Perform antigen retrieval if needed

• Block with 1% BSA

• Incubate with anti-CD14 antibody (e.g., clone 1H5D8)

• Use appropriate secondary antibody and visualization system

• Quantify using imaging software (e.g., ImageJ)

4. Flow cytometry:

• Prepare single-cell suspensions from tissues

• Block Fc receptors

• Stain with FITC-conjugated anti-mouse CD14 antibody (e.g., Biolegend #137006)

• Use FITC-conjugated IgG as control

• Analyze using flow cytometry software (e.g., Cell Quest)

Each method offers different advantages: qRT-PCR provides sensitive mRNA quantification, ELISA measures soluble CD14, IHC reveals spatial distribution in tissues, and flow cytometry allows analysis at the single-cell level.

How can flow cytometry be optimized for detecting CD14 on different mouse leukocyte populations?

Optimizing flow cytometry for CD14 detection on mouse leukocytes requires careful consideration of several parameters:

Sample preparation optimizations:

• For peritoneal macrophages:

  • Harvest cells by peritoneal lavage with cold PBS

  • Centrifuge cells and resuspend in 2% FCS-PBS

  • Filter through 70μm mesh to remove clumps

• For blood leukocytes:

  • Collect blood in EDTA tubes

  • Lyse red blood cells using commercial RBC lysis buffer

  • Wash cells twice in PBS containing 2% FCS

• For tissue-resident macrophages:

  • Digest tissues with collagenase and DNase

  • Filter through 100μm and then 40μm strainers

  • Perform density gradient separation if needed

Staining protocol optimization:

• Block Fc receptors with anti-CD16/CD32 for 10 minutes at 4°C

• Surface staining:

  • Use FITC-conjugated anti-mouse CD14 antibody (1μg per 10^6 cells)

  • Include appropriate lineage markers: F4/80 for macrophages, Ly6G for neutrophils, CD11c for dendritic cells

  • Incubate for 30 minutes at 4°C in the dark

• Wash twice with 2% FCS-PBS

• Fix cells with 1% paraformaldehyde if not analyzing immediately

Gating strategy for accurate identification:

• Gate on live cells using viability dye

• Exclude doublets using FSC-H vs. FSC-A

• Gate on leukocyte populations based on FSC/SSC properties

• Identify specific subpopulations using lineage markers

• Analyze CD14 expression within each subpopulation

• Use fluorescence-minus-one (FMO) controls to set accurate gates

Additional considerations:

• CD14 expression can be upregulated following stimulation (e.g., with LPS or glimepiride)

• Include positive controls such as RAW 264.7 or J774A.1 cells

• For detection of low CD14 expression, consider using brighter fluorochromes (PE instead of FITC)

• When assessing expression changes, report both percentage of positive cells and mean fluorescence intensity

How does CD14 contribute to bacterial infection outcomes in mouse models?

CD14's role in bacterial infection outcomes varies by pathogen type and infection site, often exhibiting a dual nature:

Gram-negative bacterial infections:

Mycobacterial infections:

• In Mycobacterium tuberculosis infection, CD14 contributes to pulmonary inflammation

• CD14 knockout mice showed significantly reduced lung inflammation during chronic M. tuberculosis infection

• While bacterial loads were similar between wild-type and CD14 KO mice, the survival rate was dramatically improved in CD14 KO mice (100% survival vs. 43% in wild-type)

• Key differences in CD14 KO mice included:

  • Reduced inflammatory cell recruitment to lungs

  • Decreased percentage of inflamed lung tissue

  • Lower lung inflammation scores (9.5 ± 0.7 vs. 13.9 ± 0.4 in wild-type)

  • Total leukocyte counts in lungs at 5 weeks post-infection were 28 ± 4 × 10^5/ml in CD14 KO vs. 62 ± 10 × 10^5/ml in wild-type mice

Pulmonary bacterial infections:

• In Achromobacter xylosoxidans pulmonary infection models, CD14 deficiency protected mice from lung injury and edema

• CD14-deficient mice showed reduced mortality despite similar bacterial loads compared to wild-type mice

• Mechanisms involved decreased lung inflammation, reduced hemorrhagic foci, and less pronounced lung edema

These findings suggest that while CD14 plays an important role in bacterial recognition, its absence often leads to better outcomes by limiting excessive inflammatory responses that cause collateral tissue damage.

What role does CD14 play in regulating macrophage polarization in mouse disease models?

CD14 serves as a critical regulator of macrophage polarization, particularly influencing the balance between classical (M1) and alternative (M2) activation states:

Parasite infection models:

• During Schistosoma mansoni infection, CD14-deficient mice displayed significantly increased alternative activation of macrophages in liver granulomas

• Expression of M2 markers including Ym1, RELMα, and Arg1 was increased 2-3 fold in infected CD14^-/- mice compared to wild-type controls

• Conversely, expression of Nos2 (iNOS), associated with classical M1 activation, was decreased in CD14^-/- mice

• Immunohistochemical staining for M2 markers in hepatic granulomas showed pronounced differences visible even at low magnification

Mechanistic insights:

• CD14 appears to regulate STAT6 activation, a key transcription factor in the IL-4Rα pathway that drives M2 polarization

• CD14^-/- mice showed increased STAT6 activation in vivo

• Adoptive transfer experiments demonstrated that wild-type macrophages transferred into CD14^-/- mice normalized the M2/CD4+ Th cell balance to levels closer to infected wild-type mice

• This suggests that CD14 expression on macrophages directly influences their polarization state

Implications for T cell responses:

• The macrophage polarization shift in CD14^-/- mice was accompanied by changes in T cell responses

• Splenocytes from infected CD14^-/- mice exhibited increased production of CD4+-specific IL-4, IL-5, and IL-13

• There was also an increase in CD4+Foxp3+IL-10+ regulatory T cells

• These findings suggest that CD14 on macrophages influences subsequent adaptive immune responses

The regulatory role of CD14 in macrophage polarization has significant implications for therapeutic approaches, as modulating CD14 function could potentially shift macrophage phenotypes to promote resolution of inflammation or enhance pathogen clearance depending on the disease context.

How does CD14 contribute to sterile inflammation in mouse models of tissue injury?

CD14 plays a significant role in sterile inflammation during tissue injury in mouse models, particularly in hepatic ischemia-reperfusion (I/R) injury:

Hepatic ischemia-reperfusion injury:

• CD14 expression is upregulated in reperfused livers, with increased soluble CD14 (sCD14) in circulation

• CD14 knockout mice subjected to hepatic I/R showed markedly reduced liver damage compared to wild-type controls:

  • Significantly lower alanine aminotransferase (ALT) levels at both 6 and 24 hours post-reperfusion

  • Decreased IL-6 levels, indicating reduced inflammatory responses

  • Substantially less liver necrosis by histological analysis

  • Reduced apoptosis at 24 hours as demonstrated by TUNEL staining

Mechanistic insights:

These findings indicate that CD14 contributes to sterile inflammation by:

• Acting as a co-receptor for damage-associated molecular patterns (DAMPs) released during tissue injury

• Amplifying inflammatory signaling through TLR4-dependent pathways

• Promoting recruitment and activation of inflammatory cells

• Enhancing production of pro-inflammatory cytokines

• Contributing to tissue damage through necrosis and apoptosis

The protective effect of CD14 deficiency in sterile inflammation models suggests that therapeutic targeting of CD14 might be beneficial in clinical scenarios involving ischemia-reperfusion injury, such as organ transplantation, myocardial infarction, or stroke.

What are the most effective methods for manipulating CD14 expression in mouse macrophages in vitro?

Several effective methods exist for manipulating CD14 expression in mouse macrophages for in vitro experiments:

RNA interference approaches:

• siRNA transfection:

  • Commercially available mouse CD14-specific siRNAs (typically 21-23 nucleotides)

  • Transfection using lipid-based reagents (e.g., Lipofectamine)

  • Optimal for short-term knockdown (3-5 days)

  • Particularly effective in cell lines like RAW264.7

  • Has been shown to inhibit release of TNF-α, IL-6, and nitric oxide production following LPS exposure

• shRNA expression:

  • Lentiviral or retroviral vectors expressing CD14-targeted shRNAs

  • Provides longer-term knockdown compared to siRNA

  • Can be used to create stable CD14-knockdown cell lines

  • Study identified effective shRNA sequence (shRNA-674) targeting bovine CD14 that showed conservation with mouse CD14

CRISPR-Cas9 gene editing:

• Design guide RNAs targeting exons of mouse CD14 gene

• Deliver Cas9 and guide RNA using plasmid transfection or lentiviral vectors

• Screen clones for complete knockout using Western blot or flow cytometry

• Provides permanent knockout compared to RNA interference methods

Pharmacological manipulation:

• Glimepiride treatment (5 μM) can induce release of CD14 from macrophage cell membranes

• Flow cytometry analysis showed significant reduction in cellular CD14 with corresponding increase in supernatant CD14 after 1-hour treatment of RAW 264 cells

Overexpression approaches:

• Transfection with mouse CD14 expression plasmids

• Lentiviral transduction for stable overexpression

• Addition of recombinant soluble CD14 to culture medium (can act as an inflammatory co-ligand)

Efficiency monitoring:

• Validate knockdown/overexpression by Western blot, qRT-PCR, and flow cytometry

• Functional validation using LPS stimulation assays measuring cytokine production

These methods can be selected based on experimental needs, with siRNA providing rapid but temporary knockdown, shRNA and CRISPR offering longer-term solutions, and pharmacological approaches allowing for more dynamic manipulation of CD14 levels.

How can researchers effectively address contradictory findings regarding CD14's role across different mouse disease models?

Addressing contradictory findings regarding CD14's role across different mouse disease models requires systematic approaches:

1. Standardize experimental conditions:

• Use mice with identical genetic backgrounds for knockout comparisons

• Control for age, sex, and housing conditions

• Standardize pathogen strains, doses, and administration routes

• Apply consistent timepoints for analysis across studies

2. Consider context-dependent factors:

• Pathogen type (Gram-negative bacteria vs. Gram-positive bacteria vs. parasites)

• Infection site (e.g., lung vs. liver vs. central nervous system)

• Acute vs. chronic disease stages

• Local vs. systemic effects

3. Analyze both protective and detrimental roles:

• Evaluate early pathogen recognition and clearance mechanisms

• Assess late-stage inflammation and tissue damage parameters

• Examine both innate and adaptive immune responses

• Consider the balance between pathogen control and immunopathology

4. Employ complementary approaches:

• Compare complete knockout models with conditional or tissue-specific knockouts

• Use both genetic (knockout) and pharmacological (antibody blocking) approaches

• Combine in vivo models with in vitro cell-specific studies

• Perform adoptive transfer experiments to identify cell-specific contributions

5. Integrate molecular mechanisms:

• Analyze downstream signaling pathways (TLR4, STAT6)

• Examine interactions with co-receptors and adaptor molecules

• Distinguish between membrane-bound and soluble CD14 effects

• Consider interactions with other pattern recognition receptors

Case example resolving contradictions:
In Schistosoma mansoni infection, CD14 deficiency led to smaller liver granulomas but increased fibrosis , seemingly contradictory outcomes. Researchers resolved this by:

• Demonstrating altered macrophage polarization (increased M2)

• Showing enhanced CD4+ Th2 responses

• Performing adoptive transfer experiments with wild-type macrophages

• Examining STAT6 activation pathways

This comprehensive approach revealed that CD14 regulates the IL-4Rα-STAT6 pathway, explaining how absence of CD14 could simultaneously reduce granuloma size but increase fibrotic responses through enhanced M2 polarization .

What are the critical considerations when designing experiments comparing wild-type and CD14-deficient mice?

When designing experiments comparing wild-type and CD14-deficient mice, researchers should consider several critical factors:

1. Genetic background considerations:

• Use appropriate background-matched controls (e.g., C57BL/6 for most commercially available CD14 KO mice)

• Consider using littermate controls when possible

• Account for potential genetic drift in long-established knockout colonies

• Verify CD14 deficiency in experimental animals by genotyping and protein expression analysis

2. Experimental design parameters:

• Determine appropriate sample sizes through power analysis based on expected effect sizes

• Include both male and female mice to account for sex differences

• Use age-matched mice (typically 8-12 weeks old for most studies)

• Consider both acute and chronic timepoints (as seen in M. tuberculosis studies where differences were more pronounced at later timepoints)

• Include multiple timepoints for analysis (e.g., 2, 5, and 32 weeks in tuberculosis models)

3. Phenotypic analysis considerations:

• Examine multiple parameters:

  • Bacterial/pathogen burden

  • Cellular infiltration (total counts and differential analyses)

  • Tissue damage and pathology scores

  • Cytokine/chemokine profiles

  • Cell activation states

• Use complementary techniques:

  • Histology and immunohistochemistry for tissue architecture

  • Flow cytometry for cellular phenotyping

  • qRT-PCR for gene expression

  • ELISA for protein quantification

  • Functional assays specific to the model system

4. Data interpretation nuances:

• Differentiate between direct CD14 effects and compensatory mechanisms

• Consider potential redundancy in pattern recognition receptors

• Account for both local tissue and systemic responses

• Evaluate both protective and pathological aspects of immune responses

5. Controls and validation experiments:

• Include positive controls for CD14-dependent responses (e.g., LPS stimulation)

• Consider rescue experiments through adoptive transfer of wild-type cells or addition of recombinant CD14

• Include antibody neutralization experiments to compare with genetic knockout approaches

• When possible, validate key findings in CD14 conditional knockout models to address developmental compensation concerns

6. Technical considerations for specific models:

• For bacterial infections, standardize inoculum preparation and verification

• For sterile inflammation models like ischemia-reperfusion, standardize surgical techniques

• For parasite models, control for parasite life cycle stage and burden

• For all models, standardize housing conditions, diet, and microbiome status

Attention to these considerations will enhance experimental rigor and facilitate meaningful comparisons between studies, helping to resolve apparent contradictions in CD14's roles across different disease models.