Recombinant Mouse C-C motif chemokine 22 protein (Ccl22)

What is Recombinant Mouse CCL22 and what are its key biological functions?

Recombinant Mouse CCL22, also known as Chemokine (C-C Motif) Ligand 22 or MDC, is a chemokine primarily secreted by dendritic cells and macrophages. It exerts its effects by interacting with cell surface chemokine receptors, particularly CCR4 . Biologically, CCL22 functions as a selective chemoattractant for Th2 cytokine-producing cells and serves as an important activator of eosinophils after their migration into tissues . It also plays significant roles in the trafficking of activated/effector T-lymphocytes to inflammatory sites and demonstrates chemotactic activity for monocytes, dendritic cells, and natural killer cells .

Recently, CCL22 has been identified as a crucial mediator in adipose tissue thermogenesis and metabolism, particularly in promoting the beiging of white adipose tissue (WAT) in response to cold exposure . This expanded understanding of CCL22's functions highlights its potential significance beyond immune regulation and into metabolic homeostasis.

What are the molecular characteristics of Recombinant Mouse CCL22?

Recombinant Mouse CCL22 is typically produced using either E. coli or Pichia expression systems . The recombinant protein corresponds to amino acids 25-92 of the native sequence (Accession #NP_033163.1 or O88430) . The predicted molecular weight is approximately 7.8 kDa , and recombinant preparations typically achieve >97% purity as determined by SDS-PAGE and silver stain analysis .

When producing the recombinant protein, manufacturers may include various tags (such as His-tags) for purification purposes, though tag-free versions are also available . The protein is commonly supplied in lyophilized form, often containing stabilizers like trehalose (typically 8%) . The biological activity of Recombinant Mouse CCL22 is assessed through chemotactic assays, with typical ED₅₀ values ranging from 5-20 ng/ml for human T-lymphoblastoid CEM-NKR cells and 0.5-3 ng/ml for mouse BaF/3 cells transfected with hCCR4 .

How should Recombinant Mouse CCL22 be reconstituted and stored for optimal stability?

For proper reconstitution of lyophilized Recombinant Mouse CCL22, researchers should:

• Centrifuge the vial before opening to ensure the lyophilized protein is at the bottom of the vial .

• Reconstitute to a concentration of 0.1-0.5 mg/mL using sterile distilled water .

• Avoid vortexing or vigorous pipetting, which can denature the protein .

For long-term stability, consider these practices:

• Add a carrier protein or stabilizer (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose) to minimize protein degradation .

• Aliquot the reconstituted solution to minimize freeze-thaw cycles, which can significantly reduce biological activity .

• Store the lyophilized protein at -20°C to -80°C for up to 1 year from receipt .

• After reconstitution, the protein solution remains stable at -20°C for approximately 3 months or at 2-8°C for up to 1 week .

• For experimental use, reconstituted protein can be diluted in buffers containing carrier proteins to maintain stability during handling.

What are the optimal methods for evaluating CCL22's chemotactic activity in vitro?

To evaluate the chemotactic activity of Recombinant Mouse CCL22, researchers can employ several methodological approaches:

• Transwell Migration Assay:

  • Use chambers with appropriate pore sizes (3-5 μm for lymphocytes and 5-8 μm for larger cells).

  • Place different concentrations of CCL22 (typically starting at 0.1-100 ng/ml) in the lower chamber.

  • Add cells of interest to the upper chamber (e.g., T-lymphoblastoid CEM-NKR cells or CCR4-transfected BaF/3 cells) .

  • Incubate for 2-4 hours at 37°C with 5% CO₂.

  • Quantify migrated cells by flow cytometry or cell counting.

• Cell-Specific Migration Assessment:

  • For T-lymphocytes: CCL22 exhibits differential attraction between chronically activated versus resting T-lymphocytes .

  • For dendritic cells and natural killer cells: Assess migration using specific markers for these populations .

  • Controls should include cells exposed to buffer alone (negative control) and cells exposed to known chemoattractants (positive control).

• Dose-Response Analysis:

  • Typical ED₅₀ values for CCL22-induced migration range from 0.5-20 ng/ml, depending on the target cell type .

  • Establish complete dose-response curves to identify optimal concentrations for specific cell types.

How can Recombinant Mouse CCL22 be used to study adipose tissue beiging in vitro?

For studying CCL22's role in adipose tissue beiging in vitro, researchers can follow this methodological approach:

• Isolation of Stromal Vascular Fraction (SVF) cells:

  • Isolate SVF cells from inguinal white adipose tissue (iWAT) of mice.

  • Seed SVF cells in appropriate culture plates with complete media .

• CCL22 Treatment Protocol:

  • Pretreat SVF cells with recombinant CCL22 (10 ng/ml is effective) for approximately 4 days before inducing differentiation into beige adipocytes .

  • For control groups, maintain parallel cultures without CCL22 supplementation.

• Beige Adipocyte Differentiation:

  • After CCL22 pretreatment, induce differentiation using standard adipogenic cocktails with beiging-specific inducers.

  • Monitor differentiation progress over 7-10 days.

• Assessment of Beiging Phenotype:

  • Analyze UCP1 expression using immunofluorescence staining and Western blotting .

  • Perform qPCR analysis of thermogenic genes (e.g., Ucp1, Pgc1α, Cidea) .

  • Assess mitochondrial content and function using MitoTracker staining or Seahorse analysis.

This protocol has been shown to effectively promote beiging in both normal SVF cells and those from lymph node-removed (LNR) mice, demonstrating CCL22's direct role in beige adipogenesis .

How does CCL22 mediate communication between immune cells and adipose tissue metabolism?

CCL22 serves as a critical communicator between immune cells and adipose tissue metabolism, particularly in the context of cold-induced thermogenesis. This cross-talk operates through a sophisticated signaling network:

• Source of CCL22 in Adipose Tissue:

  • Cold exposure triggers upregulation of CCL22 expression primarily in M2 macrophages within adipose tissue .

  • While both dendritic cells and M2 macrophages produce CCL22, the most pronounced cold-induced increase occurs in M2 macrophages .

  • This was confirmed through experiments showing that silencing CCL22 in M2 macrophages abolished their capacity to restore beiging in lymph node removal (LNR) models .

• CCL22-CCR4 Signaling Axis:

  • CCL22 exerts its effects by binding to the CCR4 receptor on target cells, particularly eosinophils .

  • This interaction leads to recruitment and activation of eosinophils, which then secrete IL-4 .

  • IL-4 signaling subsequently activates thermogenic programming in adipocyte precursors.

• Lymph Node-Adipose Tissue Relationship:

  • Local lymph nodes regulate CCL22 production in adjacent adipose tissue .

  • Lymph node removal abolishes cold-induced CCL22 expression in iWAT, suggesting lymphatic communication .

  • Supplementation with recombinant CCL22 restores beiging capacity in lymph node-removed models, confirming CCL22's mediator role .

This communication pathway represents a novel immune-metabolic circuit that integrates environmental cues (cold) with immune signaling to regulate energy homeostasis through adaptive thermogenesis.

What are the methodological considerations for in vivo supplementation of Recombinant Mouse CCL22?

When designing experiments involving in vivo supplementation of Recombinant Mouse CCL22, researchers should consider these methodological aspects:

• Dosage and Administration:

  • Effective dosing: 20 μg/kg per day has been demonstrated to be effective in mouse models .

  • Administration routes: Local injection into target adipose tissue (e.g., iWAT) is commonly used for tissue-specific effects .

  • Treatment duration: 14-day supplementation protocols have shown significant physiological effects .

• Experimental Design Considerations:

  • Environmental conditions: Combined CCL22 supplementation with cold exposure (6°C) enhances thermogenic effects .

  • Dietary conditions: CCL22 supplementation shows pronounced effects when combined with high-fat diet (HFD) challenges .

  • Controls: Include vehicle-injected controls under identical environmental and dietary conditions.

• Assessment Parameters:

  • Physiological measurements:

    • Core body temperature using rectal probes

    • Blood glucose levels

    • Body weight and body composition (fat mass)

    • Energy expenditure and respiratory exchange ratio using metabolic cages

  • Tissue-specific analyses:

    • Histological examination (H&E staining)

    • Immunofluorescence staining for UCP1

    • Western blotting for thermogenic proteins

    • qPCR analysis of thermogenic gene expression

  • Metabolic function tests:

    • Glucose tolerance test (GTT)

    • Insulin tolerance test (ITT)

These methodological considerations ensure robust and reproducible results when studying CCL22's effects on adipose tissue biology and systemic metabolism in vivo.

How can the therapeutic potential of CCL22 be evaluated in obesity and metabolic disease models?

To evaluate CCL22's therapeutic potential in obesity and metabolic disease models, researchers should implement comprehensive assessment protocols:

• Animal Model Selection:

  • Diet-induced obesity (DIO) models using high-fat diet feeding .

  • Genetic models of obesity (e.g., ob/ob or db/db mice) for mechanism-specific investigations.

  • Age considerations: Middle-aged mice (10-12 weeks old) are typically used to study adult-onset obesity .

• Intervention Strategies:

  • Preventive approach: Begin CCL22 administration (20 μg/kg daily) concurrently with high-fat diet introduction .

  • Therapeutic approach: Initiate CCL22 treatment after establishing obesity to evaluate reversal potential.

  • Combined interventions: Evaluate CCL22 in combination with cold exposure (6°C) for enhanced thermogenic activation .

• Comprehensive Outcome Assessment:

  • Metabolic parameters:

    • Weight gain trajectories and body composition analysis

    • Glucose homeostasis (fasting glucose, GTT, ITT)

    • Lipid metabolism (serum triglycerides, cholesterol profiles)

    • Energy expenditure and substrate utilization (indirect calorimetry)

  • Tissue-specific changes:

    • Adipose tissue morphology and UCP1 expression

    • Liver steatosis assessment

    • Brown adipose tissue activation

    • Circulating adipokines and inflammatory markers

  • Molecular mechanisms:

    • CCL22-CCR4 signaling axis activity

    • Downstream effectors including IL-4 production

    • Thermogenic gene program activation (UCP1, PGC1α, CIDEA)

• Translational Considerations:

  • Corroborate findings with human adipose tissue samples

  • Analyze correlations between serum CCL22 levels and obesity parameters in clinical cohorts

  • Evaluate CCL22 expression changes during weight loss interventions in humans

Research has already demonstrated that CCL22 supplementation protects mice from diet-induced obesity and improves glucose tolerance, suggesting significant therapeutic potential . Additionally, human studies have revealed negative correlations between CCL22 levels and body weight/fat mass, with increased CCL22 levels observed during successful weight loss interventions .

How do findings from mouse CCL22 studies translate to human biology?

Translational research suggests significant parallels between mouse and human CCL22 biology, despite some species-specific differences:

• Clinical Correlations:

  • Human studies have demonstrated a negative correlation between serum CCL22 levels and body weight/fat mass percentage, mirroring findings in mice .

  • In a cohort of obese adults (BMI > 30 kg/m²), lower CCL22 levels were associated with higher body weight and fat mass .

  • Weight loss interventions in humans, specifically alternate day fasting (ADF) combined with low carbohydrate diets, were associated with significant increases in circulating CCL22 levels .

• Functional Conservation:

  • Human adipose-derived stromal vascular fraction (SVF) cells respond to recombinant CCL22 treatment with increased UCP1 expression and thermogenic gene activation, similar to mouse cells .

  • Ex vivo experiments with human subcutaneous adipose tissues demonstrated that CCL22 treatment promotes beiging under cold conditions (31°C), suggesting conserved thermogenic mechanisms .

• Methodological Considerations for Human Studies:

  • Human adipose tissue samples require different isolation and culture conditions than mouse samples.

  • Temperature parameters for inducing "cold" responses in human adipocytes (31°C) differ from those used in mouse studies (6°C) .

  • Longer culture periods may be necessary for human cells (20 days) compared to mouse cells .

These translational findings support the potential relevance of CCL22-targeted approaches for human metabolic disorders, although further clinical studies are needed to fully establish safety and efficacy profiles.

What methodological approaches are recommended for studying CCL22's effects on human adipose tissue?

When investigating CCL22's effects on human adipose tissue, researchers should consider these specialized methodological approaches:

• Human Adipose Tissue Processing:

  • Obtain subcutaneous adipose tissue samples through surgical procedures or liposuction with appropriate ethical approval.

  • Process samples within 1-3 hours of collection to maintain cell viability.

  • Perform collagenase digestion with human-specific enzyme concentrations and centrifugation protocols to isolate the SVF fraction.

• In Vitro Culture System:

  • Culture isolated human SVF cells with recombinant CCL22 (10 ng/ml recommended) for 4 days prior to differentiation .

  • Induce adipogenic differentiation using human-specific differentiation cocktails.

  • Maintain cultures at standard temperature (37°C) followed by mild cold exposure (31°C) for 20 days to induce beiging .

• Analytical Methods:

  • Assess UCP1 protein levels through Western blotting and immunofluorescence staining .

  • Perform qPCR analysis of human thermogenic genes (UCP1, PGC1α, CIDEA, TBX1) .

  • Evaluate mitochondrial content and function using appropriate staining techniques and respirometry.

  • Consider single-cell or bulk RNA sequencing to capture the full spectrum of transcriptional changes.

• Clinical Correlations:

  • Measure circulating CCL22 levels in human subjects using validated ELISA kits .

  • Correlate CCL22 levels with metabolic parameters, including:

    • Body weight and BMI

    • Body composition (fat mass percentage)

    • Glucose metabolism markers

    • Energy expenditure measurements

  • Monitor changes in CCL22 levels during weight loss interventions .

These approaches have successfully demonstrated that CCL22 promotes thermogenic programming in human adipose tissue, supporting its potential translational significance for metabolic health interventions .

What are common challenges when working with Recombinant Mouse CCL22 and how can they be addressed?

Researchers may encounter several challenges when working with Recombinant Mouse CCL22. Here are common issues and recommended solutions:

• Protein Stability Issues:

  • Challenge: Loss of bioactivity during storage or handling.

  • Solutions:

    • Minimize freeze-thaw cycles by preparing small aliquots upon reconstitution .

    • Add carrier proteins (0.1% BSA, 5% HSA) to prevent surface adsorption and increase stability .

    • Store reconstituted protein at -20°C for medium-term storage (up to 3 months) .

• Inconsistent Biological Activity:

  • Challenge: Variable chemotactic responses in cell migration assays.

  • Solutions:

    • Validate protein activity using positive control cells (e.g., CCR4-expressing BaF/3 cells) .

    • Establish complete dose-response curves (0.1-100 ng/ml) for each cell type.

    • Ensure consistent cell densities and passage numbers in chemotaxis assays.

• Reconstitution Difficulties:

  • Challenge: Incomplete solubilization or protein aggregation.

  • Solutions:

    • Centrifuge the vial before opening to collect all lyophilized material .

    • Avoid vortexing; instead, gently rotate or rock to dissolve .

    • If aggregation occurs, try reconstituting in buffer with higher pH (7.4-8.0).

• Experimental Variability in Beiging Studies:

  • Challenge: Inconsistent adipocyte differentiation and UCP1 induction.

  • Solutions:

    • Standardize SVF isolation protocols and cell seeding densities.

    • Ensure consistent timing of CCL22 treatment (4 days pre-differentiation is optimal) .

    • Include positive controls (e.g., norepinephrine-treated cultures) in each experiment.

• In Vivo Delivery Challenges:

  • Challenge: Ensuring effective delivery to target tissues.

  • Solutions:

    • Consider local injection directly into adipose tissue for targeted effects .

    • Maintain consistent injection sites to minimize variability.

    • Confirm tissue distribution using labeled protein or by measuring local CCL22 concentrations.

Addressing these challenges through careful experimental design and technique optimization will improve the reliability and reproducibility of CCL22-related research.

How can researchers optimize CCL22-based experimental designs for different research questions?

Optimizing CCL22-based experimental designs requires tailoring approaches to specific research questions. Here are strategic recommendations for different research contexts:

• For Immunological Studies:

  • Cell Migration Assays:

    • Optimize CCL22 concentrations (typically 0.5-20 ng/ml) for different target cell populations .

    • Include appropriate controls: CCR4 antagonists as negative controls, other CCR4 ligands as positive controls.

    • For differential migration studies, compare responses of resting vs. activated T-lymphocytes, as CCL22 preferentially attracts activated cells .

  • Receptor Signaling Studies:

    • Employ CCR4-transfected cell lines alongside wild-type controls to confirm receptor specificity .

    • Monitor downstream signaling events at appropriate time points (calcium flux: seconds; kinase activation: minutes; gene expression: hours).

• For Adipose Tissue Beiging Research:

  • In Vitro Optimization:

    • Timing: Apply CCL22 (10 ng/ml) for 4 days before adipogenic induction for optimal effects .

    • Control for eosinophil presence in SVF cultures, as these cells mediate CCL22's effects via IL-4 secretion .

    • Include IL-4 blocking antibodies as negative controls to confirm mechanism.

  • In Vivo Optimization:

    • Dosage: 20 μg/kg per day for 14 days shows significant effects .

    • Environmental conditions: Combine CCL22 treatment with cold exposure (6°C) to enhance thermogenic activation .

    • Consider tissue-specific delivery methods for targeted effects.

• For Metabolic Disease Models:

  • Prevention vs. Treatment:

    • For prevention studies: Begin CCL22 administration concurrently with high-fat diet introduction .

    • For treatment studies: Establish obesity first (typically 8-12 weeks of HFD), then initiate CCL22 intervention.

  • Comprehensive Phenotyping:

    • Implement metabolic cage studies to capture energy expenditure changes (key for thermogenesis) .

    • Include glucose and insulin tolerance tests separated by recovery periods .

    • Monitor both acute (hours to days) and chronic (weeks) responses to distinguish immediate vs. adaptive effects.

• For Translational Research:

  • Human Sample Studies:

    • Account for donor variability by increasing sample size or using paired designs.

    • Adapt cold exposure protocols for human cells (31°C instead of 6°C) .

    • Extend culture periods for human adipocytes (up to 20 days) compared to mouse protocols .

These optimization strategies will enhance experimental rigor and increase the likelihood of generating meaningful, reproducible results across different CCL22 research applications.

What are emerging areas of CCL22 research beyond established immunological functions?

CCL22 research is expanding beyond its classical immunological roles into several emerging areas that represent promising frontiers for future investigation:

• Metabolic Regulation and Energy Homeostasis:

  • Recent discoveries have established CCL22 as a key regulator of adipose tissue beiging and energy expenditure .

  • Future research should explore CCL22's role in brown adipose tissue activation and whole-body energy homeostasis.

  • Investigating CCL22's interactions with other metabolic hormones and regulators (leptin, adiponectin, FGF21) would provide insights into integrated metabolic networks.

• Neuro-Immune-Adipose Interactions:

  • The connection between lymph nodes and adjacent adipose tissue suggests potential neuro-immune regulation of metabolism .

  • Future studies should examine whether CCL22 mediates communication between the nervous system and adipose tissue.

  • Investigating how environmental stimuli (stress, temperature) modulate CCL22 production could reveal novel adaptive pathways.

• Therapeutic Applications in Metabolic Diseases:

  • Given CCL22's ability to counteract diet-induced obesity and improve glucose metabolism, its therapeutic potential warrants systematic exploration .

  • Development of CCL22 analogs or CCR4 agonists with enhanced stability or tissue specificity represents an important avenue for drug discovery.

  • Combination approaches with established anti-obesity or anti-diabetic therapies may yield synergistic benefits.

• Aging and Senescence:

  • Age-related changes in CCL22 signaling and its impact on immunometabolism during aging remain largely unexplored.

  • Investigating whether CCL22 supplementation can counteract age-related metabolic dysfunction would be valuable for geroscience research.

• Exercise Physiology:

  • The relationship between exercise, muscle-adipose crosstalk, and CCL22 signaling represents an unexplored area.

  • Whether exercise-induced metabolic benefits involve CCL22-mediated pathways deserves investigation.

These emerging research directions highlight CCL22's increasingly recognized role at the intersection of immunity and metabolism, suggesting its potential significance in integrative physiology and disease intervention.

What novel methodological approaches could advance CCL22 research in the coming years?

Advancing CCL22 research will likely depend on implementing innovative methodological approaches that overcome current limitations and address complex research questions:

• Advanced Genetic Models:

  • Cell-specific conditional knockout systems: Developing models with inducible, tissue-specific deletion of CCL22 or CCR4 (e.g., adipocyte-specific, macrophage-specific) would enable precise dissection of cell-autonomous functions.

  • CRISPR-Cas9 base editing: Rather than complete gene deletion, introducing specific mutations to modify CCL22 structure or regulation could reveal function-specific domains.

  • Humanized mouse models: Creating mice expressing human CCL22/CCR4 would improve translational relevance.

• High-Resolution Imaging Technologies:

  • Intravital microscopy: Real-time visualization of CCL22-mediated immune cell trafficking in living tissues.

  • CLARITY or iDISCO tissue clearing: Three-dimensional imaging of CCL22 distribution and cell interactions within intact adipose tissues.

  • PET imaging with labeled CCL22: Non-invasive tracking of CCL22 biodistribution and receptor binding in vivo.

• Single-Cell and Spatial Omics:

  • Single-cell RNA sequencing: Identifying cell-specific responses to CCL22 treatment within heterogeneous tissues.

  • Spatial transcriptomics: Mapping CCL22 signaling networks within the tissue microenvironment.

  • Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics to comprehensively characterize CCL22 signaling pathways.

• Advanced Delivery Systems:

  • Controlled-release formulations: Developing biomaterials for sustained CCL22 delivery to overcome its short half-life.

  • Tissue-targeted nanoparticles: Directing CCL22 to specific tissues through targeted delivery systems.

  • Optogenetic or chemogenetic control: Creating systems for spatiotemporally controlled CCL22 release or CCR4 activation.

• Translational Research Tools:

  • Organoids and microphysiological systems: Developing 3D culture systems incorporating multiple cell types to model complex CCL22-mediated interactions.

  • Patient-derived xenografts: Implanting human adipose tissue into immunodeficient mice to study CCL22 effects in a more relevant context.

  • Digital biomarkers: Using wearable technology to correlate CCL22 levels with real-time metabolic parameters in human studies.

These methodological innovations would enable researchers to address more sophisticated questions about CCL22 function and potentially accelerate the translation of basic findings into clinical applications.