Recombinant Mouse C-C motif chemokine 6 protein (Ccl6)

What is mouse CCL6 and what are its fundamental structural properties?

Mouse CCL6 (also known as C10, SCYA6, and MRP-1) is a small cytokine belonging to the CC chemokine family. It is a member of the N6 subfamily of beta-chemokines that have an N-terminal extension relative to other beta-chemokines. The mouse CCL6 cDNA encodes a 116 amino acid (aa) precursor that includes a 21 aa signal sequence . The mature protein has a predicted molecular weight of 10.7 kDa . CCL6 belongs to the same subfamily as CCL9, CCL15, and CCL23, characterized by their N-terminal extensions .

Structurally significant is that removal of an additional 20 amino acids at the N-terminal results in an 8 kDa protein (aa 42-116), which transitions from a weak CCR1 agonist to a dramatically more potent and efficacious macrophage chemoattractant . This truncation is functionally relevant as the N-terminal region can be cleaved by inflammation-associated proteases such as chymase, cathepsin G, and elastase .

What are the primary biological functions of CCL6 in the immune system?

CCL6 functions primarily as a chemoattractant in the immune system. It is particularly potent in attracting macrophages to sites of inflammation, but can also attract B cells, CD4+ lymphocytes, and eosinophils . The protein is upregulated in activated macrophages at sites of inflammation and functions to recruit additional macrophages .

Beyond its chemotactic properties, recent research has revealed a role for CCL6 in viral defense. Studies have shown that CCL6 can directly decrease human norovirus (HuNoV) replication in HG23 cells, and might indirectly regulate the immune response against NoV infection . Vitamin A treatment significantly increases CCL6 expression, which correlates with enhanced antiviral activity against norovirus .

In inflammation cascades, IL-13 stimulates CCL6 production, which then induces the release of CCL2, CCL3, and proteases involved in tissue repair . The protein might also be an important mediator in the development of lung injury .

What are the optimal conditions for storage and reconstitution of recombinant mouse CCL6?

Recombinant mouse CCL6 is typically provided in lyophilized form and requires proper storage and reconstitution to maintain activity. Based on manufacturer specifications, the following protocol is recommended:

Storage conditions:

• Store lyophilized protein desiccated at -20°C to -70°C

• Properly stored, lyophilized CCL6 is stable for six to twelve months

Reconstitution protocol:

• Reconstitute using sterile water or buffer (specific to your experimental needs)

• After aseptic reconstitution, store at 2°C to 8°C for up to one month

• For longer storage, aliquot and store at -20°C to -70°C in a manual defrost freezer

• Avoid repeated freeze-thaw cycles as these significantly reduce protein activity

How can CCL6 knockdown be effectively achieved in experimental settings?

Based on the research protocols described in the literature, CCL6 knockdown can be achieved through siRNA-mediated gene silencing. The following methodology has been validated in previous studies:

• Selection of appropriate siRNA:

  • Commercial siRNAs targeting CCL6 are available (e.g., FlexiTube GeneSolution GS20305 from Qiagen)

  • Multiple concentrations should be tested (5, 10, and 25 nM have been successfully used)

• Transfection procedure:

  • Seed cells (e.g., RAW 264.7) at appropriate density (1 × 10^5 cells per well has been used successfully)

  • Allow cells to adhere and grow for approximately 6 hours before transfection

  • Prepare transfection complexes using siRNA and an appropriate transfection reagent (e.g., HiPerFect Transfection Reagent)

  • Incubate transfection complexes for 10 minutes at room temperature

  • Add complexes dropwise to cells and incubate for 30 hours to achieve gene silencing

• Validation of knockdown:

  • Confirm knockdown efficiency using quantitative RT-PCR with CCL6-specific primers and probes

  • Example primers for mouse CCL6 (NM_009139.3):

    • Forward primer: 5'-TTATTAGGGTAGTCTTTGGGGCTTTG-3'

    • Reverse primer: 5'-ACAACTGGGAACCCACAAAGCT-3'

    • Probe: 5'-FAM-GTGTCTGGTTCTGATACAAGCTTAAGCCGGGT-BHQ1-3'

What experimental systems are available for studying CCL6's antiviral effects?

To study CCL6's antiviral effects, particularly against noroviruses, researchers have employed several experimental systems:

• HG23 cell system for human norovirus:

  • HG23 cells are Huh-7-based NoV replicon-bearing cells that can be used to assess direct antiviral effects against human norovirus

  • Protocol: Apply recombinant murine CCL6 (1, 10, and 100 ng) to HG23 cells

  • Quantify NoV replicon replication 72 hours after treatment

• RAW 264.7 cell system for murine norovirus:

  • For MNV replication studies, treat cells with CCL6 twice: 12 hours prior to and after MNV infection

  • Compare CCL6 effects with positive controls such as recombinant IFN-β

• CCL6 knockout models in RAW 264.7 cells:

  • Develop CCL6 knockout cell lines using siRNA (as described above)

  • Infect with different murine norovirus strains (e.g., MNV-1.CW1 or MNV-1.CR6) at MOI 0.01

  • Quantify viral replication by plaque assay at 24 hours post-infection

How does the N-terminal truncation of CCL6 affect its functional properties?

The N-terminal region of CCL6 plays a critical role in modulating its biological activity. Research has demonstrated significant differences between full-length and truncated forms:

The truncated form of CCL6 (aa 42-116) demonstrates significantly enhanced biological activity compared to the full-length protein. This truncation occurs naturally in inflammatory environments, as the N-terminal region of CCL6 can be cleaved following incubation with synovial fluid from inflamed joints or by inflammation-associated proteases such as chymase, cathepsin G, and elastase .

This structural modification represents an important regulatory mechanism for CCL6 activity, allowing for context-dependent modulation of its chemotactic potency. Researchers should consider which form is most appropriate for their specific experimental questions, as the biological responses may differ substantially .

What is the mechanism by which vitamin A regulates CCL6 expression and function?

Vitamin A (particularly its active form, retinoic acid or RA) significantly impacts CCL6 expression and function through several mechanisms:

• Transcriptional regulation:

  • Microarray analysis reveals that retinol treatment significantly increases CCL6 expression (4.75-fold) compared to control conditions

  • This increase is further enhanced during MNV infection (6.40-fold), suggesting synergistic effects

• In vivo regulation:

  • In mouse models, RA administration (1 mg/kg/day) significantly increases CCL6 expression in ileum tissues

  • This effect is particularly pronounced in the absence of viral infection

• Functional consequences:

  • The retinol-induced increase in CCL6 correlates with decreased norovirus replication

  • Treatment with 100 ng of recombinant CCL6 significantly reduced human norovirus genome replication in HG23 cells to 71.3% of control levels

• Differential regulation of immune factors:

  • While increasing CCL6, RA administration simultaneously decreases expression of other antiviral factors like RIG-I, MDA-5, and IFN-β

  • This suggests a complex immunomodulatory role for vitamin A that may preferentially activate CCL6-mediated antiviral mechanisms while downregulating other pathways

How can researchers differentiate between direct and indirect antiviral effects of CCL6?

Differentiating between direct and indirect antiviral effects of CCL6 requires carefully designed experimental approaches:

• Direct antiviral effect assessment:

  • Use cell systems that support viral replication but lack complete immune response capability

  • Example: HG23 cells harboring a human NoV replicon can be used to measure direct effects of CCL6 on viral genome replication

  • Treatment with CCL6 directly reduced NoV genome replication in HG23 cells to 71.3%

• Indirect (immune-mediated) effect assessment:

  • Compare antiviral effects in immunocompetent vs. immunodeficient systems

  • Measure changes in immune factors (cytokines, chemokines) following CCL6 administration

  • Examine effects of CCL6 on immune cell recruitment and activation

• Comparative analysis approach:

  • Compare CCL6 effects with well-characterized direct antivirals (nucleoside analogs) and immune modulators (interferons)

  • In studies with IFN-β, it showed more significant anti-NoV effects in immunocompetent RAW 264.7 cells than in HG23 cells, indicating its primary mechanism is immune-mediated

  • In contrast, CCL6 showed significant effects on HG23 cells, suggesting a direct antiviral component

• Knockdown validation:

  • Use CCL6 knockout or knockdown systems to confirm specificity of effects

  • In CCL6 knockout RAW 264.7 cells, relative replication levels of MNV-1.CW1 and MNV-1.CR6 were significantly increased, confirming CCL6's antiviral role

What are the critical quality control parameters for evaluating recombinant CCL6 preparations?

When working with recombinant mouse CCL6, several quality control parameters should be assessed to ensure experimental reproducibility:

• Purity assessment:

  • Commercial recombinant CCL6 should have >97% purity as determined by SDS-PAGE and silver stain

  • Visual inspection of a single band at the expected molecular weight (~10.7 kDa) confirms protein integrity

• Endotoxin testing:

  • Endotoxin levels should be <1.0 EU/μg as determined by the LAL method

  • High endotoxin levels can confound immunological experiments by triggering non-specific inflammation

• N-terminal sequence verification:

  • Confirmation of the correct N-terminal sequence (typically starting with Gly22 in the mature protein)

  • This ensures proper processing of the signal peptide

• Functional activity testing:

  • Chemotactic activity in standard migration assays using appropriate target cells (macrophages)

  • The ED50 for chemotactic activity should be approximately 0.5-2 ng/mL

• Batch-to-batch consistency:

  • Always refer to lot-specific datasheets for technical information

  • Use lot-specific storage instructions as these may vary slightly between preparations

What statistical approaches are most appropriate for analyzing CCL6-related experimental data?

Based on the literature, the following statistical approaches are recommended for analyzing CCL6-related experimental data:

• For gene expression analysis:

  • Use the 2^-ΔΔCt relative quantification method for qPCR data:

    • ΔΔCt = (Ct.Target − Ct.GAPDH)Group1 − (Ct.Target − Ct.GAPDH)Group2

  • This normalizes target gene expression to an internal control (GAPDH) and allows comparison between experimental groups

• For group comparisons:

  • One-way analysis of variance (ANOVA) followed by Duncan's post hoc test is appropriate for comparing multiple groups

  • Statistical significance is typically set at P values < 0.05

  • Perform analyses using standard statistical software such as SPSS Statistics software

• Data representation:

  • Express all features as means and standard deviations

  • For complex datasets, consider hierarchical clustering analysis to identify patterns of gene expression, as was done with microarray data for CCL6 and related genes

• Sample size considerations:

  • For in vivo experiments, groups of 5-6 mice have provided sufficient statistical power in previous CCL6 studies

  • For in vitro experiments, a minimum of three independent replicates is recommended

What are promising areas for further investigation of CCL6 functions in disease models?

Based on current knowledge, several promising research directions merit further investigation:

• Expanded viral defense studies:

  • Given CCL6's demonstrated role in norovirus inhibition, its potential activity against other viral pathogens should be explored

  • The differential effects on acute (MNV-1.CW1) versus persistent (MNV-1.CR6) norovirus strains suggest CCL6 may have virus-specific mechanisms worth investigating

• Mechanistic studies of CCL6's direct antiviral activity:

  • The molecular mechanisms by which CCL6 directly inhibits viral replication remain poorly understood

  • Investigating potential interactions with viral proteins or effects on cellular factors required for viral replication could yield important insights

• CCL6 in lung injury and repair:

  • Given its potential role as a mediator in lung injury development, CCL6's function in acute respiratory distress syndrome and other pulmonary conditions warrants investigation

  • The interplay between CCL6 and tissue repair mechanisms could provide therapeutic targets

• Vitamin A and CCL6 in mucosal immunity:

  • The vitamin A-CCL6 axis appears important in intestinal antiviral defense

  • Exploring this relationship in other mucosal tissues could reveal tissue-specific immune regulatory mechanisms

• Therapeutic potential of modified CCL6:

  • The enhanced activity of truncated CCL6 suggests that engineered variants might have therapeutic applications

  • Structure-function studies to optimize CCL6's beneficial activities while minimizing potential inflammatory side effects could lead to novel biotherapeutics