CCL28 Rat

What is rat CCL28 and what are its primary structural features?

Rat CCL28 (Chemokine C-C motif Ligand 28) is a chemokine with a molecular weight of approximately 13.1 kDa, comprising 116 amino acids. Its structure features six conserved cysteine residues, with two positioned adjacently, which is distinctive for this chemokine. The protein is expressed primarily in epithelial cells at mucosal sites, including salivary glands and colon tissues . When working with recombinant rat CCL28, researchers should note that it's typically produced in Escherichia coli expression systems with >96% purity as confirmed by SDS-PAGE and HPLC analyses .

What are the primary physiological roles of CCL28 in rat models?

Rat CCL28 functions primarily as a chemoattractant for several immune cell types, including resting T-cells, eosinophils, and IgA antibody-secreting cells (ASCs). It plays a crucial role in regulating IgA ASC accumulation in mammary glands, which consequently affects the transfer of IgA antibodies from mother to infant . Recent studies have also demonstrated that CCL28 modulates neutrophil responses during infection, promoting neutrophil accumulation to the gut during Salmonella enterica serovar Typhimurium (STm) infection and to the lung during multidrug-resistant Acinetobacter baumannii (Ab) infection . Additionally, CCL28 displays antimicrobial activity under low-salt conditions against Candida albicans and both gram-negative and gram-positive bacteria .

How can researchers effectively generate CCL28 knockout rat models?

Creating CCL28 knockout rat models can be accomplished using CRISPR/Cas9 technology. Based on established protocols, researchers should select appropriate target sites (such as exons 1 and 3) and design two pairs of gRNA targeting vectors that should be confirmed by sequencing. The gRNA and Cas9 mRNA should be generated through in vitro transcription and co-injected into fertilized eggs. The resulting pups (F0 founders) require genotyping by PCR and sequence confirmation. To establish a stable knockout line, breed F0 founders with wild-type rats and test for germline transmission in the F1 generation . For genotyping, researchers can use the following primers:

• Forward: 5′-TCATATACAGCACCTCACTCTTGCCC-3′

• Reverse: 5′-GCCTCTCAAAGTCATGCCAGAGTC-3′

• He/Wt-Reverse: 5′-AGGGTGTGAGGTGTCCTTGATGC-3′

The expected PCR product size is 466 bp for the wild-type allele and 700 bp for the knockout allele .

What methodologies are optimal for measuring CCL28 expression during inflammation?

For quantitative assessment of CCL28 expression in inflammatory conditions, enzyme-linked immunosorbent assay (ELISA) has proven effective. In particular, this method successfully detected a ~fourfold increase of CCL28 in feces from wild-type mice at day 4 post-infection with Salmonella Typhimurium relative to uninfected controls . For tissue-specific expression, researchers should consider quantitative PCR to measure transcript levels of CCL28 along with complementary immunohistochemistry to localize protein expression within specific cell types. Flow cytometry can be employed to analyze CCL28 receptor (CCR3 and CCR10) expression on specific immune cell populations. When studying inflammation-induced expression, researchers should monitor CCL28 levels after stimulation with proinflammatory cytokines such as IL-1α, IL-1β, or TNF-α, as these have been demonstrated to upregulate CCL28 production in epithelial cells .

How does CCL28 deficiency affect bacterial dissemination in different infection models?

CCL28 deficiency produces notably different outcomes depending on the infection model and site. In gastrointestinal Salmonella Typhimurium infection, CCL28-/- mice showed significantly higher bacterial dissemination to extraintestinal tissues by day 3 post-infection compared to wild-type littermates. Specifically, higher bacterial colony-forming units (CFU) were recovered from Peyer's patches, mesenteric lymph nodes, bone marrow, and spleen in CCL28-/- mice, suggesting that CCL28 is essential for limiting extraintestinal Salmonella dissemination .

In contrast, when bypassing the gut by infecting mice intraperitoneally with Salmonella, no significant differences in bacterial burden were observed between wild-type and CCL28-/- mice in the spleen, liver, or blood at day 4 post-infection, despite a ~fourfold increase in serum CCL28 levels . This indicates that CCL28's protective role against Salmonella is primarily localized to the gut mucosa.

The impact of CCL28 deficiency appears inverted in pulmonary Acinetobacter baumannii infection, where CCL28-/- mice exhibited substantially improved survival (88% survival through 10 days post-infection) compared to wild-type mice (75% mortality within 48 hours) . This striking difference in outcomes across infection models highlights the context-dependent roles of CCL28 in host defense.

What is the relationship between CCL28 and neutrophil function during mucosal infections?

CCL28 significantly influences neutrophil function during mucosal infections through multiple mechanisms. Research shows that CCL28 promotes neutrophil accumulation at infection sites, enhances neutrophil antimicrobial activity, increases production of reactive oxygen species (ROS), and augments formation of neutrophil extracellular traps (NETs) .

In Salmonella infection, CCL28-/- mice exhibited reduced neutrophil recruitment to the cecum compared to wild-type mice, correlating with decreased expression of proinflammatory cytokines IFNγ and IL-1β . Similarly, in Acinetobacter baumannii lung infection, CCL28-/- mice showed decreased neutrophil accumulation in bronchoalveolar lavage fluid and lung tissue .

Mechanistically, these effects appear to be mediated primarily through the CCR3 receptor on neutrophils. Flow cytometry analysis revealed that neutrophils isolated from infected mucosal sites express CCL28 receptors, particularly CCR3. Approximately 20% of gut neutrophils expressed CCR3 during Salmonella infection, compared to only ~5% of blood neutrophils and ~4% of bone marrow neutrophils . This suggests that CCR3 expression is upregulated on neutrophils at sites of inflammation, potentially increasing their responsiveness to CCL28.

How can researchers reconcile the contradictory outcomes of CCL28 deficiency in different infection models?

The contradictory outcomes of CCL28 deficiency in Salmonella (increased pathology) versus Acinetobacter (improved survival) infection models present an intriguing research question that can be approached through several methodologies:

• Tissue-specific analysis: Researchers should perform comparative transcriptomic and proteomic analyses of infected tissues (gut versus lung) in wild-type and CCL28-/- animals to identify differentially regulated pathways that might explain the opposing outcomes.

• Neutrophil phenotyping: Comprehensive characterization of neutrophil populations in different tissues, focusing on activation status, receptor expression profiles, and effector functions, may reveal tissue-specific neutrophil programming that responds differently to CCL28.

• Pathogen-specific interactions: Investigate whether CCL28 interacts directly with different pathogens or influences pathogen-specific virulence factor expression.

• Temporal dynamics: Time-course experiments examining CCL28 expression, neutrophil recruitment, and bacterial burden could reveal critical windows where CCL28 switches from protective to pathological roles.

The apparent contradiction likely reflects the double-edged nature of neutrophil responses - while neutrophils control infection, their excessive activation can cause collateral tissue damage. In Salmonella infection, the protective antimicrobial effects of CCL28-mediated neutrophil recruitment appear to outweigh any tissue damage, while in Acinetobacter pneumonia, CCL28-driven neutrophil activation may primarily contribute to immunopathology rather than pathogen control .

What experimental approaches can determine the molecular mechanisms by which CCL28 enhances neutrophil antimicrobial functions?

To elucidate the molecular mechanisms underlying CCL28's enhancement of neutrophil antimicrobial functions, researchers should employ the following approaches:

• Receptor antagonism studies: Use specific antagonists against CCR3 (e.g., SB328437) and CCR10 (e.g., BI-6901) to determine which receptor mediates specific neutrophil responses to CCL28. Research has shown that CCR3 antagonism significantly reduces CCL28-induced neutrophil extracellular trap formation, suggesting CCR3 as the primary mediator .

• Intracellular signaling analysis: Examine CCL28-triggered signaling cascades in neutrophils through phosphorylation studies (Western blot or phospho-flow cytometry) focusing on MAPK, PI3K/Akt, and calcium signaling pathways.

• Functional assays: Implement the following assays to quantify specific neutrophil functions:

  • ROS production using dihydrorhodamine 123 or similar fluorescent probes

  • Phagocytosis assays using fluorescently labeled bacteria

  • NET formation assays using DNA-staining dyes like DAPI and HELIX, combined with immunofluorescence for neutrophil markers

  • Quantification of DNA-MPO complexes as markers of NET formation

• Transcriptional profiling: Perform RNA-seq analysis of neutrophils before and after CCL28 stimulation to identify transcriptional programs activated by this chemokine.

In experimental designs, researchers should include appropriate controls, such as neutrophils from CCL28-/- animals, receptor antagonists, and inhibitors of specific signaling pathways to establish causality in observed effects .

What are the optimal storage and handling protocols for rat CCL28 to maintain biological activity?

For optimal preservation of rat CCL28 biological activity, researchers should store the protein desiccated at -20°C . When working with lyophilized recombinant rat CCL28, reconstitution should be performed using sterile techniques with appropriate buffers that maintain protein stability. After reconstitution, aliquoting the protein to minimize freeze-thaw cycles is recommended, as repeated freezing and thawing can reduce biological activity.

To confirm biological activity before experimental use, a chemotaxis bioassay using human lymphocytes can be employed. Active rat CCL28 typically demonstrates chemotactic activity in a concentration range of 5-50 ng/ml . Prior to experimental application, purity should be verified via SDS-PAGE and HPLC analyses, with preparations demonstrating >96% purity being suitable for most research applications .

What considerations should researchers account for when designing CCL28 functional studies across different inflammatory conditions?

When designing functional studies of CCL28 across various inflammatory conditions, researchers should consider several critical factors:

• Expression dynamics: CCL28 expression varies by tissue and inflammatory stimulus. Baseline expression occurs in epithelial cells at mucosal sites, but is upregulated by specific proinflammatory stimuli including IL-1α, IL-1β, TNF-α, and following bacterial infection . Design time-course experiments to capture both early and late expression patterns.

• Context-dependent effects: CCL28 demonstrates distinct and sometimes contradictory roles depending on the tissue environment and infection model. For instance, CCL28 deficiency increases susceptibility to Salmonella gut infection but improves survival in Acinetobacter pneumonia . Therefore, parallel studies in multiple tissues or disease models are necessary for comprehensive understanding.

• Receptor expression analysis: The distribution and density of CCL28 receptors (CCR3 and CCR10) on target cells significantly influences responses. Flow cytometry analysis has shown that neutrophils at infected mucosal sites have increased CCR3 expression (approximately 20% of gut neutrophils during Salmonella infection) compared to neutrophils in circulation (~5%) or bone marrow (~4%) . Include receptor expression analysis in experimental designs.

• Dose-response relationships: The concentration of CCL28 significantly impacts its biological effects. At high concentrations (1 μM), CCL28 exhibits direct antimicrobial activity, while lower concentrations mediate chemotactic effects . Include appropriate concentration ranges in experimental designs.

• Interaction with other inflammatory mediators: Consider potential synergistic or antagonistic interactions between CCL28 and other cytokines or chemokines present in inflammatory microenvironments.

How might CCL28-targeted therapies be developed for inflammatory or infectious diseases?

Development of CCL28-targeted therapies represents a promising research direction, but requires careful consideration of CCL28's context-dependent effects. Potential therapeutic approaches include:

• Receptor-specific antagonists: Based on experimental evidence showing that CCR3 antagonism with SB328437 reduces CCL28-induced neutrophil extracellular trap formation , development of CCR3-specific antagonists could potentially modulate excessive neutrophil activation in conditions where this contributes to pathology, such as acute respiratory distress syndrome or inflammatory bowel disease.

• Context-selective targeting: Given the opposing roles of CCL28 in different infection models - protective in Salmonella gut infection but detrimental in Acinetobacter pneumonia - therapeutic approaches need to be tailored to specific disease contexts. For example, CCL28 augmentation might benefit gastrointestinal infections while CCL28 inhibition could improve outcomes in certain respiratory infections.

• Cell-specific delivery systems: Development of delivery systems that target CCL28 modulators to specific cell types or tissues could enhance therapeutic efficacy while minimizing off-target effects.

• Antimicrobial peptide derivatives: Given CCL28's direct antimicrobial activity against various pathogens including Candida albicans and both gram-positive and gram-negative bacteria under low-salt conditions , structure-function studies could identify peptide derivatives with enhanced antimicrobial properties for therapeutic development.

Research should proceed with caution given CCL28's multiple functions in immunity, including its role in regulating IgA antibody-secreting cell accumulation in mammary glands and subsequent antibody transfer from mother to infant .

What are the key unresolved questions regarding CCL28 regulation of neutrophil function?

Despite recent advances, several critical questions regarding CCL28's regulation of neutrophil function remain unresolved:

• Receptor trafficking dynamics: While research has shown that CCR3 can be rapidly mobilized to the neutrophil surface upon stimulation with proinflammatory molecules or in response to phagocytosis , the molecular mechanisms controlling this receptor trafficking remain poorly understood. Future studies should investigate the intracellular storage compartments for CCR3 and the signaling pathways that trigger its surface expression.

• Differential regulation of neutrophil effector functions: CCL28 enhances multiple neutrophil functions, including ROS production and NET formation , but whether these effects are regulated through distinct signaling pathways or represent a unified response remains unclear. Detailed signaling studies combined with specific pathway inhibitors could address this question.

• Strain and species differences: Most CCL28 research has been conducted in C57BL/6 mouse models, but potential strain-specific and species-specific differences in CCL28 function have not been systematically explored.

• Long-term consequences of CCL28-mediated neutrophil activation: While acute effects of CCL28 on neutrophil function have been described, the long-term consequences for tissue homeostasis, repair, and resolution of inflammation require further investigation.

• Intersection with adaptive immunity: How CCL28-mediated neutrophil responses influence subsequent adaptive immune responses, particularly at mucosal sites, remains to be fully elucidated.

Addressing these questions will require integrated approaches combining in vivo models with advanced imaging techniques, single-cell analysis, and systems biology approaches to capture the complexity of CCL28-neutrophil interactions in different physiological and pathological contexts.