CCL28 Mouse

What is CCL28 and where is it expressed in mice?

CCL28 is a chemokine highly expressed in mucosal tissues of mice. It has been detected in the gut mucosa and lung tissue, where it plays roles during infection . Additionally, CCL28 shows wide expression throughout the mouse central nervous system (CNS), including the cerebrum, cerebellum, brain stem, and spinal cord. Within the hippocampus, CCL28 expression is localized primarily in pyramidal cells of the CA area, granular cells of the dentate gyrus, and certain interneurons in both the CA area and hilus . This distribution pattern suggests CCL28 has diverse functions across multiple organ systems in mice.

What receptors interact with CCL28 in mouse models?

In mice, CCL28 primarily signals through two receptors: CCR3 and CCR10. Neutrophils isolated from infected mucosal tissues express both receptors, with CCR3 showing higher expression levels compared to CCR10 . Interestingly, unstimulated neutrophils store pre-formed intracellular CCR3 that can be rapidly mobilized to the cell surface following phagocytosis or in response to inflammatory stimuli, allowing for quick functional responses to CCL28 . This receptor dynamics enables rapid neutrophil responsiveness during infection.

What are the primary biological functions of CCL28 in mice?

CCL28 serves several critical functions in mice:

• Promotion of neutrophil accumulation in infected mucosal tissues, such as the gut during Salmonella infection and the lungs during Acinetobacter infection

• Enhancement of neutrophil antimicrobial activity, including increased production of reactive oxygen species (ROS) and formation of neutrophil extracellular traps (NETs)

• Regulation of pathogen clearance, with effects that vary depending on the specific pathogen and infection site

• Potential roles in CNS function, particularly in hippocampal interneurons where it may influence neuronal inhibition

The tissue-specific and pathogen-specific nature of CCL28's effects makes it an important regulator of immune responses at mucosal surfaces.

How does CCL28 deficiency differentially impact infection outcomes in different mucosal tissues?

CCL28 deficiency leads to remarkably opposite outcomes depending on the site of infection and pathogen involved. In gut infections with Salmonella enterica serovar Typhimurium (STm), CCL28-deficient (Ccl28−/−) mice show significantly increased susceptibility, with higher bacterial dissemination to extraintestinal tissues including Peyer's patches, mesenteric lymph nodes, bone marrow, and spleen by 3 days post-infection . This suggests CCL28 is essential for controlling Salmonella at its origin in the gut mucosa.

In stark contrast, when challenged with Acinetobacter baumannii (Ab) lung infection, CCL28-deficient mice demonstrate remarkable resistance, with 88% survival compared to only 25% survival in wild-type mice . This dramatic difference occurs despite similar bacterial loads, indicating that CCL28's effects on infection outcomes are not simply related to direct antimicrobial activity but involve complex immune regulatory mechanisms that may be beneficial or detrimental depending on the context.

These divergent outcomes highlight the tissue-specific nature of CCL28's functions and suggest that targeted modulation of CCL28 activity could have therapeutic potential that needs to be carefully tailored to specific infection scenarios.

What is the relationship between CCL28 and neutrophil function in different infection models?

CCL28 significantly modulates neutrophil behavior through several mechanisms:

• Neutrophil recruitment: CCL28 promotes neutrophil accumulation in both gut and lung mucosal tissues during infection . In Acinetobacter lung infection, wild-type mice showed greater cellular infiltrates in bronchoalveolar lavage (BAL) fluid compared to Ccl28−/− mice, with neutrophils comprising the majority of these cells .

• Antimicrobial capacity: In vitro stimulation of neutrophils with CCL28 enhances their ability to kill Salmonella, but interestingly not Acinetobacter . This selective enhancement explains why CCL28 deficiency increases susceptibility to Salmonella but decreases mortality from Acinetobacter.

• Inflammatory response: CCL28 stimulation enhances neutrophil production of reactive oxygen species (ROS) and formation of neutrophil extracellular traps (NETs) . While these processes help control certain infections, they can also cause extensive tissue damage that may contribute to pathology.

• Receptor-dependent activation: The enhancement of neutrophil antimicrobial functions by CCL28 is largely dependent on CCR3 signaling , highlighting this receptor as a key mediator of CCL28's effects on neutrophil function.

These findings demonstrate that CCL28 acts as a double-edged sword in infection, promoting pathogen clearance but potentially exacerbating inflammatory damage depending on the context.

What evidence exists for CCL28's role in the central nervous system of mice?

While CCL28's immune functions have been well-studied, emerging evidence suggests important roles in the central nervous system:

• Expression pattern: CCL28 shows widespread distribution throughout the mouse CNS, including cerebrum, cerebellum, brain stem, and spinal cord .

• Cellular localization: In the hippocampus, CCL28 is expressed by multiple neuronal populations, including pyramidal cells of the CA area, granular cells of the dentate gyrus, and various interneurons. Double-labeling immunocytochemistry has revealed that most calbindin, calretinin, and parvalbumin-positive neurons (markers for specific interneuron populations) co-express CCL28 .

• Response to pathological conditions: During and after pilocarpine-induced status epilepticus (SE), CCL28 expression is down-regulated specifically in hippocampal interneurons in the CA1 area and in the hilus of the dentate gyrus .

• Functional implications: The pattern of CCL28 expression changes during epilepsy suggests it may be involved in the loss of hippocampal interneurons and subsequent disinhibition of pyramidal neurons, potentially contributing to seizure susceptibility .

These findings point to previously unappreciated non-immune functions of CCL28 in the CNS, suggesting it may serve as a neuroimmune mediator with implications for neurological disorders.

What are the recommended methods for generating CCL28-deficient mice for research?

Researchers have successfully generated CCL28-deficient (Ccl28−/−) mice using CRISPR/Cas9 technology. The methodology includes:

• Target site selection: Targeting critical exons (e.g., exons 1 and 3) to disrupt the CCL28 gene .

• gRNA design and validation: Constructing multiple gRNA targeting vectors and confirming them by sequencing to ensure specificity .

• Delivery method: Co-injecting gRNA and Cas9 mRNA (generated by in vitro transcription) into fertilized eggs to produce knockout mice .

• Founder identification: Genotyping F0 founder pups by PCR and confirming knockouts by sequencing .

• Colony establishment: Breeding F0 founders with wild-type mice to test germline transmission and generate F1 animals .

• Genotyping protocol: For routine genotyping, researchers can use specific primer sets:

  • Forward primer: 5′-TCATATACAGCACCTCACTCTTGCCC-3′

  • Reverse primer: 5′-GCCTCTCAAAGTCATGCCAGAGTC-3′

  • Heterozygote/Wild-type Reverse primer: 5′-AGGGTGTGAGGTGTCCTTGATGC-3′

With this protocol, wild-type alleles produce a 466 bp band while knockout alleles yield a 700 bp product on gel electrophoresis, allowing for clear identification of genotypes .

What ELISA methods are available for measuring mouse CCL28 in different sample types?

Several validated ELISA approaches are available for quantifying mouse CCL28:

• DuoSet ELISA Development kit (R&D Systems):

  • Contains components for sandwich ELISA to measure natural and recombinant mouse CCL28

  • Includes optimized capture and detection antibody pairings with recommended concentrations

  • Suitable for analysis of cell culture supernatants but requires optimization for complex matrices like serum and plasma

  • Components include capture antibody, detection antibody, recombinant standard, and streptavidin-HRP

• SimpleStep ELISA Kit (Abcam):

  • Designed for quantitative measurement of mouse CCL28 in serum, plasma, cell culture supernatants, and cell/tissue extracts

  • More comprehensive kit with ready-to-use components

  • Validated for a wider range of sample types

• Custom ELISA protocol for specific applications:

  • For measuring CCL28 in fecal samples: Collect fresh fecal pellets, weigh them, resuspend in 1 ml sterile PBS containing protease inhibitor cocktail, incubate at room temperature while shaking for 30 minutes, centrifuge at 9391 × g for 10 minutes, collect supernatant for analysis

  • For serum samples: Collect whole blood by cardiac puncture, allow to clot at room temperature for 30 minutes, centrifuge, and collect serum for analysis

These methods provide researchers with options for CCL28 quantification across different experimental contexts, from basic in vitro studies to complex in vivo infection models.

What infection models are appropriate for studying CCL28 function in mice?

Based on current research, several infection models have been validated for studying CCL28 function:

• Salmonella gut infection model:

  • Utilize the streptomycin-treated C57BL/6 mouse model of colitis

  • Pretreat mice with streptomycin before oral infection with Salmonella enterica serovar Typhimurium (STm)

  • This model allows assessment of CCL28's role in controlling gut infection and preventing dissemination to extraintestinal sites

  • Key timepoints: Significant differences between wild-type and CCL28-deficient mice observed at 3-4 days post-infection

  • Readouts: Bacterial CFU in gastrointestinal contents, Peyer's patches, mesenteric lymph nodes, bone marrow, and spleen; CCL28 levels in feces

• Acinetobacter lung infection model:

  • Intratracheal infection with Acinetobacter baumannii (Ab)

  • Appropriate for studying CCL28's role in lung immunity and neutrophil-mediated inflammation

  • Key timepoints: Survival differences evident within 48 hours; neutrophil recruitment peaks at 1 day post-infection

  • Readouts: Survival, bacterial CFU in bronchoalveolar lavage (BAL) fluid, lung tissue, and blood; cellular infiltrates in BAL fluid; histopathology; inflammatory mediator levels

• Neurological models:

  • Pilocarpine-induced status epilepticus model to study CCL28's role in the CNS

  • Evaluates changes in CCL28 expression in specific neuronal populations during pathological conditions

  • Readouts: Immunohistochemistry for CCL28 expression in hippocampal interneurons and pyramidal cells

These diverse models enable comprehensive investigation of CCL28's functions across different organ systems and disease states.

How should researchers interpret apparently contradictory outcomes of CCL28 deficiency in different infection models?

The divergent outcomes observed in CCL28-deficient mice—increased susceptibility to Salmonella gut infection versus protection from Acinetobacter lung infection—require careful interpretation:

• Consider tissue-specific immune environments:

  • Different mucosal tissues have unique immunological milieux that interact distinctly with CCL28 signaling

  • The balance between protective immunity and immunopathology differs between gut and lung

• Evaluate pathogen-specific factors:

  • CCL28 stimulation enhances neutrophil killing of Salmonella but not Acinetobacter

  • Different pathogens trigger distinct inflammatory programs that may be differentially regulated by CCL28

• Assess relative contributions of direct versus indirect effects:

  • Direct antimicrobial activity: CCL28 shows modest direct antimicrobial activity against Acinetobacter at high concentrations in vitro, but this likely doesn't account for in vivo effects given the high bacterial loads observed

  • Immunomodulatory effects: CCL28's primary impact appears to be through neutrophil recruitment and activation rather than direct pathogen killing

• Consider the damage-response framework:

  • In Salmonella infection, CCL28-mediated neutrophil accumulation and activation aids pathogen clearance, with benefits outweighing inflammatory damage

  • In Acinetobacter lung infection, excessive neutrophil recruitment and activation may cause collateral tissue damage that contributes to mortality, even if it helps control infection

These interpretative frameworks help reconcile seemingly contradictory findings and highlight the context-dependent nature of chemokine functions in infection.

What methodological considerations are important when analyzing CCL28 expression in tissue samples?

When analyzing CCL28 expression in tissues, researchers should consider several methodological factors:

• Sample preparation techniques:

  • For protein analysis in fecal samples: Immediate processing with protease inhibitors is crucial to prevent degradation

  • For RNA analysis: Rapid tissue preservation is essential to maintain RNA integrity

• Selection of appropriate detection methods:

  • ELISA: Provides quantitative measurement of protein levels in biological fluids and tissue homogenates

  • Immunohistochemistry/immunofluorescence: Allows for cellular and subcellular localization of CCL28 expression

  • qRT-PCR: Enables quantification of CCL28 mRNA levels for transcriptional analysis

• Validation strategies:

  • Use multiple detection methods to confirm findings

  • Include appropriate positive and negative controls, including CCL28-deficient tissues

  • Perform double-labeling studies to identify specific cell populations expressing CCL28

• Temporal considerations:

  • CCL28 expression changes dynamically during infection or disease progression

  • In Salmonella infection, significant increases in CCL28 are observed by day 4 post-infection

  • During status epilepticus, CCL28 expression is down-regulated in specific neuronal populations

• Regional specificity:

  • In CNS studies, precise neuroanatomical localization is critical given the heterogeneity of CCL28 expression across different regions and cell types

  • In gut studies, consider sampling multiple intestinal segments to account for regional variations

Adhering to these methodological considerations ensures reliable and reproducible analysis of CCL28 expression across different experimental contexts.

How can researchers distinguish between direct and indirect effects of CCL28 in mouse models?

Distinguishing direct from indirect effects of CCL28 requires systematic experimental approaches:

• In vitro versus in vivo comparisons:

  • Direct effects can be assessed in purified systems (e.g., recombinant CCL28 with isolated neutrophils or pathogens)

  • Comparison with in vivo findings helps identify potential indirect mechanisms mediated by other cell types or factors

• Receptor blocking studies:

  • Use of receptor-specific blocking antibodies or receptor-deficient cells can determine whether effects are mediated directly through CCL28 receptors (CCR3 or CCR10)

  • For instance, CCL28-enhanced neutrophil antimicrobial activity was shown to be largely CCR3-dependent

• Temporal analysis:

  • Immediate responses (minutes to hours) are more likely to reflect direct effects

  • Delayed responses may involve indirect mechanisms requiring intermediate cellular or molecular events

• Cell-specific conditional knockouts:

  • Generation of cell type-specific CCL28 or CCL28 receptor knockout mice can help delineate the contribution of specific cellular sources or targets

• Adoptive transfer experiments:

  • Transfer of wild-type neutrophils to CCL28-deficient mice (or vice versa) can help determine whether phenotypes are due to direct effects on neutrophils or indirect effects on the tissue environment

• Mechanistic dissection:

  • For antimicrobial effects, compare direct CCL28 antimicrobial activity against pathogens with its effects on immune cell killing capacity

  • For example, high concentrations of CCL28 (1 μM) exhibited direct antimicrobial activity against low inocula of Acinetobacter in vitro, but this effect was lost with higher bacterial loads and likely doesn't explain in vivo phenotypes

These approaches allow researchers to distinguish between direct CCL28 effects and those mediated through complex cellular interactions or secondary mediators.

What are potential therapeutic applications of CCL28 research in mouse models?

CCL28 research in mouse models suggests several potential therapeutic applications:

• Targeted modulation in infectious diseases:

  • Enhancing CCL28 activity could potentially improve host defense against gut pathogens like Salmonella

  • Inhibiting CCL28 activity might reduce mortality in severe respiratory infections like Acinetobacter pneumonia by limiting excessive neutrophil-mediated inflammation

• Neurological applications:

  • Given CCL28's expression in hippocampal neurons and its altered regulation during epilepsy, it may represent a novel target for seizure disorders

  • Further investigation of CCL28's role in neuronal function could reveal applications in other neurological conditions

• Biomarker development:

  • Changes in CCL28 levels during infection suggest potential as a diagnostic or prognostic biomarker

  • Different expression patterns in various tissues could allow for site-specific disease monitoring

• Immune modulation strategies:

  • Selective targeting of CCL28 receptors (CCR3 vs. CCR10) could provide precision in modulating specific aspects of immune responses

  • The pre-formed intracellular pool of CCR3 in neutrophils represents a unique target for rapid intervention in acute inflammatory conditions

Development of these therapeutic approaches will require careful translation from mouse models to human applications, with consideration of species-specific differences in CCL28 biology.

What methodological advances would improve CCL28 research in mouse models?

Several methodological advances could significantly enhance CCL28 research:

• Improved detection tools:

  • Development of highly sensitive and specific antibodies for different applications (flow cytometry, immunohistochemistry, functional blocking)

  • Creation of reporter mouse strains expressing fluorescent proteins under the CCL28 promoter to enable real-time visualization of expression

• Advanced genetic models:

  • Conditional and inducible CCL28 knockout systems to study temporal and tissue-specific effects

  • Knock-in mice with tagged CCL28 to facilitate tracking of endogenous protein

  • Receptor-specific knockout mice to dissect differential roles of CCR3 versus CCR10 signaling

• Enhanced imaging techniques:

  • Intravital microscopy approaches to visualize CCL28-dependent neutrophil recruitment and behavior in live animals

  • Multiphoton imaging of labeled CCL28 in the CNS to understand its dynamics in neuronal function

• Single-cell analysis:

  • Application of single-cell RNA-sequencing to identify all cell populations expressing CCL28 and its receptors across tissues

  • Single-cell proteomics to correlate CCL28 expression with cellular phenotypes and functions

• Standardized assay systems:

  • Development of standardized protocols for measuring CCL28 in different sample types to improve cross-study comparability

  • Validated reference standards for quantification across different experimental platforms

These methodological advances would enable more sophisticated investigation of CCL28's diverse functions across organ systems and disease states.