GO:0071644 negative regulation of chemokine (C-C motif) ligand 4 production: Immune Regulation, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0071644 describes any process that stops, prevents, or reduces the production of chemokine (C-C motif) ligand 4 (CCL4), also known as MIP-1β.
• CCL4 is a CC chemokine that attracts immune cells and is implicated in inflammatory and autoimmune diseases such as rheumatoid arthritis and lupus.
• Negative regulation of CCL4 production can occur through microRNAs, transcription factors, and epigenetic modifiers, as shown in monocytes and mast cells.
• Dysregulated CCL4 production contributes to pathology in rheumatoid arthritis, lupus, and cardiovascular conditions.
• Key experimental approaches to study this process include knockout, knockdown, overexpression, and CRISPR-based editing of regulatory genes.
• Understanding negative regulation of CCL4 production may reveal therapeutic targets for inflammatory diseases.
Description
Chemokine (C-C motif) ligand 4 (CCL4), also known as macrophage inflammatory protein-1β (MIP-1β), is a CC chemokine that plays a critical role in immune cell recruitment and activation. The production of CCL4 is tightly regulated at multiple levels to prevent excessive inflammation. GO:0071644, negative regulation of chemokine (C-C motif) ligand 4 production, encompasses any process that stops, prevents, or reduces the frequency, rate, or extent of CCL4 production. This regulation is essential for maintaining immune homeostasis and preventing inflammatory damage. Dysregulation of CCL4 production has been implicated in autoimmune diseases such as rheumatoid arthritis and lupus, as well as in cardiovascular conditions. Therefore, understanding the mechanisms that negatively regulate CCL4 production is of significant research and therapeutic interest.
negative regulation of chemokine (C-C motif) ligand 4 production At A Glance
| GO ID | GO:0071644 |
|---|---|
| GO term | negative regulation of chemokine (C-C motif) ligand 4 production |
| Ontology | biological_process |
| Synonym | negative regulation of CCL4 production; negative regulation of macrophage inflammatory protein production; negative regulation of MIP-1b production |
| Major function | Inhibition of the production of CCL4, a chemokine involved in immune cell recruitment and inflammation |
| Related chemokine | CCL4 (MIP-1β) |
| Cellular context | Monocytes, macrophages, T cells, mast cells, and other immune cells |
| Disease relevance | Rheumatoid arthritis, lupus, cardiovascular inflammation, and other inflammatory disorders |
What Is GO:0071644?
GO:0071644 is a biological process term defined as any process that stops, prevents, or reduces the frequency, rate, or extent of production of chemokine (C-C motif) ligand 4. It includes mechanisms that inhibit the synthesis, processing, or release of CCL4, also known as MIP-1β.
Why Is negative regulation of chemokine (C-C motif) ligand 4 production Important in Cell Biology?
Negative regulation of CCL4 production is crucial for controlling inflammation and preventing autoimmune pathology. Excessive CCL4 contributes to the recruitment of inflammatory cells and tissue damage in diseases such as rheumatoid arthritis and lupus. Understanding the molecular players that suppress CCL4 production can identify new therapeutic targets and biomarkers for inflammatory diseases.
• Controls immune cell trafficking and prevents excessive inflammation.
• Dysregulation is linked to autoimmune diseases like rheumatoid arthritis.
• Implicated in lupus pathogenesis, where EZH2 inhibition reduces CCL4.
• MicroRNA-155 regulates monocyte chemokine expression, including CCL4, in rheumatoid arthritis.
• ELK4 modulates cytokine/chemokine production in mast cells.
• VISTA influences myeloid cell chemokine responsiveness.
• CCL4 levels are altered during pediatric heart surgery with cardiopulmonary bypass.
• The Pseudomonas plecoglossicida fliL gene affects immune response and chemokine production in fish.
• CCR5 and MIP-1α regulation in rheumatoid arthritis T cells highlights chemokine network complexity.
• Th1-like effector memory cells in tuberculosis produce chemokines, including CCL4.
What Happens During negative regulation of chemokine (C-C motif) ligand 4 production?
Transcriptional repression of CCL4
In simple terms: The cell reduces the reading of the CCL4 gene into messenger RNA.
Negative regulation of CCL4 production often begins with transcriptional repression. For example, the transcription factor ELK4 can exert opposite roles in cytokine/chemokine production in activated mast cells, suggesting context-dependent regulation. Epigenetic modifiers such as EZH2 can also suppress CCL4 transcription, as inhibition of EZH2 ameliorates lupus-like disease and reduces chemokine production.
Post-transcriptional regulation by microRNAs
In simple terms: Small RNA molecules can bind to CCL4 mRNA and prevent it from being translated into protein.
MicroRNAs are key post-transcriptional regulators. MicroRNA-155 regulates monocyte chemokine and chemokine receptor expression in rheumatoid arthritis, including CCL4, thereby dampening inflammatory responses. This represents a direct mechanism to reduce CCL4 production at the mRNA level.
Inhibition of CCL4 secretion
In simple terms: Even if CCL4 protein is made, the cell can block its release outside.
Negative regulation can also occur at the level of secretion. Although specific mechanisms are less defined, studies on myeloid cell chemokine responsiveness, such as those involving VISTA, suggest that surface receptors can modulate chemokine release. In the context of pediatric heart surgery, continuous ultrafiltration altered inflammatory mediator profiles, including chemokines, indicating that secretion can be influenced by external interventions.
Regulation of CCL4 in specific cell types
In simple terms: Different immune cells have unique ways to control CCL4 production.
In CD4+ T cells from rheumatoid arthritis patients, CCR5 expression and MIP-1α production are dysregulated, highlighting cell-type-specific regulation of chemokines. Resident Th1-like effector memory cells in pulmonary recall responses to Mycobacterium tuberculosis produce chemokines, and their regulation may involve negative feedback loops. In fish infected with Pseudomonas plecoglossicida, the fliL gene influences immune response and chemokine production, demonstrating evolutionary conservation of regulatory mechanisms.
Key Genes Involved in GO:0071644 negative regulation of chemokine (C-C motif) ligand 4 production
The following genes and proteins have been experimentally implicated in the negative regulation of CCL4 production or related chemokine pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ELK4 | Transcription factor that can modulate cytokine/chemokine production in mast cells | Shows opposite roles in cytokine/chemokine production and degranulation |
| EZH2 | Epigenetic modifier that suppresses chemokine production | Inhibition ameliorates lupus-like disease and reduces CCL4 |
| miR-155 | MicroRNA that regulates monocyte chemokine expression | Regulates CCL4 and chemokine receptors in rheumatoid arthritis |
| VISTA | Immune checkpoint regulator on myeloid cells | Defines signature of myeloid cell chemokine responsiveness |
| CCR5 | Chemokine receptor that binds CCL4 | Regulation linked to MIP-1α production in rheumatoid arthritis T cells |
| fliL | Bacterial flagellar gene influencing host immune response | Affects chemokine production in infected groupers |
| CCL4 | Chemokine (C-C motif) ligand 4 (MIP-1β) | Target of negative regulation; involved in inflammation |
| CCL3 | Chemokine (C-C motif) ligand 3 (MIP-1α) | Co-regulated with CCL4 in rheumatoid arthritis |
| CCL5 | Chemokine (C-C motif) ligand 5 (RANTES) | Often co-expressed with CCL4 in inflammatory responses |
| IL-6 | Pro-inflammatory cytokine | Modulated alongside chemokines in heart surgery |
| TNF-α | Pro-inflammatory cytokine | Regulated in parallel with chemokines in various models |
| IFN-γ | Th1 cytokine | Associated with Th1-like effector memory cells producing chemokines |
| NF-κB | Transcription factor driving chemokine expression | Central to inflammatory chemokine regulation |
| STAT1 | Transcription factor in IFN signaling | May mediate negative regulation of chemokines |
| SOCS1 | Suppressor of cytokine signaling | Potential negative regulator of chemokine production |
| TGF-β | Anti-inflammatory cytokine | Can suppress chemokine production |
| IL-10 | Anti-inflammatory cytokine | Inhibits chemokine production in monocytes |
| Foxp3 | Regulatory T cell transcription factor | Associated with suppression of inflammatory chemokines |
How Is negative regulation of chemokine (C-C motif) ligand 4 production Regulated?
Negative regulation of CCL4 production is controlled by a network of transcription factors, microRNAs, and epigenetic modifiers. MicroRNA-155 directly targets chemokine mRNAs in monocytes, reducing CCL4 production. Epigenetic silencing by EZH2 suppresses CCL4 transcription, and its inhibition leads to reduced chemokine levels in lupus models. ELK4 can act as a negative regulator of chemokine production in mast cells under certain conditions. Additionally, anti-inflammatory cytokines such as IL-10 and TGF-β can downregulate chemokine production.
negative regulation of chemokine (C-C motif) ligand 4 production and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| miR-155 | Rheumatoid arthritis | Knockout or overexpression in monocyte cell lines |
| EZH2 | Lupus | CRISPR knockout in MRL/lpr mice or human cell lines |
| ELK4 | Mast cell-related inflammation | Knockdown in mast cell lines (e.g., HMC-1) |
| CCR5 | Rheumatoid arthritis | Point mutation or knockout in CD4+ T cells |
| fliL | Bacterial infection in fish | Knockout in Pseudomonas plecoglossicida |
Rheumatoid Arthritis
In rheumatoid arthritis, microRNA-155 regulates monocyte chemokine and chemokine receptor expression, including CCL4, contributing to synovial inflammation. Dysregulated CCR5 and MIP-1α production in CD4+ T cells further highlights the role of chemokines in disease pathogenesis. Negative regulation of CCL4 production is therefore a potential therapeutic strategy to dampen joint inflammation.
Systemic Lupus Erythematosus
In lupus, inhibition of EZH2 ameliorates disease in MRL/lpr mice and reduces chemokine production, including CCL4. This suggests that epigenetic reprogramming can negatively regulate CCL4 and other inflammatory mediators, offering a targeted approach for lupus therapy.
Cardiovascular Inflammation
Pediatric heart surgery with cardiopulmonary bypass and continuous ultrafiltration alters inflammatory mediator profiles, including chemokines. Negative regulation of CCL4 production may be relevant to controlling postoperative inflammation and improving outcomes.
Infectious Diseases
In Mycobacterium tuberculosis infection, resident Th1-like effector memory cells produce chemokines, and their regulation is critical for granuloma formation and bacterial control. Similarly, in fish infected with Pseudomonas plecoglossicida, the fliL gene influences chemokine production, indicating a conserved role in host defense.
From negative regulation of chemokine (C-C motif) ligand 4 production-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does gene X negatively regulate CCL4 production? | CRISPR knockout of gene X in monocytes/macrophages, measure CCL4 by ELISA |
| Does a specific point mutation in gene Y affect CCL4 repression? | CRISPR point mutation knock-in in cell lines, compare CCL4 levels |
| Does overexpression of gene Z reduce CCL4? | Lentiviral overexpression in primary immune cells or cell lines |
| Does epigenetic modifier EZH2 directly repress CCL4? | CRISPR knockout or pharmacological inhibition, ChIP-qPCR for histone marks |
| Does microRNA-155 target CCL4 3'UTR? | Luciferase reporter with wild-type vs mutant 3'UTR, miRNA mimic/inhibitor |
| Does VISTA modulate CCL4 in myeloid cells? | CRISPR knockout of VISTA in myeloid cell lines, chemokine array |
How to Study the negative regulation of chemokine (C-C motif) ligand 4 production Process
| Method | What It Measures | Typical Application |
|---|---|---|
| ELISA | CCL4 protein concentration | Quantify negative regulation in cell supernatants |
| RT-qPCR | CCL4 mRNA levels | Assess transcriptional repression |
| RNA-seq | Global gene expression | Identify pathways co-regulated with CCL4 |
| ChIP-qPCR | Transcription factor binding and histone marks | Study epigenetic repression of CCL4 |
| Luciferase reporter | MicroRNA targeting of CCL4 3'UTR | Validate miR-155 binding |
| CRISPR knockout screen | Genes whose loss increases CCL4 | Discover novel negative regulators |
| Flow cytometry | Intracellular CCL4 staining | Single-cell analysis of CCL4 production |
| Luminex | Multiplex cytokine/chemokine profiling | Measure CCL4 alongside other mediators |
Quantifying CCL4 Production
ELISA and Luminex assays are standard for measuring CCL4 protein levels in cell culture supernatants and serum. These methods are quantitative and can be used to assess the impact of genetic perturbations on negative regulation.
Transcriptional Analysis
RT-qPCR and RNA-seq can measure CCL4 mRNA levels to determine if negative regulation occurs at the transcriptional level. Chromatin immunoprecipitation (ChIP) can identify transcription factor binding and histone modifications at the CCL4 promoter.
MicroRNA Target Validation
Luciferase reporter assays with the CCL4 3'UTR are used to confirm direct targeting by microRNAs such as miR-155. Mutating the predicted binding site abolishes repression, providing causal evidence.
CRISPR Screening
Genome-wide CRISPR knockout or activation screens can identify novel negative regulators of CCL4 production. Cells are stimulated, and CCL4 levels are measured by flow cytometry or ELISA, followed by sequencing to identify enriched sgRNAs.
How CRISPR Can Be Used to Study GO:0071644 negative regulation of chemokine (C-C motif) ligand 4 production
Knockout
CRISPR knockout of candidate negative regulators (e.g., EZH2, ELK4) can be used to test whether their loss increases CCL4 production. This approach provides causal evidence for gene function in the regulatory pathway.
Point Mutation
Introducing specific point mutations in transcription factor binding sites within the CCL4 promoter or in microRNA seed regions can dissect the precise sequences required for negative regulation. This is useful for understanding disease-associated variants.
Knock-in
Knock-in of tagged versions of regulatory proteins (e.g., HA-tagged EZH2) allows for chromatin immunoprecipitation and proteomic studies to identify interaction partners at the CCL4 locus. Reporter knock-ins can also monitor CCL4 promoter activity in real time.
Overexpression
Overexpression of putative negative regulators (e.g., miR-155, SOCS1) via lentiviral vectors can suppress CCL4 production, confirming their repressive role. This is particularly useful for microRNAs and anti-inflammatory cytokines.
How EDITGENE Supports negative regulation of chemokine (C-C motif) ligand 4 production Research
Researchers studying negative regulation of chemokine (C-C motif) ligand 4 production-related genes often need to determine whether a candidate gene is causally involved in suppressing CCL4. EDITGENE provides a comprehensive suite of CRISPR services to enable such investigations, from gene knockout to precise point mutations and overexpression models.
Contact EDITGENE today to design your custom CRISPR model for negative regulation of chemokine (C-C motif) ligand 4 production research.
Frequently Asked Questions About negative regulation of chemokine (C-C motif) ligand 4 production
What is GO:0071644?
GO:0071644 is a Gene Ontology term for negative regulation of chemokine (C-C motif) ligand 4 production, describing any process that stops, prevents, or reduces the production of CCL4 (MIP-1β).
What genes are involved in negative regulation of CCL4 production?
Key genes include EZH2, ELK4, microRNA-155, and VISTA, which have been shown to modulate CCL4 production in various immune cells.
How is CCL4 production negatively regulated?
It can be regulated at transcriptional, post-transcriptional, and secretory levels by transcription factors, microRNAs, and epigenetic modifiers.
What diseases are associated with dysregulated CCL4 production?
Rheumatoid arthritis, lupus, cardiovascular inflammation, and infectious diseases like tuberculosis have been linked to altered CCL4 regulation.
What is the role of microRNA-155 in CCL4 regulation?
MicroRNA-155 regulates monocyte chemokine expression, including CCL4, in rheumatoid arthritis, acting as a negative regulator.
How can CRISPR be used to study negative regulation of CCL4?
CRISPR knockout, point mutation, knock-in, and overexpression models can test the causal role of candidate genes in suppressing CCL4 production.
What cell types are relevant for studying CCL4 negative regulation?
Monocytes, macrophages, T cells, and mast cells are commonly used, as they produce CCL4 and respond to regulatory signals.
What methods measure CCL4 production?
ELISA, Luminex, RT-qPCR, RNA-seq, and flow cytometry are standard methods to quantify CCL4 at protein and mRNA levels.
Is EZH2 a negative regulator of CCL4?
Yes, inhibition of EZH2 reduces chemokine production, including CCL4, in lupus models, suggesting EZH2 suppresses CCL4.
What is the difference between CCL4 and MIP-1β?
They are the same protein; CCL4 is the systematic name, and MIP-1β is a common alias.
Conclusion
Negative regulation of chemokine (C-C motif) ligand 4 production (GO:0071644) is a critical process for controlling inflammation and preventing autoimmune pathology. Key regulators such as microRNA-155, EZH2, and ELK4 have been identified, and their dysfunction contributes to diseases like rheumatoid arthritis and lupus. CRISPR-based models offer powerful tools to dissect these mechanisms and identify new therapeutic targets. EDITGENE provides end-to-end services to support such research, from knockout to library screening.
References
- 1. Huang Y et al.. 2023. ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.. Front Immunol 14:1171380 PMID: 37529050
- 2. Broughton TWK et al.. 2019. Defining the Signature of VISTA on Myeloid Cell Chemokine Responsiveness.. Front Immunol 10:2641 PMID: 31803182
- 3. Elmesmari A et al.. 2016. MicroRNA-155 regulates monocyte chemokine and chemokine receptor expression in Rheumatoid Arthritis.. Rheumatology (Oxford) 55(11):2056-2065 PMID: 27411480
- 4. Bierer J et al.. 2023. Novel inflammatory mediator profile observed during pediatric heart surgery with cardiopulmonary bypass and continuous ultrafiltration.. J Transl Med 21(1):439 PMID: 37408044
- 5. Shi L et al.. 2024. Role of the Pseudomonas plecoglossicida fliL gene in immune response of infected hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).. Front Immunol 15:1415744 PMID: 39026675
- 6. Wang CR et al.. 2003. Regulation of CCR5 expression and MIP-1alpha production in CD4+ T cells from patients with rheumatoid arthritis.. Clin Exp Immunol 132(2):371-8 PMID: 12699431
- 7. Walrath J et al.. 2005. Resident Th1-like effector memory cells in pulmonary recall responses to Mycobacterium tuberculosis.. Am J Respir Cell Mol Biol 33(1):48-55 PMID: 15778493
- 8. Rohraff DM et al.. 2019. Inhibition of EZH2 Ameliorates Lupus-Like Disease in MRL/lpr Mice.. Arthritis Rheumatol 71(10):1681-1690 PMID: 31106974
