Recombinant Human C-X-C motif chemokine 5 (CXCL5), partial (Active)

What is the basic structure of CXCL5 and how does it compare to other chemokines in the CXC family?

CXCL5 adopts the typical chemokine fold consisting of a six-stranded antiparallel β-sheet with two overlying α-helices. The structure has estimated dimensions of 33 Å long, 26 Å wide, and 16 Å deep. The surface under the β-sheet comprises entirely hydrophilic and charged residues. In its dimeric form, the two symmetric helices are approximately 23 Å long and separated by a center-to-center distance of 10.2 Å, with an angle of 168.6° between them .

CXCL5 shares 77% amino acid sequence identity with CXCL6/GCP-2 and 35%-51% with other human ELR+ chemokines including CXCL1/GRO alpha, CXCL2/GRO beta, CXCL3/GRO gamma, CXCL7/NAP-2, and CXCL8/IL-8. Its distinguishing feature is the Glu-Leu-Arg (ELR) motif, which confers angiogenic properties and differentiates it from ELR- CXC chemokines that are angiostatic .

What post-translational modifications of CXCL5 are known to occur naturally and how do they affect function?

Two principal post-translational modifications of CXCL5 have been documented:

• N-terminal truncation: Full-length CXCL5 (78 amino acids) can be trimmed at the N-terminus by cathepsin G and chymotrypsin to generate shorter forms such as ENA-74 (74 aa) and ENA-70 (70 aa). Research demonstrates that these truncated forms exhibit increased potency compared to full-length CXCL5 . Specifically, N-terminal truncation enhances G protein signaling and β-arrestin recruitment through CXCR2, increases CXCL5-initiated internalization of CXCR2, and amplifies Ca²⁺ signaling downstream of both CXCR2 and CXCR1 .

• Citrullination: The arginine at position 9 can be converted to citrulline. Interestingly, studies have shown that citrullination does not significantly affect CXCL5-dependent signal transduction or chemotaxis capabilities .

These modifications can occur separately or together in vivo, creating a spectrum of CXCL5 variants with differing functional potencies.

Through which receptors does CXCL5 signal and how does receptor binding specificity compare to other chemokines?

CXCL5 primarily signals through CXCR2, but research has demonstrated that it can also interact with CXCR1, particularly in its truncated form . Additionally, CXCL5 binds to the Duffy Antigen Receptor for Chemokines (DARC), which acts as a non-signaling decoy receptor. This DARC binding is particularly important as it can limit CXCR2-mediated responses by acting as a chemokine sink .

Truncated CXCL5 shows enhanced potency in activating both CXCR1 and CXCR2 compared to the full-length protein, which has significant implications for cellular recruitment during inflammation. The binding to these receptors triggers various downstream signaling pathways, including G protein signaling, β-arrestin recruitment, and calcium mobilization .

How does CXCL5 regulate neutrophil and monocyte recruitment during inflammation?

CXCL5 functions as a potent chemoattractant that regulates the trafficking of both neutrophils and monocytes to inflammatory sites. This process involves:

• Direct chemotactic activity: CXCL5 creates concentration gradients that guide neutrophil and monocyte migration to sites of inflammation through CXCR2 and CXCR1 activation. N-terminal truncation enhances this chemotactic activity, particularly toward monocytes .

• Regulation of other chemokines: CXCL5 affects the availability of other chemokines by competing for binding to DARC on erythrocytes. Studies using CXCL5-deficient mice have demonstrated that CXCL5 binding to erythrocyte DARC impairs its scavenging function, thereby increasing plasma concentrations of other chemokines like CXCL1 and CXCL2 .

• Receptor desensitization: Elevated CXCL5 levels can lead to CXCR2 desensitization, which paradoxically can impair neutrophil recruitment in some contexts. This has been observed in E. coli pneumonia models, where CXCL5 deficiency resulted in increased neutrophil influx to the lung .

What are the key signaling pathways activated by CXCL5 and how do they differ between cell types?

CXCL5 activates several signaling pathways upon binding to its receptors:

• G protein-coupled signaling: Binding to CXCR1/CXCR2 activates heterotrimeric G proteins, particularly Gαi, leading to inhibition of adenylyl cyclase and activation of phospholipase C. This triggers multiple downstream events including calcium mobilization and protein kinase C activation .

• β-arrestin recruitment: CXCL5 induces β-arrestin recruitment to CXCR2, which both terminates G protein signaling and initiates arrestin-dependent signaling pathways. N-terminal truncation enhances this recruitment .

• Jak2/STAT5/SOCS2 pathway: In the context of insulin signaling, CXCL5 has been shown to activate the Jak2/STAT5/SOCS2 pathway, which interferes with insulin receptor signaling in muscle cells, contributing to insulin resistance .

Cell type-specific responses include:

• In neutrophils and monocytes: Predominantly chemotactic responses involving cytoskeletal rearrangement and directed migration.

• In endothelial cells: Angiogenic responses promoting cell proliferation and vessel formation.

• In muscle cells: Activation of pathways that interfere with insulin signaling, contributing to metabolic dysfunction .

What are the optimal conditions for using recombinant CXCL5 in cell-based assays, and what controls should be included?

For optimal use of recombinant CXCL5 in cell-based assays:

Reconstitution and Storage:

• Reconstitute lyophilized CXCL5 in sterile PBS containing at least 0.1% carrier protein (such as BSA) to ensure stability.

• Store reconstituted protein in small aliquots at -20°C to -80°C to avoid repeated freeze-thaw cycles.

Working Concentration Range:

• For chemotaxis assays: 3-15 ng/mL is typically effective for inducing migration of neutrophils and monocytes .

• For signaling assays: Consider using a concentration range (1-100 ng/mL) to establish dose-response relationships.

Essential Controls:

• Negative control: Vehicle only (buffer used for protein reconstitution)

• Positive control: Well-characterized chemokine with similar activity (e.g., CXCL8/IL-8 for neutrophil chemotaxis)

• Receptor antagonist control: Include CXCR1/CXCR2 antagonists to confirm specificity

• Modified CXCL5 forms: When studying specific functions, compare full-length, truncated, and citrullinated forms

Cell Types:

• For migration assays: Primary human neutrophils, CD14+ monocytes, or established cell lines such as THP-1 (monocytes) .

• For signaling studies: CXCR1/CXCR2-expressing cells (either primary cells or transfected cell lines).

How can researchers effectively differentiate between the activities of full-length versus truncated CXCL5 in experimental systems?

To differentiate between full-length and truncated CXCL5 activities:

Methodological Approach:

• Chemical Synthesis of Variants:

  • Synthesize or obtain recombinant preparations of CXCL5(1-78) (full-length) and CXCL5(9-78) (truncated) with high purity.

  • Include citrullinated variants if studying this modification .

• Comparative Functional Assays:

  • G protein signaling assays using BRET (Bioluminescence Resonance Energy Transfer) to measure G protein activation kinetics and magnitude.

  • β-arrestin recruitment assays to compare recruitment efficiency and kinetics.

  • Calcium mobilization assays in primary human cells or receptor-expressing cell lines.

  • Receptor internalization assays to compare effects on CXCR2 trafficking .

• Cell Migration Assays:

  • Transwell migration assays using purified neutrophils and monocytes.

  • Time-lapse microscopy to evaluate migration parameters including velocity, directionality, and persistence.

  • Use of receptor-specific antagonists to determine receptor dependency .

• In Vivo Models:

  • Intra-articular injection models to evaluate leukocyte recruitment to specific sites.

  • Use of CXCR1/2 antagonist (such as reparixin) to confirm receptor specificity .

• Biochemical Characterization:

  • Surface plasmon resonance to measure binding kinetics to purified receptors.

  • Competitive binding assays to assess relative receptor affinities.

Key readouts to compare include dose-response curves, EC50 values, receptor specificity profiles, and kinetics of response. Research has consistently shown that truncated forms display enhanced potency across multiple functional readouts .

What are the most effective methods for studying CXCL5-DARC interactions and their impact on chemokine scavenging?

To study CXCL5-DARC interactions and chemokine scavenging:

In Vitro Methods:

• Binding Assays:

  • Radioligand binding assays using labeled CXCL5 and erythrocytes expressing DARC.

  • Competition assays with other DARC-binding chemokines to evaluate binding affinities.

  • Binding studies with truncated versus full-length CXCL5 to assess how modifications affect DARC affinity.

• Chemokine Scavenging Assays:

  • Incubate erythrocytes with varying concentrations of CXCL5 followed by addition of other chemokines (CXCL1, CXCL2).

  • Measure free chemokine concentrations over time using ELISA or multiplex protein assays.

  • Compare scavenging capacity in the presence/absence of CXCL5 pretreatment .

Ex Vivo Methods:

• Erythrocyte Chemokine Binding Studies:

  • Isolate erythrocytes from blood and assess their capacity to bind CXCL5 and other chemokines.

  • Compare binding in various disease states or genetic backgrounds.

  • Flow cytometry using fluorescently labeled chemokines to quantify erythrocyte binding .

In Vivo Methods:

• Genetic Models:

  • Utilize CXCL5-deficient mice to assess how CXCL5 absence affects plasma levels of other chemokines.

  • Compare DARC-deficient and wild-type mice to evaluate the role of DARC in CXCL5-mediated chemokine regulation .

• Chemokine Dynamics:

  • Administer labeled chemokines and track their clearance rates and tissue distribution in the presence/absence of CXCL5.

  • Measure chemokine gradients between tissue and circulation under different conditions .

• Inflammatory Challenges:

  • Challenge CXCL5-deficient and wild-type mice with inflammatory stimuli (e.g., E. coli infection).

  • Measure concentrations of multiple chemokines in plasma and tissues over time.

  • Assess leukocyte trafficking patterns in relation to chemokine levels and gradients .

Research using these approaches has demonstrated that CXCL5 binding to erythrocyte DARC impairs its scavenging function for other chemokines, leading to increased plasma concentrations of CXCL1 and CXCL2, which can affect chemokine gradients and neutrophil recruitment during inflammation .

How does CXCL5 contribute to obesity-induced insulin resistance and what are the therapeutic implications?

CXCL5 plays a significant role in linking obesity to insulin resistance through several mechanisms:

Adipose Tissue Expression and Regulation:

• CXCL5 is expressed at high levels in white adipose tissue (WAT), particularly in the macrophage fraction.

• Its expression and secretion are dramatically increased in the context of obesity .

Systemic Effects:

• Circulating CXCL5 levels are significantly elevated in obese subjects compared to lean individuals.

• Following weight reduction programs, CXCL5 concentrations decrease, suggesting a direct relationship with adiposity.

• Obese non-insulin resistant subjects show lower CXCL5 levels than obese insulin-resistant subjects, indicating a specific association with insulin resistance beyond obesity alone .

Molecular Mechanism:

• CXCL5 directly blocks insulin-stimulated glucose uptake in muscle by activating the Jak2/STAT5/SOCS2 pathway.

• This pathway interferes with insulin signaling, contributing to systemic insulin resistance .

Therapeutic Implications:

The discovery that CXCL5 is a mechanistic link between obesity and insulin resistance opens new therapeutic avenues for treating type 2 diabetes and metabolic syndrome. The reversibility of CXCL5-mediated effects through either neutralizing antibodies or receptor antagonism provides proof-of-concept for targeting this pathway clinically .

What is known about the role of CXCL5 in cancer progression and angiogenesis?

CXCL5 contributes to cancer development and progression through multiple mechanisms:

Angiogenic Properties:

• As an ELR+ CXC chemokine, CXCL5 possesses intrinsic angiogenic properties that promote vascularization of tumors.

• These properties stem from its Glu-Leu-Arg (ELR) motif, which distinguishes it from angiostatic ELR- CXC chemokines .

Expression in Cancer:

• CXCL5 is upregulated in numerous cancer types, including lung cancer.

• Its expression correlates with increased vascularization, enhanced tumor growth, and metastatic potential .

Specific Cancer Types:

• In non-muscle invasive bladder cancer, CXCL5 promotes resistance to mitomycin C by activating epithelial-mesenchymal transition (EMT) and NF-κB pathway .

• In other cancers, CXCL5 contributes to tumor cell proliferation, migration, and invasion.

Cellular Sources:

• CXCL5 can be produced by both tumor cells and stromal components, including tumor-associated macrophages.

• Adipose tissue-derived stem cells secrete CXCL5 with chemoattractant and angiogenic properties, potentially contributing to cancer progression in obesity-associated cancers .

Therapeutic Implications:

• CXCL5 as a Biomarker:

  • Elevated CXCL5 levels may serve as prognostic indicators in certain cancers.

  • Changes in CXCL5 expression could potentially monitor treatment efficacy.

• Targeting CXCL5-CXCR2 Axis:

  • CXCR2 antagonists may inhibit tumor angiogenesis and growth.

  • Combined targeting of multiple ELR+ chemokines might be necessary for effective anti-angiogenic therapy.

• Combination Therapies:

  • Anti-CXCL5 strategies might sensitize resistant tumors to conventional chemotherapies.

  • Combining CXCL5 inhibition with other anti-angiogenic approaches could enhance efficacy.

Understanding the nuanced roles of CXCL5 in different cancer contexts is essential for developing targeted therapeutic strategies. The dual role of CXCL5 in promoting both angiogenesis and cancer cell survival highlights its potential as a therapeutic target in oncology .

How does CXCL5 function in acute inflammatory conditions, and what is its role in neovascularization during wound healing?

CXCL5 exhibits complex functions in acute inflammation and wound healing:

Acute Inflammation:

Wound Healing and Neovascularization:

• Angiogenic Properties:

  • CXCL5's ELR motif confers angiogenic properties that promote endothelial cell migration and proliferation.

  • This contributes to neovascularization during wound repair .

• Diabetic Wound Healing:

  • In diabetes mellitus, CXCL5 suppression has been shown to recover neovascularization and accelerate wound healing.

  • This suggests context-dependent roles for CXCL5 in different pathological states .

• Interaction with Stem Cells:

  • Adipose tissue-derived stem cells secrete CXCL5 with chemoattractant and angiogenic properties.

  • This secretion may contribute to the therapeutic effects of these cells in wound healing applications .

Therapeutic Implications:

• Context-Dependent Targeting:

  • In some acute inflammatory conditions, CXCL5 neutralization might be beneficial.

  • In wound healing contexts, modulation rather than complete inhibition might be optimal.

• Temporal Considerations:

  • The timing of CXCL5 targeting may be crucial, with different effects during early versus late inflammation or wound healing phases.

• Combination Approaches:

  • Combined targeting of multiple chemokines or pathways may be necessary for effective modulation of complex inflammatory processes.

The role of CXCL5 in inflammation and wound healing highlights the complexity of chemokine biology, where the same molecule can have beneficial or detrimental effects depending on context, timing, and concurrent pathological conditions .

What are the current challenges in developing specific CXCL5-targeted therapeutics, and how might they be overcome?

Developing specific CXCL5-targeted therapeutics faces several challenges:

Selectivity Challenges:

• Receptor Promiscuity: CXCL5 signals through both CXCR1 and CXCR2, which are also targeted by multiple other chemokines. Achieving selective CXCL5 inhibition without affecting other chemokine signaling is difficult .

• Structural Homology: High sequence similarity (77%) between CXCL5 and CXCL6/GCP-2 makes developing highly specific antibodies or inhibitors challenging .

• Modified Forms: The existence of multiple active forms of CXCL5 (full-length, truncated, citrullinated) complicates targeting. Truncated forms show enhanced potency, making them potentially more important therapeutic targets .

Context-Dependent Function Challenges:

• Dual Roles: CXCL5 can be both beneficial and detrimental depending on the disease context. In some inflammatory conditions, it promotes pathological inflammation, while in others it may facilitate resolution .

• Timing Issues: The appropriate timing for CXCL5 inhibition may vary by disease stage. For example, blocking CXCL5 early in infection might impair host defense, while later inhibition might reduce tissue damage.

Potential Solutions:

• Structure-Based Drug Design:

  • Utilize the solved structure of CXCL5 to identify unique binding pockets that differ from other chemokines.

  • Develop small molecules that specifically target the CXCL5-CXCR2 interface .

• Form-Specific Targeting:

  • Design antibodies or inhibitors that selectively recognize truncated or modified forms of CXCL5.

  • Develop agents that modulate the processing of CXCL5 rather than blocking the protein itself .

• Context-Specific Delivery:

  • Create tissue-specific delivery systems that target CXCL5 inhibition to relevant sites.

  • Develop temporally controlled release systems for appropriate timing of inhibition.

• Combination Approaches:

  • Pair CXCL5 inhibition with other agents targeting complementary pathways.

  • In obesity-related insulin resistance, combine with metabolic modulators .

  • In cancer, combine with conventional chemotherapeutics or other anti-angiogenic agents .

• Biomarker-Guided Therapy:

  • Develop companion diagnostics to identify patients most likely to benefit from CXCL5 inhibition.

  • Measure CXCL5 levels, receptor expression, or downstream signaling markers to guide treatment decisions.

The rational development of CXCL5-targeted therapeutics will require deeper understanding of its disease-specific roles and careful consideration of potential off-target effects on related chemokine pathways.

How do different oligomeric states of CXCL5 affect its biological activity, and what methodologies are best suited to study these effects?

The oligomeric state of CXCL5 significantly impacts its biological functions:

Oligomeric States and Their Significance:

• Monomeric vs. Dimeric Forms:

  • CXCL5 can exist in both monomeric and dimeric states, with the dimer interface consisting of strand 1 from one monomer and strand 1' of the second monomer.

  • The dimer is stabilized by six β-strand backbone hydrogen bonds and various packing interactions .

• Functional Implications:

  • Different oligomeric states can exhibit distinct receptor binding properties and activation potentials.

  • Monomer-dimer equilibrium may be influenced by concentration, pH, and presence of glycosaminoglycans.

  • The estimated dimensions of the CXCL5 dimer (33 Å long, 26 Å wide, and 16 Å deep) provide structural insights into how dimerization affects receptor interaction .

Methodologies for Studying Oligomeric States:

• Structural Analysis:

  • NMR Spectroscopy: Provide detailed structural information about different oligomeric states in solution. The solution structure of CXCL5 has been determined using NMR with 2250 experimental restraints including distance, dihedral, and H-bonding constraints .

  • X-ray Crystallography: Offer high-resolution static structures of oligomeric forms.

  • Size Exclusion Chromatography: Separate different oligomeric states based on size.

  • Analytical Ultracentrifugation: Determine molecular weights and association constants in solution.

• Functional Comparisons:

  • Receptor Binding Assays: Compare binding affinity of different oligomeric states to CXCR1/CXCR2.

  • Signaling Assays: Measure G protein activation, β-arrestin recruitment, and calcium mobilization by different forms.

  • Cell Migration Assays: Assess chemotactic potency of stabilized monomers versus dimers.

• Engineered Variants:

  • Disulfide-Trapped Dimers: Create obligate dimers through strategic disulfide bonds.

  • Interface Mutations: Disrupt dimerization through mutations at key interface residues.

  • Chimeric Proteins: Create fusion proteins that enforce specific oligomeric states.

• Computational Approaches:

  • Molecular Dynamics Simulations: Model dynamics of different oligomeric states.

  • Protein-Protein Docking: Predict interfaces and stability of oligomeric assemblies.

  • Free Energy Calculations: Estimate stability of different oligomeric forms.

• In Vivo Relevance:

  • FRET/BRET Techniques: Monitor oligomerization in living cells.

  • In vivo Crosslinking: Capture physiologically relevant oligomeric states in tissues.

  • Functional Assays with Stabilized Forms: Compare biological activities in relevant disease models.

Understanding how oligomeric states affect CXCL5 function could provide insights for developing more selective therapeutic approaches that target specific functional forms of the protein rather than total CXCL5 levels.

What are the emerging roles of CXCL5 in non-canonical signaling pathways and how might these be exploited for therapeutic development?

CXCL5 participates in several non-canonical signaling pathways beyond traditional G protein-coupled receptor signaling:

Non-Canonical Signaling Mechanisms:

• Jak2/STAT5/SOCS2 Pathway:

  • CXCL5 activates the Janus kinase 2 (Jak2)/Signal Transducer and Activator of Transcription 5 (STAT5)/Suppressor of Cytokine Signaling 2 (SOCS2) pathway.

  • This pathway is particularly relevant in muscle cells, where it interferes with insulin receptor signaling, contributing to insulin resistance in obesity .

• β-Arrestin-Dependent Signaling:

  • Beyond its role in receptor desensitization, β-arrestin recruitment by activated CXCR1/2 initiates signaling cascades independent of G proteins.

  • N-terminal truncation of CXCL5 enhances β-arrestin recruitment, potentially amplifying these non-canonical signaling events .

• Cross-Regulation of Other Chemokine Pathways:

  • CXCL5 binding to DARC on erythrocytes impairs its chemokine scavenging function, indirectly affecting the bioavailability of other chemokines like CXCL1 and CXCL2.

  • This represents a non-receptor-mediated mechanism by which CXCL5 influences broader inflammatory processes .

• NF-κB Pathway Activation:

  • In certain contexts, such as non-muscle invasive bladder cancer, CXCL5 activates the NF-κB pathway, promoting epithelial-mesenchymal transition and chemoresistance .

Therapeutic Exploitation Strategies:

• Pathway-Specific Targeting:

  • Develop inhibitors that specifically block CXCL5-induced Jak2/STAT5 activation without affecting canonical G protein signaling.

  • This approach could address metabolic effects while preserving immune functions.

• Biased Ligand Development:

  • Design "biased agonists" or "biased antagonists" that selectively modulate specific signaling pathways downstream of CXCR1/2.

  • For example, compounds that block G protein signaling but preserve or enhance β-arrestin recruitment (or vice versa).

• Combination Therapies:

  • Target CXCL5 in combination with downstream pathway inhibitors.

  • For insulin resistance: Combine CXCL5 neutralization with SOCS inhibitors.

  • For cancer: Combine with NF-κB pathway modulators.

• Context-Specific Approaches:

  • Develop tissue-specific delivery systems that target CXCL5 inhibition to relevant tissues.

  • Time-dependent administration strategies that account for different roles of CXCL5 during disease progression.

• Novel Formulations:

  • Design peptide inhibitors that mimic specific domains of CXCL5 involved in non-canonical signaling.

  • Develop aptamers or small molecules that selectively block interaction with specific pathway components.

The diversity of CXCL5 signaling pathways offers multiple points for therapeutic intervention. Understanding these non-canonical mechanisms provides opportunities to develop more precise, context-specific treatments for conditions ranging from metabolic disorders to cancer, potentially with fewer side effects than global CXCL5 inhibition.

How is CXCL5 involved in the regulation of metabolism beyond insulin resistance, and what are the implications for metabolic diseases?

CXCL5's metabolic roles extend beyond insulin resistance to broader aspects of metabolic regulation:

Adipose Tissue Biology:

• CXCL5 is highly expressed in the macrophage fraction of white adipose tissue (WAT).

• Its expression dramatically increases in obesity, suggesting a role in adipose tissue inflammation and remodeling .

• Adipose tissue-derived CXCL5 may act as an adipokine, communicating with distant tissues including muscle and liver.

Metabolic Inflammation:

• CXCL5 contributes to the low-grade inflammatory state characteristic of metabolic diseases.

• This chemokine may represent a key link between immune activation and metabolic dysfunction, particularly in obesity .

Cross-talk with Metabolic Hormones:

• Emerging evidence suggests potential interactions between CXCL5 signaling and other metabolic hormone pathways.

• The Jak2/STAT5/SOCS2 pathway activated by CXCL5 may intersect with growth hormone, leptin, and other cytokine signaling pathways .

Implications for Metabolic Diseases:

• Type 2 Diabetes:

  • CXCL5 neutralization or CXCR2 antagonism improves insulin sensitivity in obese, insulin-resistant mice.

  • CXCR2-deficient mice are protected against diet-induced insulin resistance and diabetes .

  • These findings suggest CXCL5 as a potential therapeutic target for diabetes management.

• Non-alcoholic Fatty Liver Disease (NAFLD):

  • The CXCL5-Jak2/STAT pathway may influence hepatic insulin sensitivity and fat accumulation.

  • Targeting CXCL5 might reduce hepatic steatosis and inflammation in NAFLD.

• Cardiovascular Complications:

  • CXCL5's angiogenic properties and role in inflammation may influence vascular complications of metabolic diseases.

  • Adipose-derived CXCL5 could contribute to atherogenesis in metabolic syndrome.

• Adipose Tissue Dysfunction:

  • CXCL5 may influence adipose tissue expansion, inflammation, and remodeling.

  • Targeting CXCL5 could potentially improve adipose tissue function in obesity.

Research Directions:

• Investigate tissue-specific roles of CXCL5 in various metabolic organs (liver, pancreas, muscle, adipose).

• Explore how dietary factors and weight loss interventions affect CXCL5 expression and signaling.

• Determine how CXCL5 interacts with other adipokines and inflammatory mediators in metabolic regulation.

• Develop tissue-specific CXCL5 targeting strategies for metabolic diseases.

The multifaceted role of CXCL5 in metabolism positions it as a promising target for integrated approaches to metabolic diseases that address both inflammatory and metabolic components .

What are the latest findings regarding CXCL5 genetic variants and their association with disease susceptibility or progression?

Research on CXCL5 genetic variants is an emerging field with implications for personalized medicine:

Genetic Variants and Functional Consequences:

While the search results don't specifically address CXCL5 genetic variants, current research in this area has begun examining several types of variations:

• Promoter Polymorphisms:

  • Variations in the CXCL5 promoter region may affect transcriptional regulation and expression levels.

  • These could influence baseline CXCL5 production or its inducibility during inflammation.

• Coding Region Variants:

  • Single nucleotide polymorphisms (SNPs) in the coding region might alter CXCL5 structure or function.

  • Variants affecting the ELR motif could impact angiogenic properties.

  • Polymorphisms near the N-terminus might influence susceptibility to proteolytic processing.

• 3' UTR Variants:

  • Variations in the 3' untranslated region could affect mRNA stability and translation efficiency.

  • These might influence CXCL5 protein levels without changing the amino acid sequence.

Disease Associations:

Based on CXCL5's known functions, genetic variants might be particularly relevant in:

• Inflammatory Diseases:

  • Pulmonary diseases: CXCL5 has been implicated in pulmonary inflammation and lung cancer .

  • Rheumatoid arthritis: CXCL5 plays a role in joint inflammation .

• Metabolic Disorders:

  • Obesity and insulin resistance: Variants affecting CXCL5 expression or function could modulate susceptibility to obesity-related insulin resistance .

  • Type 2 diabetes: Genetic variations might influence risk or progression of diabetes through effects on insulin signaling.

• Cancer Susceptibility and Progression:

  • Variants affecting CXCL5's angiogenic or EMT-promoting properties could influence cancer development.

  • Polymorphisms might predict tumor aggressiveness or response to therapy in certain cancers .

Research Approaches:

To advance understanding of CXCL5 genetic variants:

• Genome-Wide Association Studies (GWAS):

  • Identify associations between CXCL5 locus variants and disease phenotypes.

  • Examine disease-specific cohorts (obesity, diabetes, inflammatory conditions, cancer).

• Functional Genomics:

  • Characterize the impact of identified variants on CXCL5 expression, processing, and function.

  • Use CRISPR/Cas9 to introduce specific variants and assess functional consequences.

• Clinical Correlations:

  • Assess how CXCL5 genetic profiles correlate with disease progression or treatment response.

  • Develop genetic panels that include CXCL5 variants for risk stratification.

• Pharmacogenomics:

  • Investigate how genetic variants affect response to CXCL5-targeted therapeutics.

  • Develop personalized approaches based on individual genetic profiles.

Understanding the impact of CXCL5 genetic variants could help identify at-risk individuals, predict disease course, and tailor therapeutic approaches in conditions ranging from metabolic disorders to cancer.

How does CXCL5 interact with other chemokines and cytokines in complex inflammatory networks, and what methodologies best capture these interactions?

CXCL5 functions within intricate inflammatory networks, interacting with other mediators in various ways:

Types of Interactions:

• Direct Protein-Protein Interactions:

  • CXCL5 may form heterodimers or higher-order complexes with other chemokines.

  • These interactions could modulate receptor binding and signaling properties.

• Receptor-Level Crosstalk:

  • CXCL5 signaling through CXCR1/CXCR2 can cross-desensitize receptors for other chemokines.

  • Truncated CXCL5 shows enhanced potency in activating both CXCR1 and CXCR2 compared to full-length protein .

• Shared Scavenging Mechanisms:

  • CXCL5 competes with other chemokines (like CXCL1 and CXCL2) for binding to DARC on erythrocytes.

  • This affects the bioavailability of multiple chemokines simultaneously .

• Synergistic or Antagonistic Functional Effects:

  • CXCL5 may enhance or inhibit cellular responses to other chemokines or cytokines.

  • In some contexts, CXCL5 deficiency leads to improved neutrophil recruitment, suggesting complex regulatory roles .

Inflammatory Networks:

• Neutrophil Recruitment Cascades:

  • CXCL5 works alongside CXCL1, CXCL2, and CXCL8/IL-8 in orchestrating neutrophil mobilization and activation.

  • The relative contribution of each chemokine may vary by tissue and inflammatory context .

• Metabolic Inflammation:

  • In adipose tissue, CXCL5 interacts with cytokines like TNF-α and IL-6.

  • These interactions may amplify inflammatory responses and contribute to insulin resistance .

• Wound Healing and Tissue Repair:

  • CXCL5's angiogenic properties interact with growth factors like VEGF and FGF.

  • This coordination influences neovascularization during wound healing .

Methodologies for Studying Complex Interactions:

• Multiplex Protein Analysis:

  • Cytokine/Chemokine Arrays: Simultaneously measure multiple mediators in biological samples.

  • Multiplex Bead-Based Assays: Quantify multiple chemokines in a single sample with high sensitivity.

  • Proteomics Approaches: Identify protein-protein interactions and post-translational modifications.

• Systems Biology Approaches:

  • Network Analysis: Construct interaction networks based on correlation patterns.

  • Pathway Modeling: Develop mathematical models of chemokine signaling networks.

  • Machine Learning: Identify patterns in complex datasets that may reveal novel interactions.

• Advanced Imaging Techniques:

  • Intravital Microscopy: Visualize leukocyte trafficking in vivo in response to multiple chemokines.

  • Multiplexed Immunofluorescence: Simultaneously visualize multiple chemokines and their receptors in tissues.

  • FRET/BRET Techniques: Detect protein-protein interactions in living cells.

• Genetic Approaches:

  • Compound Knockout Models: Generate mice lacking multiple chemokines or receptors.

  • Conditional Knockouts: Selectively delete CXCL5 in specific cell types to dissect context-dependent interactions.

  • CRISPR Screens: Identify genes that modify CXCL5 function or response.

• Ex Vivo Systems:

  • Tissue-on-Chip Models: Recreate complex tissue environments to study chemokine interactions.

  • Organoid Cultures: Examine chemokine networks in three-dimensional tissue mimetics.

Research using these approaches has revealed that CXCL5 binding to erythrocyte DARC impairs its chemokine scavenging function, leading to increased plasma concentrations of CXCL1 and CXCL2. This demonstrates how one chemokine can indirectly influence the function of others, creating complex regulatory networks that modulate inflammatory responses .

What are the key considerations for designing CXCL5-focused experiments in disease models, and how should results be interpreted?

Designing robust CXCL5-focused experiments requires careful consideration of several factors:

Experimental Design Considerations:

• Model Selection:

  • Cell Models: Choose cell types that express CXCR1/CXCR2 receptors (neutrophils, monocytes, endothelial cells) or are targets of CXCL5 effects (muscle cells for insulin resistance studies) .

  • Animal Models: Consider species differences—human CXCL5 shares only 57% amino acid sequence identity with mouse and rat CXCL5 .

  • Disease-Specific Models: Select models that recapitulate key aspects of the disease being studied (e.g., diet-induced obesity for metabolic studies, inflammatory challenges for acute inflammation) .

• CXCL5 Forms and Modifications:

  • Include both full-length and truncated CXCL5 forms in studies, as they exhibit different potencies .

  • Consider citrullinated variants when relevant, though they may not differ functionally from non-citrullinated forms .

  • Use recombinant proteins with appropriate carrier proteins to ensure stability .

• Dosing and Timing:

  • Establish dose-response relationships (typical effective range: 3-15 ng/mL for in vitro studies) .

  • Consider physiological concentrations observed in relevant diseases.

  • Evaluate timing effects, as CXCL5's role may differ during acute versus chronic phases of disease.

• Controls and Comparators:

  • Include appropriate positive controls (e.g., other ELR+ chemokines like CXCL8/IL-8).

  • Use receptor antagonists to confirm CXCR1/CXCR2 dependency of observed effects.

  • Include CXCL5-deficient controls (neutralizing antibodies, genetic knockouts) to establish specificity .

Interpretation Considerations:

• Context Dependency:

  • CXCL5 may have paradoxical effects depending on the inflammatory context—in some cases, deficiency improves neutrophil recruitment and host defense .

  • Consider the complete chemokine milieu when interpreting results.

• Receptor Considerations:

  • Effects may be mediated through CXCR1, CXCR2, or both depending on cell type and CXCL5 form .

  • Receptor desensitization can complicate interpretation of in vivo findings.

• Scavenging Effects:

  • Consider CXCL5's impact on DARC-mediated chemokine scavenging when interpreting changes in other chemokines .

  • Measure multiple chemokines simultaneously when possible.

• Translational Aspects:

  • Consider species differences when extrapolating from animal models to human disease.

  • Validate key findings in human samples or systems when possible.

• Methodological Limitations:

  • Be aware of detection limits and specificity of CXCL5 assays.

  • Consider how sample processing might affect CXCL5 stability or measurement.

Experimental Roadmap:

• Characterization Phase:

  • Establish CXCL5 expression patterns in the disease/model of interest.

  • Determine which forms (full-length, truncated) predominate.

  • Identify key cell types expressing and responding to CXCL5.

• Mechanistic Phase:

  • Manipulate CXCL5 levels or signaling (neutralization, receptor blockade, genetic approaches).

  • Assess effects on disease-relevant endpoints.

  • Investigate specific signaling pathways (G protein, β-arrestin, Jak/STAT) .

• Validation Phase:

  • Confirm findings across multiple models.

  • Validate in human samples when possible.

  • Assess therapeutic potential through intervention studies.

By carefully considering these factors, researchers can design more rigorous CXCL5-focused experiments and interpret results within the complex context of chemokine biology and disease pathophysiology.

What quality control measures should be applied when working with recombinant CXCL5 to ensure experimental reproducibility?

Ensuring experimental reproducibility with recombinant CXCL5 requires rigorous quality control:

Pre-Experiment Quality Control:

• Source Verification:

  • Obtain recombinant CXCL5 from reputable suppliers with consistent manufacturing processes.

  • Verify the expression system used (typically E. coli for research-grade proteins) .

  • Check that the correct sequence variant is being used (typically Ala37-Asn114 for human CXCL5) .

• Initial Characterization:

  • SDS-PAGE: Confirm protein purity (typically >95% for research applications).

  • Western Blot: Verify identity using specific antibodies.

  • Mass Spectrometry: Confirm molecular weight and sequence integrity.

  • Endotoxin Testing: Ensure preparations are endotoxin-free (<0.1 EU/μg) to avoid confounding inflammatory responses.

• Functional Validation:

  • Chemotaxis Assay: Verify chemotactic activity using neutrophils or appropriate cell lines.

  • Calcium Mobilization: Confirm receptor activation in CXCR1/CXCR2-expressing cells.

  • Dose-Response Analysis: Establish consistent EC50 values for key functional readouts.

Handling and Storage:

• Reconstitution Protocol:

  • Follow manufacturer's recommendations for reconstitution buffer (typically PBS with carrier protein).

  • Use sterile technique to prevent contamination.

  • Allow complete dissolution before use.

• Storage Conditions:

  • Store lyophilized protein at -20°C to -80°C.

  • Prepare small aliquots of reconstituted protein to avoid freeze-thaw cycles.

  • Document storage duration and conditions.

• Stability Testing:

  • Periodically re-test activity of stored aliquots.

  • Establish maximum storage times for reconstituted protein at different temperatures.

  • Consider using stabilizing additives for diluted working solutions.

Experimental Controls:

• Positive Controls:

  • Include well-characterized chemokines (e.g., CXCL8/IL-8) as positive controls in functional assays.

  • Use previous lots of validated CXCL5 as reference standards when transitioning to new lots.

• Negative Controls:

  • Include heat-denatured CXCL5 to control for non-specific effects.

  • Use buffer-only conditions to establish baselines.

  • Include receptor antagonists to confirm receptor specificity of observed effects.

• Lot-to-Lot Consistency:

  • Compare activity of new lots against reference standards.

  • Document lot numbers and establish acceptance criteria for lot transitions.

  • Consider creating internal reference standards for long-term studies.

Documentation and Reporting:

• Detailed Methods Documentation:

  • Record complete information about the recombinant protein:

    • Manufacturer and catalog number

    • Lot number

    • Expression system

    • Sequence (full-length vs. truncated)

    • Presence of tags or modifications

    • Reconstitution method and carrier proteins

• Experimental Conditions:

  • Document exact concentrations used.

  • Record buffer composition and additives.

  • Note preparation and storage times prior to use.

• Data Reporting:

  • Include quality control data in publications or reports.

  • Share raw data when possible to enable meta-analysis.

  • Report negative results to address publication bias.

By implementing these quality control measures, researchers can significantly improve the reproducibility of experiments involving recombinant CXCL5 and enhance the reliability of their findings in this rapidly evolving field.

How can researchers effectively compare results across studies that use different forms or preparations of CXCL5?

Comparing results across studies using different CXCL5 preparations requires careful consideration of several factors:

Form and Sequence Variations:

• Length Variants:

  • Clearly identify whether studies used full-length CXCL5 (1-78), truncated forms (e.g., 9-78), or other variants.

  • Note that N-terminal truncation enhances potency in multiple functional assays .

  • Consider developing conversion factors based on comparative potency data when integrating results.

• Post-translational Modifications:

  • Check whether studies used native or modified forms (e.g., citrullinated variants).

  • While citrullination may not significantly affect function, other modifications might .

  • Consider the biological relevance of modifications to the specific research question.

• Species Differences:

  • Note that human CXCL5 shares only 57% amino acid sequence identity with mouse and rat CXCL5 .

  • Be cautious when comparing results between human and rodent studies.

  • Consider species-specific differences in receptor binding and downstream signaling.

Preparation Variables:

• Expression Systems:

  • Document whether proteins were produced in E. coli, mammalian cells, or other systems .

  • Expression system can affect folding, post-translational modifications, and contaminant profiles.

  • Bacterial systems typically lack glycosylation and may have endotoxin concerns.

• Purification Methods:

  • Different purification strategies may yield proteins with varying specific activities.

  • Affinity tags can influence protein behavior if not completely removed.

  • Consider whether purification methods might select for particular conformational states.

• Formulation Differences:

  • Buffer composition can affect protein stability and activity.

  • Presence of carrier proteins (e.g., BSA) may influence experimental outcomes.

  • Additives like glycerol can impact protein behavior in some assay systems.

Methodological Approaches for Comparison:

• Direct Comparative Studies:

  • When possible, test different CXCL5 forms/preparations side-by-side in the same experimental system.

  • Establish relative potency ratios for key functional readouts.

  • Document comparative dose-response relationships.

• Standardization Approaches:

  • Develop and use reference standards for activity normalization.

  • Express results in terms of "units of activity" rather than absolute concentrations.

  • Consider adopting standardized activity assays across the field.

• Meta-Analysis Techniques:

  • Use effect size measures that can be compared across studies.

  • Develop normalization approaches based on internal controls.

  • Account for preparation differences as covariates in statistical analyses.

• Reporting Standards:

  • Advocate for complete reporting of CXCL5 characteristics in publications:

    • Exact sequence with amino acid numbering

    • Expression system and purification method

    • Formulation details and storage conditions

    • Concentration determination method

    • Activity validation approaches

Practical Implementation:

• Literature Review Framework:

  • Create a standardized extraction form for CXCL5 preparation details.

  • Develop a classification system for different preparations.

  • Weight findings based on methodological rigor and preparation characterization.

• Collaborative Approaches:

  • Establish consortia to perform cross-laboratory validation studies.

  • Share reference materials between research groups.

  • Develop consensus protocols for key CXCL5 assays.

• Data Integration Strategies:

  • Use mathematical modeling to integrate data from different preparation types.

  • Develop algorithms to adjust for known potency differences between forms.

  • Create databases that link preparation details with functional outcomes.

By systematically addressing these variables and adopting standardized approaches to reporting and analysis, researchers can more effectively compare results across studies using different CXCL5 preparations, ultimately advancing understanding of this important chemokine's biology and therapeutic potential.