Recombinant Macaca mulatta C-X-C motif chemokine 10 protein (CXCL10)

What is Macaca mulatta CXCL10 and how does it compare to human CXCL10?

Macaca mulatta CXCL10 is a chemokine belonging to the CXC subfamily, characterized by the presence of four conserved cysteine residues. Similar to its human counterpart, rhesus macaque CXCL10 functions as a chemoattractant for activated T-lymphocytes and plays roles in immune cell migration and angiogenesis inhibition. Human CXCL10 consists of a 98 amino acid precursor protein that is cleaved to form a 77 amino acid secreted protein, with significant homology to the rhesus macaque version . The amino acid sequence maintains the crucial cysteine residues that determine the protein's tertiary structure and receptor-binding properties, though species-specific variations exist that may affect certain functional aspects in experimental contexts .

What are the primary biological functions of CXCL10 in rhesus macaque models?

In rhesus macaque models, CXCL10 demonstrates several critical biological functions that parallel its human counterpart. These include stimulation of monocytes, natural killer cell and T-cell migration, regulation of T-cell and bone marrow progenitor maturation, modulation of adhesion molecule expression, and inhibition of angiogenesis . CXCL10 is particularly important in SIV infection models, where it plays a role in immune cell trafficking and inflammatory responses . Like its human homolog, Macaca mulatta CXCL10 signals through the CXCR3 receptor, mediating its chemotactic effects on multiple immune cell populations . These functions make CXCL10 an important target for studying inflammatory and infectious disease processes in non-human primate models.

How is CXCL10 expression regulated in Macaca mulatta tissues?

CXCL10 expression in Macaca mulatta is primarily induced by interferon-gamma, similar to what is observed in humans. Additionally, other inflammatory stimuli including LPS, IL-1 beta, TNF-alpha, IL-12, and viral infections can trigger CXCL10 expression . In rhesus macaque models of SIV infection, CXCL10 expression increases in various tissues, contributing to inflammatory responses . Cell types known to produce CXCL10 in response to these stimuli include monocytes, fibroblasts, endothelial cells, activated T-lymphocytes, keratinocytes, osteoblasts, astrocytes, and smooth muscle cells . The regulation of CXCL10 expression is tightly controlled through multiple signaling pathways, primarily those activated by type I and type II interferons, making it an important biomarker for inflammation and immune activation in rhesus macaque studies.

What are the recommended protocols for reconstitution and storage of recombinant Macaca mulatta CXCL10?

For optimal results with recombinant Macaca mulatta CXCL10, follow these methodological guidelines:

• Reconstitution: Reconstitute lyophilized protein at 100 μg/mL in sterile PBS. For cell culture applications, including at least 0.1% carrier protein (such as bovine or human serum albumin) is recommended to enhance stability .

• Storage conditions: Store the lyophilized protein at -20°C to -80°C. After reconstitution, prepare single-use aliquots and store at -20°C to -80°C to avoid repeated freeze-thaw cycles which can compromise protein activity .

• Handling precautions: Thaw aliquots on ice immediately before use and keep the reconstituted protein on ice during experimental setup. Avoid exposing the protein to room temperature for extended periods .

• Quality control verification: Before experimental use, verify protein integrity by SDS-PAGE analysis under reducing conditions, where Macaca mulatta CXCL10 should appear as a single band of approximately 8-9 kDa, similar to human CXCL10 .

How can I validate the biological activity of recombinant Macaca mulatta CXCL10?

Validation of recombinant Macaca mulatta CXCL10 biological activity can be performed through several complementary approaches:

• Chemotaxis assay: Using cells expressing CXCR3 (such as activated T cells or CXCR3-transfected cell lines like BaF3), measure migration in response to a concentration gradient of the recombinant protein. Effective concentrations typically range from 0.03-0.18 μg/mL based on human CXCL10 studies .

• Calcium flux assay: Measure intracellular calcium mobilization in CXCR3-expressing cells loaded with calcium-sensitive dyes upon stimulation with the recombinant protein.

• Signal transduction analysis: Assess phosphorylation of downstream signaling molecules including ERK1/2, Akt, and PI3K pathways through Western blotting or phospho-flow cytometry.

• Receptor binding assays: Confirm specific binding to CXCR3 through competition assays with labeled CXCL10 or cross-competition with other CXCR3 ligands.

• Functional assays: Evaluate inhibition of angiogenesis using endothelial cell tube formation assays, which provides a physiologically relevant assessment of CXCL10 functionality .

What methods are effective for detecting and quantifying CXCL10 in Macaca mulatta biological samples?

For accurate detection and quantification of CXCL10 in Macaca mulatta samples, consider these methodological approaches:

• ELISA: Develop or utilize species-specific or cross-reactive ELISA kits validated for use with rhesus macaque samples. Human CXCL10 ELISA kits may cross-react with Macaca mulatta CXCL10 due to sequence homology, but validation is essential .

• Multiplex bead-based assays: These allow simultaneous quantification of multiple cytokines including CXCL10 in limited sample volumes, which is particularly valuable for precious non-human primate samples.

• Quantitative PCR: For mRNA expression analysis, design primers specific to Macaca mulatta CXCL10 sequence to ensure accurate quantification. Normalization to appropriate housekeeping genes is critical.

• Immunohistochemistry/Immunofluorescence: For tissue localization studies, use antibodies that cross-react with Macaca mulatta CXCL10 or develop species-specific antibodies. Validation through positive and negative controls is essential.

• Flow cytometry: For intracellular detection of CXCL10 in specific cell populations, perform intracellular cytokine staining following appropriate stimulation protocols .

How does Macaca mulatta CXCL10 compare functionally to human and mouse CXCL10 in experimental systems?

Functional comparisons between Macaca mulatta, human, and mouse CXCL10 reveal important species-specific considerations:

• Receptor interaction: All three species' CXCL10 proteins interact with CXCR3, but with potentially different binding affinities. Human CXCL10 is approximately 67% homologous to mouse CXCL10 (Crg-2), while Macaca mulatta CXCL10 demonstrates higher homology to human CXCL10 .

• Chemotactic potency: When testing chemotaxis using the BaF3 mouse pro-B cell line transfected with human CXCR3, human CXCL10 demonstrates effective doses (ED50) of 0.03-0.18 μg/mL. Macaca mulatta CXCL10 likely exhibits similar potency due to high sequence homology with human CXCL10, while mouse CXCL10 may show different potency profiles .

• Cross-reactivity: Human CXCL10 assays and detection methods may cross-react with Macaca mulatta CXCL10 but are less likely to detect mouse CXCL10. This is an important consideration when selecting reagents for multi-species studies.

• Molecular weight: Human CXCL10 appears as a 9-10 kDa protein in SDS-PAGE analysis, mouse CXCL10 (Crg-2) is approximately 8.7 kDa, and Macaca mulatta CXCL10 has similar molecular characteristics to human CXCL10 .

• In vivo half-life and distribution: Species-specific differences in protein stability and tissue distribution may affect experimental outcomes when comparing models across species.

What are the challenges and considerations for studying CXCL10 in SIV infection models using rhesus macaques?

Studying CXCL10 in SIV infection models using rhesus macaques presents several methodological challenges:

• Experimental design: When designing studies involving SIV infection, careful consideration must be given to baseline assessments, including screening for pulmonary arterial pressure (should be less than 25 mm Hg) and absence of pulmonary or cardiac abnormalities .

• Infection protocol: For SIV infection, protocols typically involve sedation with ketamine (10-15 mg/kg IM), intravenous catheterization, and administration of SIV ΔB670 (TCID50, 2.6 × 105; 1:100 dilution of viral stock). Some studies may involve additional mucosal exposure .

• Sample collection: Serial blood and bronchoalveolar lavage fluid (BALF) samples should be obtained throughout infection for assessment of viral load, CXCL10 levels, and flow cytometric analysis .

• Imaging considerations: Advanced imaging techniques such as PET-CT scans may be employed to evaluate disease progression. Protocols typically involve overnight fasting, sedation with ketamine, injection with appropriate tracers, and maintenance of anesthesia during scanning procedures .

• Longitudinal monitoring: CXCL10 expression changes dynamically throughout SIV infection, necessitating time-course studies with multiple sampling points to capture the complete picture of CXCL10 involvement in disease progression.

How can I design experiments to study CXCL10-mediated chemotaxis in rhesus macaque cell populations?

Designing robust experiments to study CXCL10-mediated chemotaxis in rhesus macaque cells requires careful methodological planning:

• Cell isolation and preparation:

  • Isolate peripheral blood mononuclear cells (PBMCs) from rhesus macaque blood using density gradient centrifugation

  • For specific subpopulations, employ magnetic or fluorescence-activated cell sorting

  • Activate T cells with appropriate stimuli (anti-CD3/CD28 or PHA/IL-2) to upregulate CXCR3 expression

• Chemotaxis assay optimization:

  • Use modified Boyden chambers (transwell systems with 5-8 μm pore size depending on cell type)

  • Establish a CXCL10 concentration gradient (0.001-1 μg/mL) based on effective doses observed in human studies (ED50 = 0.03-0.18 μg/mL)

  • Include appropriate positive controls (SDF-1/CXCL12 for general chemotaxis) and negative controls

  • Optimize incubation time (typically 2-4 hours for lymphocytes)

• Quantification methods:

  • Flow cytometry for precise enumeration of migrated cells and phenotypic analysis

  • Fluorescent labeling of cells prior to chemotaxis for simplified quantification

  • Image-based analysis for morphological assessment of migration

• Validation approaches:

  • Confirm specificity using neutralizing antibodies against CXCL10 or CXCR3

  • Employ CXCR3 antagonists to confirm receptor-mediated effects

  • Compare responses to other CXCR3 ligands (CXCL9, CXCL11) to determine ligand specificity

• Data analysis considerations:

  • Calculate chemotactic index (ratio of cells migrating toward chemokine versus random migration)

  • Determine dose-response relationships

  • Apply appropriate statistical analyses for between-group comparisons

What is the role of CXCL10 in rhesus macaque models of pulmonary arterial hypertension?

In rhesus macaque models of pulmonary arterial hypertension (PAH), CXCL10 plays several key roles that can be studied through specific methodological approaches:

• Expression pattern analysis: CXCL10 expression is altered in pulmonary tissues during PAH development. This can be assessed through immunohistochemistry of lung sections, qPCR of lung tissue samples, and ELISA of bronchoalveolar lavage fluid .

• Correlation with disease parameters: CXCL10 levels can be correlated with clinical parameters of PAH, including:

  • Pulmonary arterial pressure measurements

  • Right ventricular hypertrophy (RV:LV ratio)

  • PET-CT imaging findings, particularly standard uptake values (SUV) in the right and left ventricles

• Cell-specific production: Multiple cell types in the lung may produce CXCL10 during PAH development. Flow cytometric analysis of intracellular CXCL10 in cell suspensions from digested lung tissue can identify the primary cellular sources.

• Functional assessment: The contribution of CXCL10 to PAH pathogenesis can be studied through:

  • Administration of neutralizing antibodies against CXCL10

  • CXCR3 receptor antagonists

  • Evaluation of downstream effects on vascular remodeling and inflammatory cell recruitment

• Integration with SIV infection models: Since PAH can develop as a complication of SIV/HIV infection, combined models can provide insights into the role of CXCL10 in infection-associated PAH, requiring longitudinal monitoring over 10-12 months post-infection .

How can I overcome common technical issues when working with recombinant Macaca mulatta CXCL10?

Researchers working with recombinant Macaca mulatta CXCL10 frequently encounter technical challenges that can be addressed through these methodological solutions:

• Protein aggregation:

  • Add carrier protein (0.1% BSA or HSA) to reconstitution buffers

  • Filter solutions through 0.2 μm filters before use

  • Maintain protein concentrations below 100 μg/mL to minimize aggregation

  • Consider carrier-free formulations for applications where BSA might interfere

• Loss of biological activity:

  • Minimize freeze-thaw cycles by preparing single-use aliquots

  • Store reconstituted protein at -80°C rather than -20°C for long-term storage

  • Add protease inhibitors to protein solutions when working with complex biological samples

  • Validate activity periodically using functional assays (chemotaxis or receptor binding)

• Specificity issues in detection assays:

  • Validate antibodies specifically for Macaca mulatta CXCL10 recognition

  • Perform cross-reactivity tests when using human CXCL10 detection reagents

  • Include appropriate positive and negative controls in all assays

  • Consider generating Macaca mulatta-specific reagents for critical applications

• Variability in functional responses:

  • Standardize cell culture conditions, particularly for CXCR3-expressing cells

  • Normalize chemotaxis data to control for inter-experiment variability

  • Use internal standards when comparing results across different experimental batches

  • Standardize protocols for primary cell isolation from rhesus macaque samples

What are the latest methodological advances in studying CXCL10-CXCR3 interactions in non-human primate models?

Recent methodological advances have significantly enhanced our ability to study CXCL10-CXCR3 interactions in non-human primate models:

• Real-time imaging technologies:

  • Intravital microscopy allows visualization of CXCL10-mediated cell recruitment in living tissues

  • Fluorescent protein tagging of CXCL10 and CXCR3 enables tracking of ligand-receptor interactions

  • Bioluminescence resonance energy transfer (BRET) assays provide insights into receptor conformational changes following CXCL10 binding

• Single-cell analysis approaches:

  • Single-cell RNA sequencing reveals heterogeneity in CXCR3 expression and response to CXCL10

  • Mass cytometry (CyTOF) enables comprehensive phenotyping of CXCL10-responsive cells

  • Phospho-flow cytometry detects CXCL10-induced signaling events at the single-cell level

• Receptor signaling analysis:

  • CRISPR-Cas9 gene editing allows precise modification of CXCR3 in primary macaque cells

  • Biosensor technologies detect real-time changes in second messengers following CXCL10 stimulation

  • Proteomic approaches identify novel components of CXCL10-initiated signaling pathways

• Systems biology integration:

  • Computational modeling of CXCL10 gradient formation and cellular responses

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics data

  • Network analysis of CXCL10-regulated genes in disease states

• Translation to therapeutic applications:

  • Development of macaque-specific CXCL10 antagonists

  • Validation of humanized antibodies against CXCL10 in non-human primate models

  • Evaluation of CXCR3 modulators for therapeutic potential in inflammatory conditions

How does CXCL10 interact with other chemokines in rhesus macaque immune responses?

CXCL10 functions within a complex chemokine network in rhesus macaque immune responses, with several important interactions that can be methodologically investigated:

• Co-expression analysis:

  • Multiplex cytokine/chemokine assays can quantify CXCL10 alongside other chemokines in biological samples

  • Transcriptomic profiling reveals coordinated expression patterns of CXCL10 with other chemokines

  • Single-cell RNA sequencing identifies cell populations co-expressing multiple chemokines including CXCL10

• Receptor competition studies:

  • CXCL9 and CXCL11 compete with CXCL10 for binding to CXCR3, requiring careful consideration in experimental design

  • Differential effects of these chemokines can be studied using receptor competition assays

  • Dose-response relationships should be established for each chemokine individually and in combination

• Functional synergism and antagonism:

  • Chemotaxis assays with combinations of CXCL10 and other chemokines reveal synergistic or antagonistic effects

  • Calcium flux measurements in response to sequential chemokine stimulation demonstrate receptor desensitization

  • Signaling studies identify shared and distinct pathways activated by different chemokines

• In vivo trafficking studies:

  • Adoptive transfer of labeled cells allows tracking of migration in response to endogenous or administered chemokines

  • Chemokine receptor antagonist studies reveal the relative contribution of CXCL10 versus other chemokines

  • Tissue-specific chemokine gradients can be mapped to understand the spatial organization of chemokine networks

How is CXCL10 utilized in rhesus macaque models of HIV/SIV infection?

CXCL10 plays a critical role in rhesus macaque models of HIV/SIV infection, offering several methodological applications:

• Biomarker of disease progression:

  • CXCL10 levels in plasma correlate with viral load and CD4+ T cell decline

  • Longitudinal monitoring of CXCL10 provides insights into disease trajectory

  • Tissue-specific CXCL10 expression identifies sites of virus replication and inflammation

• Cell migration studies:

  • CXCL10-mediated chemotaxis of CXCR3+ T cells can be impaired during HIV-1 infection, which can be rescued by modulating actin polymerization

  • This phenomenon can be studied in rhesus macaque models using ex vivo migration assays

  • Comparative analysis between infected and uninfected animals reveals mechanisms of immune dysfunction

• Therapeutic target evaluation:

  • Neutralization of CXCL10 or blockade of CXCR3 in SIV-infected macaques can modulate immune cell trafficking

  • These interventions can be assessed for effects on viral control and inflammation

  • Combination approaches targeting multiple chemokines provide insights into redundant pathways

• Vaccine development applications:

  • CXCL10 levels can serve as correlates of vaccine-induced inflammation

  • Fc receptor-mediated phagocytosis, which relates to CXCL10 pathways, is relevant for preventive and therapeutic HIV vaccine strategies

  • CXCL10 expression patterns help identify sites of vaccine-induced immune activation

• Antiretroviral therapy response:

  • Changes in CXCL10 levels following antiretroviral treatment initiation predict immunological recovery

  • Persistent CXCL10 elevation despite viral suppression indicates ongoing inflammation

  • Integration with other inflammatory markers provides a comprehensive assessment of immune reconstitution

What are the implications of CXCL10 research in rhesus macaques for human immunological disorders?

Research on CXCL10 in rhesus macaques has significant translational implications for human immunological disorders:

• Inflammatory disease models:

  • Rhesus macaque CXCL10 studies provide insights into human inflammatory conditions

  • The high homology between human and macaque CXCL10 supports direct translational applications

  • Disease-specific alterations in CXCL10 expression identified in macaques frequently parallel human pathologies

• Cancer immunotherapy:

  • CXCL10's role in T cell recruitment is relevant for cancer immunotherapy approaches

  • Studies in rhesus macaques can inform human clinical trials targeting the CXCL10-CXCR3 axis

  • Understanding resistance mechanisms to kinase inhibitors in cancer can be enhanced through CXCL10-related research

• Autoimmune disease insights:

  • Experimental autoimmune models in rhesus macaques reveal CXCL10's contribution to pathogenesis

  • Therapeutic targeting of CXCL10 in these models provides proof-of-concept for human applications

  • Biomarker applications of CXCL10 for disease activity assessment translate directly to human care

• Vaccine development:

  • CXCL10 responses to vaccination in rhesus macaques predict human responses

  • Optimization of adjuvants based on CXCL10 induction profiles improves vaccine efficacy

  • Understanding geographical and demographic variations in CXCL10 responses informs personalized vaccination strategies

• Drug development pipeline:

  • Rhesus macaque studies serve as a critical preclinical step for CXCL10-targeting therapeutics

  • Pharmacokinetic and pharmacodynamic properties of these agents in macaques inform human dosing

  • Safety profiles established in macaques accelerate translation to human clinical trials

What emerging technologies are advancing Macaca mulatta CXCL10 research?

The field of Macaca mulatta CXCL10 research is being transformed by several cutting-edge technologies:

• CRISPR-based approaches:

  • Precise genome editing allows creation of CXCL10 or CXCR3 knockouts in macaque cells

  • Introduction of reporter systems at endogenous loci enables real-time monitoring of CXCL10 expression

  • Base editing technologies permit study of specific CXCL10 variants with altered functions

• Advanced imaging techniques:

  • Multiphoton intravital microscopy visualizes CXCL10-mediated immune cell dynamics in living tissues

  • Positron emission tomography with CXCR3-specific tracers maps receptor distribution in vivo

  • Super-resolution microscopy reveals nanoscale organization of CXCL10-CXCR3 interactions

• Organoid and tissue-on-chip platforms:

  • Macaque-derived organoids provide physiologically relevant systems for CXCL10 studies

  • Microfluidic devices model CXCL10 gradient formation and cellular responses

  • Co-culture systems recreate complex tissue environments for studying CXCL10 functions

• Single-cell multi-omics:

  • Integrated analysis of transcriptome, proteome, and epigenome at single-cell resolution

  • Spatial transcriptomics maps CXCL10 expression in tissue contexts with cellular resolution

  • Trajectory analysis identifies dynamic changes in CXCL10 responses during disease progression

• Computational and AI approaches:

  • Machine learning algorithms predict CXCL10 responses based on genetic and environmental factors

  • Systems biology modeling integrates CXCL10 into broader immune networks

  • Virtual screening identifies novel modulators of CXCL10-CXCR3 interactions

How can I design longitudinal studies to investigate CXCL10 dynamics in rhesus macaque disease models?

Designing robust longitudinal studies to investigate CXCL10 dynamics in rhesus macaque disease models requires careful methodological planning:

• Study design considerations:

  • Power analysis to determine appropriate sample sizes (typically 8-12 animals per group for adequate statistical power)

  • Inclusion of age-matched and sex-balanced control groups

  • Establishment of baseline measurements for each animal as its own control

  • Comprehensive health screening before study initiation, including pulmonary arterial pressure assessment

• Sampling strategy:

  • Determination of optimal sampling intervals based on disease progression (e.g., more frequent during acute phases)

  • Collection of multiple sample types (blood, BAL, lymph node biopsies, etc.) at each timepoint

  • Minimally invasive sampling techniques to reduce stress effects on immune parameters

  • Standardized collection protocols to minimize technical variability

• CXCL10 measurement approaches:

  • Selection of consistent assay platforms throughout the study duration

  • Inclusion of internal standards across batches to normalize for assay drift

  • Storage of additional sample aliquots for future analyses with emerging technologies

  • Parallel measurement of related chemokines and cytokines for network analysis

• Integrated data collection:

  • Correlation of CXCL10 measurements with clinical parameters (e.g., viral load, CD4 counts, imaging findings)

  • Documentation of treatments and interventions that might affect CXCL10 levels

  • Environmental factors and housing conditions that could influence immune parameters

  • Behavioral and physiological stress indicators that might modulate CXCL10 expression

• Advanced analytical approaches:

  • Mixed-effects modeling to account for individual variability and repeated measures

  • Trajectory analysis to identify patterns of CXCL10 dynamics associated with disease outcomes

  • Machine learning algorithms to integrate multiple parameters for predictive modeling

  • Systems biology approaches to place CXCL10 dynamics in broader immunological context