Recombinant Macaca mulatta Interleukin-6 protein (IL6)

Basic Research Questions

• What are the basic characteristics of recombinant Macaca mulatta IL6 protein?

  Recombinant Macaca mulatta IL6 is a single non-glycosylated polypeptide chain containing 186 amino acids, with a molecular weight of approximately 21.1 kDa as determined by SDS-PAGE and HPLC analyses . The protein's amino acid sequence is: MAPVLPGEDSKNVAAPHSQP LTSSERIDKH IRYILDGISA LRKETCNRSN MCESSKEALA ENNLNLPKMA EKDGCFQSGFNEDTCLVKII TGLLEFEVYL EYLQNRFESS EEQARAVQMS TKVLIQFLQK KAKNLDAITT PEPTTNASLLTKLQAQNQWL QDMTTHLILR SFKEFLQSNL RALRQM . It is typically produced in E. coli expression systems with >97% purity and endotoxin levels <0.1 ng/μg of protein .

• How should recombinant Macaca mulatta IL6 be reconstituted and stored?

  For optimal stability and activity:

  • Centrifuge the vial prior to opening to bring contents to the bottom

  • Reconstitute in sterile distilled water or appropriate aqueous buffer to a concentration of 0.1-1.0 mg/ml

  • For short-term storage (several weeks), the reconstituted protein is stable at 2-4°C

  • For long-term storage, divide into working aliquots and store at -80°C

  • Avoid repeated freeze-thaw cycles as they can compromise protein integrity

• What is the biological activity of recombinant Macaca mulatta IL6?

  The biological activity is typically measured through dose-dependent stimulation of IL-6-dependent cell proliferation. The ED50 (effective dose for 50% maximal response) for recombinant Macaca mulatta IL6 is typically <0.1 ng/ml when tested on mouse 7TD1 cells, corresponding to a specific activity of >1×10^7 units/mg . This can be compared to recombinant human IL-6, which demonstrates an ED50 of 0.2-0.8 ng/mL in the T1165.85.2.1 mouse plasmacytoma cell line proliferation assay .

Experimental Design Questions

• How does rhesus macaque IL6 compare with human IL6 in experimental settings?

  While both proteins share significant homology, there are important functional and experimental considerations:

  Parameter	Macaca mulatta IL6	Human IL6
  Amino acid sequence homology	Reference	High (>95%)
  Molecular weight	21.1 kDa	23.1 kDa (with PTMs)
  Cross-reactivity	Reacts with anti-human IL6 antibodies	Reacts with rhesus cells
  In vivo potency	Native in NHP models	Lower activity in macaque models
  Detection in flow cytometry	Detected by human IL6 antibodies like clone MQ2-6A3	Standard

  The MQ2-6A3 antibody generated against recombinant human IL-6 cross-reacts with an intracellular component of peripheral blood LPS-stimulated permeabilized monocytes of rhesus and cynomolgus macaques, though with weaker reactivity compared to human monocytes .

• What in vivo experimental designs have proven effective when studying IL6 function in rhesus macaques?

  Based on published studies, effective experimental designs include:

  • Subcutaneous administration at 3-30 μg/kg body weight/day for 11 days to study acute phase responses

  • Sequential administration protocols where IL6 is followed by other cytokines such as GM-CSF (at 5.5 μg/kg/day)

  • Blood sampling at regular intervals (typically days 0, 2, 4, 7, 11, and 14) to track changes in inflammatory markers

  • Monitoring of acute phase proteins (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) and platelet counts as primary endpoints

  These experimental parameters have demonstrated that recombinant IL6 induces dose-dependent acute phase protein responses and increases platelet counts by up to twofold above baseline levels after 2-3 days following treatment cessation .

• How can I effectively measure IL6-induced inflammation in rhesus macaque models?

  Multiple complementary approaches provide robust assessment:

  • Serum biomarkers: Measure C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin (positive acute phase proteins) and prealbumin (negative acute phase protein)

  • Hematological parameters: Monitor platelet counts, white blood cell counts, and myeloid progenitor levels

  • Cytokine profiling: Assess IL6 alongside TNF-α, IL-1RA, IL-5, and IL-8 to characterize inflammatory state

  • Flow cytometry: Analyze immune cell populations, particularly monocytes and T-cells

  • Gene expression analysis: Evaluate inflammation-associated genes, especially those in the JAK-STAT pathway

  In aging studies, baseline IL6 levels have shown significant elevation in older versus younger animals (P = 0.03), consistent with the "inflammaging" phenotype characterized by persistent elevation of pro-inflammatory cytokine levels .

Advanced Research Questions

• How do IL6 signaling pathways differ between human and Macaca mulatta models in inflammatory conditions?

  Analysis of transcriptomic data reveals important differences:

  • Both species utilize the JAK-STAT pathway for IL6 signaling, but with distinct regulatory patterns

  • In rhesus macaques (Macaca mulatta), differential expression of SOCS3 indicates upregulation of the JAK-STAT pathway but potentially impaired functionality compared to human models

  • Cross-regulation with both IL10 and IL6 suggests duplicated signaling efforts in rhesus models

  • In infection models, older rhesus macaques show sustained pro-inflammatory gene expression in immune cells at 7 days post-infection, while younger animals demonstrate better control of inflammation

  These differences are critical when translating findings between species, particularly for therapeutic interventions targeting the IL6 pathway.

• How does maternal IL6 affect offspring neurodevelopment in Macaca mulatta models?

  Longitudinal studies on Japanese macaques have provided valuable insights applicable to rhesus macaque research:

  • Elevated maternal third-trimester plasma IL6 levels associate with:

    • Smaller left amygdala volume at 4 months of age

    • More rapid amygdala growth from 4 to 36 months

    • Increased anxiety-like behavior at 11 months, mediated by reduced amygdala volumes at 4 months

  These findings establish a causal pathway where maternal inflammation affects early brain development, which subsequently influences behavioral outcomes. This relationship provides a valuable model for understanding neurodevelopmental disorders in humans .

• What statistical approaches are most appropriate for analyzing IL6 polymorphism data in relation to disease risk?

  Meta-analyses of IL6 gene polymorphisms have employed several sophisticated statistical methods:

  • Bayesian approach with Markov chain Monte Carlo simulations: Allows for multivariate linear mixed models that account for multiple variables simultaneously

  • Mahalanobis distance computation: Identifies multivariate outliers to improve data quality

  • Multivariate models with multiple-response variables: Enable analysis of complex immune parameters while controlling for confounding factors

  These approaches have proven valuable in resolving contradictory findings from different studies. For example, meta-analysis of IL6 gene SNPs (rs1800795, rs1800796, rs1800797) across 118 GWAS studies with over 115,000 samples revealed significant associations with cancer risk (p-value < 0.05) that varied by cancer type and ethnicity .

• How does social integration affect IL6 expression and immune function in free-ranging rhesus macaques?

  Research on free-ranging rhesus macaques on Cayo Santiago Island has revealed:

  • Individuals with more grooming partners (higher social integration) had lower white blood cell counts than socially isolated counterparts, indicating lower inflammation levels

  • Social status and integration were not significantly associated with anatomical markers of immunity (spleen and liver size)

  • Controlling for body mass, age, sex, and behavioral group is essential when analyzing these relationships

  • Multivariate models incorporating eigenvector centrality metrics provide the most robust assessment of social integration effects on immune parameters

  This research extends findings from captive animals and WEIRD human societies to naturalistic settings where individuals interact without medical intervention.

Technical Methodology Questions

• What are the optimal methods for detecting Macaca mulatta IL6 in biological samples?

  Multiple detection methods offer complementary advantages:

  Method	Sensitivity	Sample Type	Special Considerations
  ELISA	~10 pg/ml	Serum, plasma, cell culture supernatant	Cross-reactivity with human antibodies
  Flow cytometry	Cell-level	Whole blood, PBMCs	Requires permeabilization for intracellular detection
  Gene expression (qPCR)	Very high	Any tissue with RNA extraction	Reflects transcription, not protein levels
  Single-cell transcriptomics	Cell-type specific	BALF, whole blood	Provides cell-specific expression profiles

  For flow cytometry, the MQ2-6A3 antibody clone has demonstrated cross-reactivity with LPS-stimulated permeabilized monocytes from rhesus macaques, though with weaker reactivity than observed with human samples .

• How should researchers account for age-related differences in baseline IL6 levels when designing rhesus macaque studies?

  Based on age-stratified cohort studies:

  • Older animals (mean age 18 years) demonstrate significantly higher baseline serum IL6 concentrations compared to younger animals (mean age 3.4 years) (P = 0.03)

  • Age-specific divergence in immune responses becomes more pronounced following immune challenges

  • Recommended approaches include:

    • Age-matching experimental groups

    • Including age as a covariate in statistical analyses

    • Establishing age-specific normal ranges for baseline cytokine levels

    • Employing longitudinal sampling strategies to account for age-related progression

  These considerations are particularly important for studies of inflammation, infectious disease, and vaccine development where age can significantly influence outcomes.

• What considerations should be made when selecting rhesus macaque subspecies for IL6-related research?

  Population genomics of wild Chinese rhesus macaques has identified important subspecies differences:

  • Five genetic lineages exist on the mainland and Hainan Island that recapitulate current subspecies designations

  • These subspecies diverged 125.8 to 51.3 thousand years ago but feature recent gene flow

  • Significant adaptations exist between northern (M. m. tcheliensis) and southern (M. m. brevicaudus) subspecies

  • 118 single-nucleotide polymorphisms match human disease-causing variants with 82 being subspecies-specific

  Researchers should consider these genetic differences when selecting specific subspecies for translational studies, as they may influence IL6 expression patterns and responses to experimental interventions .

• How does the JAK-STAT signaling pathway differ in rhesus macaques compared to humans, particularly in relation to IL6 signaling?

  Comparative studies reveal:

  • The JAK-STAT pathway is central to IL6 signaling in both species

  • In rhesus macaques, differential expression of SOCS3 indicates potential differences in pathway regulation

  • Gene enrichment studies show that in response to immune challenges, M. mulatta upregulates the JAK-STAT pathway along with cross-regulation with both IL10 and IL6

  • This suggests potentially duplicated signaling efforts that might affect experimental outcomes

  • The "two-fold regulation" observed in macaques could indicate impaired functionality compared to human models

  These differences should be considered when interpreting results from rhesus macaque models, particularly when testing therapeutics targeting this pathway.

• What is the role of recombinant IL6 in studying the relationship between sociality and immune function in rhesus macaques?

  Research utilizing free-ranging rhesus macaques has demonstrated:

  • Social integration correlates with lower inflammation markers, including white blood cell counts

  • Experimental studies can use recombinant IL6 to:

    • Establish baseline cytokine responsiveness in animals of different social ranks

    • Challenge the immune system and measure differences in resolution between socially integrated versus isolated individuals

    • Correlate IL6-induced responses with natural variations in social behavior

  These approaches allow researchers to investigate how social environments modulate immune function, with implications for understanding health disparities in human populations based on social integration .