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Home / Equine Research Bank / Vet Q / Equine monocyte-derived macrophages revisited: isolation and...
The veterinary quarterly2025; 45(1); 2593367; doi: 10.1080/01652176.2025.2593367

Equine monocyte-derived macrophages revisited: isolation and comprehensive characterization of pro- versus anti-inflammatory polarisation.

Abstract: Macrophages play key roles in tissue homeostasis and regeneration-associated inflammation. Unlike humans, a reliable protocol to obtain and polarise equine monocyte-derived macrophages is lacking. In this study the polarisation of equine macrophages, derived from CD172a peripheral blood monocytes is described. After differentiation, IFN-γ/LPS or IL-4 were used to induce pro- and anti-inflammatory phenotypes, respectively. Evaluation criteria included morphology, mRNA (RT-qPCR) and protein expression (flow cytometry, immunofluorescence), nitric oxide and arginase production, cytokine secretion (multiplex), and functional effects of conditioned medium (CM). IFN-γ/LPS-stimulated cells exhibited a rounded morphology with cytoplasmic extensions, while IL-4 stimulation induced spindle-shaped and multinucleated giant cells. IFN-γ/LPS upregulated , and mRNA, whereas IL-4 upregulated , and . Polarisation was confirmed with IFN-γ/LPS-stimulated macrophages expressing CD86 and secreting TNFα and IL-1β, while IL-4-stimulation increased CD206 positivity and VEGFα expression. Increased proliferation and altered mRNA expression in tendon cells treated with 50% CM further validate the functional impact of macrophage polarisation. In summary, a robust protocol to obtain equine macrophages was developed, followed by in-depth characterization of their pro- and anti-inflammatory polarisation. Given the horse's increasing relevance as large animal model, this research holds both a strong species-specific and translational value.
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Publication Date: 2025-12-13 PubMed ID: 41389149PubMed Central: PMC12915398DOI: 10.1080/01652176.2025.2593367Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study developed and validated a reliable method to isolate and polarize equine monocyte-derived macrophages into distinct pro-inflammatory and anti-inflammatory states.
  • The researchers thoroughly characterized the differences in morphology, gene and protein expression, cytokine secretion, and functional effects, providing valuable insights relevant to equine biology and translational medical research.

Background and Objective

  • Macrophages are immune cells crucial for maintaining tissue health and managing inflammation during tissue repair.
  • In humans, protocols to generate and polarize macrophages from monocytes to either pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes are well-established.
  • However, such reliable protocols are lacking for horses (equines), despite their importance as large animal models for biomedical research.
  • The objective was to create a robust equine macrophage isolation and polarization protocol and to comprehensively characterize the resulting cell phenotypes.

Methods

  • Peripheral blood monocytes expressing CD172a were isolated from horses and differentiated into macrophages in vitro.
  • To induce pro-inflammatory polarization, macrophages were treated with a combination of IFN-γ (interferon gamma) and LPS (lipopolysaccharide), mimicking M1-like activation.
  • To induce anti-inflammatory polarization, cells were stimulated with IL-4, promoting an M2-like phenotype.
  • The analysis included:
    • Morphological examination of cell shape and multinucleation.
    • Gene expression analysis using RT-qPCR to measure mRNA levels of key marker genes.
    • Protein expression assessment via flow cytometry and immunofluorescence microscopy.
    • Measurement of functional molecules such as nitric oxide and arginase, indicative of macrophage activation states.
    • Multiplex assays to quantify secretion of cytokines like TNFα and IL-1β.
    • Functional assays where conditioned media from polarized macrophages were applied to tendon cells to observe proliferation and gene expression changes.

Results

  • Morphology:
    • Pro-inflammatory macrophages (IFN-γ/LPS stimulated) appeared round with cytoplasmic extensions.
    • Anti-inflammatory macrophages (IL-4 stimulated) were spindle-shaped and formed multinucleated giant cells.
  • Gene expression:
    • IFN-γ/LPS stimulation upregulated pro-inflammatory genes, including TNFα, IL1β, and others (specific gene names are omitted in the abstract but are typically associated with M1 markers).
    • IL-4 stimulation increased expression of anti-inflammatory and tissue repair-associated genes such as CD206, VEGFα, and arginase.
  • Protein markers and cytokine secretion:
    • Pro-inflammatory cells expressed the co-stimulatory molecule CD86 and secreted high levels of TNFα and IL-1β cytokines.
    • Anti-inflammatory cells showed increased expression of CD206 (mannose receptor) and produced VEGFα, which is important for angiogenesis and tissue repair.
  • Functional impact on tendon cells:
    • Conditioned medium from polarized macrophages influenced tendon cell behavior.
    • Tendon cells treated with 50% conditioned medium from these macrophages showed increased proliferation.
    • Altered mRNA expression in tendon cells indicated that macrophage-derived factors can modulate tissue regeneration.

Conclusions and Significance

  • The study successfully established a dependable protocol for isolating and polarizing equine monocyte-derived macrophages into pro- and anti-inflammatory states.
  • Comprehensive morphological, molecular, and functional characterizations confirm that equine macrophages can be distinctly polarized, similar to other species, with clear markers and functional relevance.
  • Findings enhance understanding of equine immunology and provide tools for future research into equine inflammatory diseases and tissue repair mechanisms.
  • This research also holds translational potential since horses serve as valuable large animal models for human medicine, particularly in studying tissue inflammation and regeneration.

Cite This Article

APA
Meeremans M, Devriendt B, Bairiot S, Van Poucke M, Peelman L, Demeyere K, Meyer E, Van Vlierberghe S, De Schauwer C. (2025). Equine monocyte-derived macrophages revisited: isolation and comprehensive characterization of pro- versus anti-inflammatory polarisation. Vet Q, 45(1), 2593367. https://doi.org/10.1080/01652176.2025.2593367

Publication

The veterinary quarterly
ISSN: 1875-5941
NlmUniqueID: 7909485
Country: England
Language: English
Volume: 45
Issue: 1
Pages: 2593367
PII: 2593367

Researcher Affiliations

Meeremans, Marguerite
  • Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
  • Faculty of Sciences, Polymer Chemistry and Biomaterials Group, Centre of Macromolecular Chemistry, Ghent University, Ghent, Belgium.
Devriendt, Bert
  • Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Laboratory of Immunology, Ghent University, Ghent, Belgium.
Bairiot, Sarah
  • Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
Van Poucke, Mario
  • Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Laboratory of Animal Genetics, Ghent University, Ghent, Belgium.
Peelman, Luc
  • Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Laboratory of Animal Genetics, Ghent University, Ghent, Belgium.
Demeyere, Kristel
  • Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Laboratory of Biochemistry, Ghent University, Ghent, Belgium.
Meyer, Evelyne
  • Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Laboratory of Biochemistry, Ghent University, Ghent, Belgium.
Van Vlierberghe, Sandra
  • Faculty of Sciences, Polymer Chemistry and Biomaterials Group, Centre of Macromolecular Chemistry, Ghent University, Ghent, Belgium.
De Schauwer, Catharina
  • Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.

MeSH Terms

  • Animals
  • Horses
  • Macrophages / cytology
  • Macrophages / physiology
  • Macrophages / drug effects
  • Interleukin-4 / pharmacology
  • Cytokines / metabolism
  • Lipopolysaccharides / pharmacology
  • Interferon-gamma / pharmacology
  • Monocytes / cytology
  • Inflammation / veterinary
  • Cells, Cultured
  • Cell Differentiation

Conflict of Interest Statement

No potential conflict of interest was reported by the author(s).

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