@inproceedings{01JW8HW88NNNGY7ZE705EDRMZY,
  abstract     = {{Isolation and phenotyping of pro- versus anti-
inflammatory equine macrophages for chronic tendinopathy
research
Marguerite Meeremans1 2, Sarah Bairiot1, Sandra Van Vlierberghe2,
Mario Van Poucke3, Luc Peelman3, Kristel Demeyere4,
Evelyne Meyer4, Bert Devriendt5, Catharina De Schauwer1
1Veterinary Stem Cell Research Unit, Department of Translational
Physiology, Infectiology and Public Health, Faculty of Veterinary
Medicine, Ghent, Belgium; 2Polymer Chemistry and Biomaterials
Group, Centre of Macromolecular Chemistry, Faculty of Sciences,
Ghent, Belgium; 3Laboratory of Animal Genetics, Department of
Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent,
Belgium; 4Laboratory of Biochemistry, Department of Veterinary
and Biosciences, Faculty of Veterinary Medicine, Ghent, Belgium;
5Laboratory of Immunology, Department of Translational
Physiology, Infectiology and Public Health, Faculty of Veterinary
Medicine, Ghent, Belgium

Introduction
Inflammation and immune cells play important roles in tissue
homeostasis and regeneration. In chronic degenerative tendino-
pathies, the balance between pro- and anti-inflammatory macro-
phages is a crucial parameter determining tendon repair
outcomes. When disturbed, fibrosis occurs impairing tendon
structure and elasticity. In both human and equine athletes ten-
don injuries are a major cause of poor performance and impaired
quality of life. In contrast to human, however, a robust protocol
to isolate equine pro- and anti-inflammatory macrophages is
lacking. Additionally, the interaction between macrophages and
tendon cells was explored to improve our understanding of the
role of macrophages in tendon repair and regeneration.
Methods
Equine peripheral blood-derived monocytes (PBMCs) were isolated
using gradient density separation (Lymphoprep� 1.077 g/mL) and
magnetic-activated cell sorting based on CD172a expression. After
4 days of culture, cells were cultured another 4 days in medium sup-
plemented with either IFN-c/LPS or IL-4 to induce pro- and anti-
inflammatory macrophage polarization, respectively. Polarization
was evaluated by assessing mRNA expression (pro: TNFA, CXCL8,
IL6, CD80, CD86, and IL1B; anti: CD206, ARG2, VEGFA, IL10,
and TGFB1). Furthermore, CD86 and CD206 protein expression
was evaluated with flow cytometry, while VEGFa protein expres-
sion was assessed by immunofluorescent staining, complemented
with a NO and an arginase activity assay. Finally, a Luminex multi-
plex cytokine assay was performed to measure the secreted cyto-
kines (TNFa, CXCL8, IL-6, IL-1b, IL-10 and TGFb1) within the
culture medium upon macrophage polarization (= conditioned
medium, CM). As proof of concept, CM was supplemented to
equine tendon cells after which their proliferation (MTT assay) was
evaluated.
Results
On average 2.6x106 monocytes/mL were isolated from venous
blood samples with a purity of 87–5% post-sorting. PBMCs treated
with IFN-c/LPS showed an enlarged cell shape with a round cell
body and numerous cytoplasmic extensions in contrast to IL-4
treated PBMCs, that demonstrated both spindle-shaped and multi-
nucleated giant cells. Increased TNFA, CXCL8, CD80 and IL1B
mRNA expression was observed after treatment with IFN-c/LPS,
while IL-4 treatment increased CD206, VEGFA and TGFB1 tran-
script levels. Flow cytometry and immunofluorescence con-
firmed this polarization. No differences in NO production or
arginase activity were observed. Cytokines upregulated in the
CM of pro-inflammatory macrophages included TNFa, CXCL8
and IL-1b, while TGFb1 was increased in anti-inflammatory
macrophages. Next, varying concentrations of the CM were
applied to equine tendon cells for 24 or 72 hours and compared
to control media, containing the same supplements but not
exposed to macrophages. Applying 50% CM (both pro- and anti-
inflammatory) for 72 hours increased cell proliferation com-
pared to control media.
Conclusions
An efficient isolation protocol to generate equine macrophages is
described, followed by the in-depth characterization of their
induced pro- versus anti-inflammatory phenotypes. As the horse
is the most relevant preclinical orthopaedic animal model, this
research holds both a strong species-specific as well as transla-
tional value. Co-culturing these cells with tendon cells will pro-
vide novel insights into chronic degenerative pathologies and
facilitate the development of regenerative therapies in both horse
and man.

Topic: Submit to SYMPOSIUM
Subtopic: Veterinary TERM – results from preclinical science, clinical
studies and patient treatments to facilitate the “One health” concept
Type: Oral presentation}},
  author       = {{Meeremans, Marguerite and Bairiot, Sarah and Van Vlierberghe, Sandra and Van Poucke, Mario and Peelman, Luc and Demeyere, Kristel and Meyer, Evelyne and Devriendt, Bert and De Schauwer, Catharina}},
  booktitle    = {{TERMIS EU 2025, Abstracts}},
  language     = {{eng}},
  location     = {{Freiburg, Germany}},
  title        = {{Isolation and phenotyping of pro- versus anti-inflammatory equine macrophages for chronic tendinopathy research}},
  url          = {{http://doi.org/10.1089/ten.tea.2025.90912.abstracts.partb}},
  year         = {{2025}},
}

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