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Frontiers in veterinary science2025; 12; 1685757; doi: 10.3389/fvets.2025.1685757

Equine adipose-derived stem cells modulate in vitro neutrophil extracellular trap release by polymorphonuclear neutrophils.

Abstract: Neutrophil extracellular trap (NET) are thin and long web-like structures composed of DNA and antimicrobial proteins released by activated polymorphonuclear neutrophils (PMN) as part of the innate immune response. Adipose-derived stem cells (ADSCs) represent an accessible, abundant and minimal invasive source of mesenchymal stem cells (MSCs), with high regenerative potential, immunomodulatory and anti-inflammatory properties. Although recognized immunomodulatory properties of ADSCs, their interaction with PMN and their role on NET formation remains poorly characterized. The present study aimed to evaluate the effects of equine ADSCs on NET formation by equine PMN. Equine ADSCs were isolated from two different sources of adipose tissue, subcutaneous and retroperitoneal adipose stores. Equine PMN were isolated from peripheral blood with a discontinuous density gradient and stimulated with phorbol 12-myristate 13-acetate (PMA) to induce NET release as positive control. Scanning electron microscopy (SEM) and immunofluorescence microscopy (IFM) analyses were performed to assess NET release by equine PMN co-cultured with ADSCs. IFM-NET quantification revealed a significant NET decrease for PMN co-cultured with ADSCs and PMA. Furthermore, extracellular DNA quantification showed that inhibition of equine NET is dependent on the ADSCs to PMN ratio, for PMA and ionomycin stimulated PMN. Moreover, our findings unveil no modulation of reactive oxygen species (ROS) production by equine PMN when co-cultured with ADSCs. In summary, our results provide evidence of ADSCs on equine PMN, particularly in their capacity to attenuate NET formation and release. These results support the potential role of ADSCs on host innate immune response and thereby maintaining immune homeostasis. Further investigation is needed to better understand the specific molecular pathways involved in NETosis via ADSCs.
Copyright © 2025 Salinas-Varas, Espinosa, Muñoz-Caro, Conejeros, Gärtner, Fey, Arnhold, Taubert and Hermosilla.
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Publication Date: 2025-10-22 PubMed ID: 41200546PubMed Central: PMC12586003DOI: 10.3389/fvets.2025.1685757Google Scholar: Lookup
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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 investigated how stem cells derived from horse fat tissue (adipose-derived stem cells or ADSCs) influence the formation of neutrophil extracellular traps (NETs) released by immune cells called polymorphonuclear neutrophils (PMNs).
  • The research found that these stem cells can reduce NET formation, suggesting a role in modulating innate immune responses and inflammation in horses.

Background

  • Neutrophil Extracellular Traps (NETs): NETs are web-like DNA structures mixed with antimicrobial proteins that PMNs release when activated, helping to trap and neutralize pathogens as part of the innate immune system.
  • Adipose-Derived Stem Cells (ADSCs): These are mesenchymal stem cells sourced from fat tissue, known for their regenerative abilities, immunomodulatory effects, and anti-inflammatory properties.
  • Despite known immune effects of ADSCs, their interaction with PMNs and impact on NET formation had been unclear before this study.

Research Objectives

  • To evaluate the effects of equine ADSCs on NET formation by equine PMNs in vitro.
  • To compare ADSCs derived from two fat tissue sources: subcutaneous and retroperitoneal adipose tissue.
  • To determine whether the presence of ADSCs affects reactive oxygen species (ROS) production by PMNs, which are involved in NET formation.

Methods

  • Isolation of Cells: ADSCs were isolated from two types of horse fat tissue; PMNs were isolated from peripheral horse blood using density gradient separation.
  • NET Induction: PMNs were stimulated using phorbol 12-myristate 13-acetate (PMA) and ionomycin to induce NET formation.
  • Co-Culture Experiments: PMNs were co-cultured with ADSCs to observe effects on NET release.
  • Visualization Techniques: Scanning electron microscopy (SEM) and immunofluorescence microscopy (IFM) were used to visualize and quantify NET release.
  • Extracellular DNA Quantification: Used as a measure of NET abundance, with analyses performed at different ADSC to PMN ratios.
  • ROS Measurement: Investigated to see if ADSCs impacted PMN oxidative burst, an important step in NETosis.

Key Findings

  • When equine PMNs were co-cultured with ADSCs and stimulated with PMA, the amount of NETs released was significantly reduced, indicating that ADSCs suppress NET formation.
  • The reduction in NET release was dependent on the ratio of ADSCs to PMNs, showing that higher numbers of ADSCs more effectively inhibited NET production.
  • This inhibitory effect was observed with both PMA- and ionomycin-stimulated PMNs.
  • No significant change was detected in the production of reactive oxygen species (ROS) by PMNs in the presence of ADSCs, suggesting ADSCs reduce NET release through mechanisms other than affecting oxidative burst.

Conclusions and Implications

  • Equine ADSCs have the ability to attenuate NET formation by PMNs, indicating they can modulate innate immune responses and potentially reduce excessive inflammation.
  • This immunomodulatory interaction highlights the therapeutic potential of ADSCs in managing immune system-related conditions in horses, especially where NETs may contribute to inflammation or tissue damage.
  • Understanding the molecular pathways by which ADSCs inhibit NETosis could reveal novel targets for controlling immune responses and improving regenerative therapies.
  • Further research is needed to dissect the exact biochemical and molecular mechanisms involved in ADSC-mediated NET regulation.

Cite This Article

APA
Salinas-Varas C, Espinosa G, Muñoz-Caro T, Conejeros I, Gärtner U, Fey K, Arnhold S, Taubert A, Hermosilla C. (2025). Equine adipose-derived stem cells modulate in vitro neutrophil extracellular trap release by polymorphonuclear neutrophils. Front Vet Sci, 12, 1685757. https://doi.org/10.3389/fvets.2025.1685757

Publication

Frontiers in veterinary science
ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 12
Pages: 1685757
PII: 1685757

Researcher Affiliations

Salinas-Varas, Constanza
  • Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
Espinosa, Gabriel
  • Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
Muñoz-Caro, Tamara
  • Escuela de Medicina Veterinaria, Facultad de Medicina Veterinaria y Recursos Naturales, Universidad Santo Tomás, Talca, Chile.
Conejeros, Iván
  • Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
Gärtner, Ulrich
  • Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany.
Fey, Kerstin
  • Equine Clinic, Internal Medicine, Justus Liebig University Giessen, Giessen, Germany.
Arnhold, Stefan
  • Institute of Veterinary Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
Taubert, Anja
  • Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
Hermosilla, Carlos
  • Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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