The equine mesenchymal stromal cell (MSC) secretome modulates neutrophils and monocyte-derived macrophages and extracellular vesicles (EVs) impact macrophage viability.

Overview

• This study investigates how secretions from equine mesenchymal stromal cells (MSCs) influence immune cell functions, specifically neutrophils and macrophages, in the context of fighting methicillin-resistant Staphylococcus aureus (MRSA) infections.
• The research found that MSC secretions differentially modulate immune cell behaviors and that extracellular vesicles (EVs) from MSCs can affect macrophage survival, suggesting potential therapeutic uses for controlling inflammation during bacterial infections.

Background and Importance

• Neutrophils and macrophages are critical components of the innate immune system, responsible for detecting and destroying bacterial pathogens such as MRSA.
• Neutrophil functions include chemotaxis (movement toward infection sites) and phagocytosis (engulfing bacteria), both vital for bacterial clearance.
• MRSA can evade immune defenses by impairing these processes and manipulating macrophage polarization and reactive oxygen species (ROS) production, thus promoting infection persistence.
• Previous work showed that equine MSC secretome reduces MRSA growth, but additional effects on innate immune cells had not been fully characterized.

Study Objectives

• To determine whether the secretome from different sources of equine MSCs (adipose tissue, bone marrow, peripheral blood) modulates neutrophil and macrophage functions related to anti-MRSA defense.
• To explore the specific fractions of the MSC secretome (complete conditioned medium, soluble components, and extracellular vesicles) responsible for these immunomodulatory effects.

Experimental Design and Methods

• Conditioned medium (CM) was collected from MSCs derived from three tissue sources: adipose tissue, bone marrow, and peripheral blood.
• Neutrophil chemotaxis was measured using transwell migration assays to assess how CM influences neutrophil movement.
• Neutrophil phagocytosis capacity and ROS production were tested after exposure to CM.
• Macrophage polarization was assessed based on expression of markers CD86 (M1-like, pro-inflammatory) and CD206 (M2-like, anti-inflammatory).
• Phagocytic capacity and ROS production in macrophages were also evaluated.
• Further fractionation of CM into soluble factors and extracellular vesicles (EVs) allowed determination of which components influenced neutrophil chemotaxis and macrophage viability.
• Macrophage viability was examined, with investigation into whether EV-induced death involved autophagy mechanisms.

Key Findings

• Neutrophil Chemotaxis:
  • CM from adipose tissue- and bone marrow-derived MSCs significantly enhanced neutrophil migration, but CM from peripheral blood-derived MSCs did not.
  • The enhanced neutrophil migration was due to factors in the complete CM rather than isolated soluble components or EVs.
• Neutrophil Phagocytosis and ROS Production:
  • CM from all three MSC sources reduced the phagocytic ability of neutrophils.
  • ROS production by neutrophils was unaffected by CM treatment.
• Macrophage Polarization:
  • CM from all MSC tissue sources promoted polarization toward both M1-like (CD86-positive) and M2-like (CD206-positive) phenotypes.
  • Despite changes in polarization, macrophage phagocytosis and ROS production remained unchanged.
• Effect of Extracellular Vesicles (EVs):
  • The primary impact of EVs isolated from the MSC secretome on macrophages was to reduce cell viability, possibly via inducing autophagy.
  • This reduction in viable macrophages could help lessen tissue damage during excessive inflammation by reducing inflammatory macrophage numbers.

Conclusions and Implications

• The equine MSC secretome modulates key functions of innate immune cells, enhancing neutrophil migration while suppressing their phagocytosis, and influencing macrophage polarization without altering their bactericidal functions.
• Extracellular vesicles from MSCs can reduce macrophage viability, potentially contributing to the resolution of harmful inflammation through autophagy mechanisms.
• These findings suggest that MSC secretions have complex immunomodulatory roles that could be harnessed therapeutically to manage bacterial infections like MRSA and control dysregulated inflammation.
• Future studies may focus on identifying specific secreted factors responsible for these effects and the therapeutic application of MSC secretome components in equine and possibly human bacterial diseases.