Uterine proteomic and cytokine profiling show that Streptococcus equi subsp. zooepidemicus induces immune-metabolic dysregulation and may predispose mares to endometrosis.

Overview

• This research investigates how a bacterial infection caused by Streptococcus equi subsp. zooepidemicus affects the protein and cytokine composition in the uterine fluid of mares, revealing immune and metabolic disruptions that could lead to uterine fibrosis (endometrosis).
• The study compares healthy mares to infected mares, identifying specific inflammatory markers and protein changes that underline the disease process.

Background and Objective

• Endometritis, an inflammation of the uterus often caused by bacterial infections such as Streptococcus equi subsp. zooepidemicus, can impact fertility in mares.
• Understanding the molecular changes during infection is crucial to identifying mechanisms that contribute to chronic uterine damage like endometrosis, which involves fibrotic tissue remodeling impairing uterine function.
• The study aimed to analyze and compare the protein and cytokine profile of uterine fluid from reproductively healthy mares and those infected with S. zooepidemicus to uncover immune and metabolic perturbations.

Methods

• Samples: Uterine flush samples were collected from 15 healthy mares and 9 mares diagnosed with S. zooepidemicus-associated endometritis during the follicular phase of their reproductive cycle.
• Proteomics: Shotgun proteomics was used to identify and quantify proteins present in uterine fluid.
• Cytokine Profiling: Multiplex cytokine assays measured multiple inflammatory mediators simultaneously.
• Bioinformatics: Equine proteins were mapped to their human equivalents to leverage greater annotation resources.
• Enrichment and Network Analysis: Multiple online tools (g:Profiler, Metascape, ToppGene, STRING/MCODE) were used to identify significant biological pathways, interactions, and networks among identified proteins.

Key Findings – Cytokine Profile

• In mares with S. zooepidemicus infection, significant increases were seen in pro-inflammatory cytokines such as IL-1α, IL-1β, GRO, IL12p70, and Eotaxin.
• Additionally, some cytokines were exclusively detected in infected mares, including IL-6, IL-8, IP10, IL-10, and TNFα, indicating an active inflammatory and fibrotic environment.
• This cytokine signature suggests a persistent proinflammatory state that could drive chronic uterine damage.

Key Findings – Proteomic Changes

• 67 proteins were differentially expressed between healthy and infected mares.
• Upregulated proteins in infected mares included:
  • Neutrophil effector proteins involved in NET (neutrophil extracellular trap) formation, a process where neutrophils release web-like structures to trap pathogens but potentially cause tissue damage.
  • Acute-phase proteins linked to inflammation.
  • Glycolytic enzymes reflecting metabolic shifts towards glycolysis, often associated with immune cell activation.
  • Chromatin-associated histones, possibly related to NETs and inflammation.
• Downregulated proteins included those with:
  • Anti-inflammatory roles (e.g., SPI2).
  • Antioxidant functions (e.g., SOD3, ALDH1A1), reducing oxidative stress defense.
  • Proteins associated with uterine muscle contractility (e.g., CALM1), potentially impairing uterine clearance mechanisms.

Biological Pathways and Network Insights

• Enrichment analyses emphasized processes such as:
  • Neutrophil degranulation and NETosis, confirming enhanced neutrophil activity in the uterine environment.
  • Metabolic reprogramming including glycolysis and gluconeogenesis, linking immune activation to altered metabolism.
  • Extracellular vesicle activity, which may mediate intercellular communication in inflammation.
  • Regulation of the actin cytoskeleton, important for cell shape, motility, and function.

Additional Considerations

• The study suggests that immune regulation diminishes with age, possibly exacerbating inflammation and fibrosis in older mares with infection.
• The persistent neutrophil-driven inflammatory state and oxidative stress likely impair the uterus’s ability to clear bacteria effectively.
• This impaired clearance may predispose mares to developing endometrosis, featuring fibrotic changes reducing uterine function.

Implications and Potential Applications

• 16 candidate biomarkers associated with chromatin remodeling, antimicrobial defense, and metabolism were identified, potentially useful for diagnostic assays.
• Therapeutic interventions targeting:
  • Dysregulated inflammation,
  • NETosis (excessive neutrophil extracellular trap formation), and
  • Standard antimicrobial treatments,

  could improve fertility outcomes in affected mares.

• Understanding these pathways provides targets to prevent progression from infection to fibrosis, preserving reproductive health.

Summary

• This study thoroughly profiles the uterine immune and metabolic environment during S. zooepidemicus infection.
• Results highlight how an overactive neutrophil response and metabolic changes create a proinflammatory and profibrotic uterine milieu.
• These molecular changes hinder effective bacterial clearance and promote uterine fibrosis, providing a mechanistic basis for fertility complications in infected mares.
• The identified biomarkers and pathways open paths for diagnostics and novel therapeutic approaches to manage infectious endometritis in horses.