Interleukin 12 on extracellular matrix-related factors and functional characteristics of equine endometrial fibroblasts.

Overview

• This study investigates how interleukin-12 (IL-12), a proinflammatory cytokine, affects extracellular matrix (ECM)-related factors and the functional behavior of equine endometrial fibroblasts, which are involved in tissue fibrosis.
• The research explores the expression of IL-12 and its receptor in different categories of mare endometrial tissue and tests how IL-12 influences markers linked to fibrosis and fibroblast characteristics like proliferation and migration.

Background

• Tissue fibrosis involves excessive accumulation of extracellular matrix components, disrupting normal tissue function.
• Fibrosis is often linked to inflammation, with fibroblasts and immune cells interacting to promote progression.
• IL-12 is a proinflammatory cytokine produced by immune cells such as macrophages, dendritic cells, and B cells.
• Previous studies have suggested that IL-12 could influence fibrosis development, motivating investigation into its role in equine endometrium (the lining of the uterus in mares).

Objectives

• Determine the expression levels of IL-12 and its receptor in various categories of mare endometrial tissue.
• Examine the effects of IL-12 treatment on the expression of ECM-related factors in endometrial fibroblasts.
• Evaluate functional characteristics of endometrial fibroblasts in response to IL-12, focusing on proliferation and migration.

Methods

• Sample collection from mare endometrium categorized according to fibrosis severity (e.g., category IIB).
• Quantitative polymerase chain reaction (qPCR) used to measure mRNA levels of IL-12 subunits, IL-12 receptor, and ECM-related factors.
• Western blotting and zymography employed to assess protein expression and enzyme activity related to matrix remodeling.
• Bromodeoxyuridine (BrdU) assays used to quantify fibroblast proliferation after exposure to IL-12.
• Scratch assays used to examine fibroblast migration capability after IL-12 treatment.

Results

• IL-12 receptor beta 2 subunit (IL-12Rβ2) mRNA was significantly upregulated in category IIB endometrium during the mid-luteal phase, compared to the follicular phase.
• IL-12 treatment increased mRNA expression of several fibrotic markers in fibroblasts:
  • COL1A1 and COL3A1 (collagen genes related to ECM)
  • ACTA2 (alpha smooth muscle actin, a marker of myofibroblast differentiation)
  • LOXL2 (enzyme involved in cross-linking collagen and ECM remodeling)
• Increased expression and gelatinolytic activity of matrix metalloproteinases (MMP-9 and MMP-3) was observed, indicating active ECM remodeling processes; pro-MMP2 and MMP9 activities were also elevated.
• IL-12 reduced fibroblast proliferation after 96 hours of treatment, indicating an inhibitory effect on cell growth.
• Fibroblast migration was not significantly affected by IL-12 treatment, suggesting selective effects on functional behaviors.

Conclusions

• IL-12 influences equine endometrial fibroblasts by modulating gene expression of ECM components and enzymes involved in fibrosis and ECM remodeling.
• Its role appears multifaceted, promoting fibrotic marker expression and MMP activity, while inhibiting fibroblast proliferation.
• These findings suggest that IL-12 may have a direct impact on extracellular matrix remodeling within the endometrium, contributing to fibrosis progression.
• Understanding IL-12’s functions could offer insights into preventing or managing uterine fibrosis in mares, with potential broader implications for fibrotic diseases.