The effect of macrophage secretome on equine endometrial fibroblast transcriptomic and functional characteristics.

Overview

• This study explores how secreted factors from different subtypes of macrophages influence gene expression and behavior of equine endometrial fibroblasts, cells involved in uterine tissue structure and repair.
• It highlights the distinct roles of pro-inflammatory (MΦ1) and anti-inflammatory (MΦ2a) macrophages in regulating fibrosis-related processes in the horse endometrium, relevant for understanding and potentially treating endometrial fibrosis.

Introduction and Background

• Endometrial fibrosis, marked by excessive extracellular matrix (ECM) buildup, is a key feature of equine endometrosis, a degenerative uterine condition.
• Macrophages (MΦ), immune cells that can adopt different functional states, are known to activate fibroblasts, the cells that produce ECM, and thus contribute to fibrosis.
• Two major macrophage phenotypes studied are pro-inflammatory MΦ1 and anti-inflammatory MΦ2; their distinct effects on equine endometrial fibroblasts were previously unclear.

Objectives

• To examine the long-term effects of secretomes (the collection of secreted proteins and signaling molecules) from in vitro-generated equine monocyte-derived MΦ1 and MΦ2a on equine endometrial fibroblasts.
• To assess changes in transcriptomic profiles (gene expression), ECM-related component expression, inflammatory marker levels, and functional characteristics such as proliferation and migration in fibroblasts exposed to these macrophage secretomes.

Methods

• Equine monocytes were differentiated in vitro into two macrophage types: MΦ1 (pro-inflammatory) and MΦ2a (anti-inflammatory).
• Endometrial fibroblasts were cultured and treated with secretomes from these macrophages over a long-term period.
• Gene expression in fibroblasts after treatment was examined to identify differentially expressed genes (DEGs).
• Functional assays measured fibroblast proliferation and migration capabilities.
• Annotation and pathway analyses were conducted to understand biological processes linked to the DEGs.

Key Findings

• Treatment with MΦ1 secretome led to 404 differentially expressed genes in fibroblasts.
• These genes were largely associated with immune-related activities such as neutrophil movement, lymphocyte migration, and proliferation and growth of connective tissue cells, indicating a role in inflammatory and tissue remodeling responses.
• Treatment with MΦ2a secretome resulted in 282 DEGs in fibroblasts.
• These genes were linked to assembly of intercellular junctions, plasma membrane formation, and development of gap junctions, suggesting promotion of tissue organization and structural functions.
• Both MΦ1 and MΦ2a secretomes increased fibroblast proliferation and migration, which are important for tissue repair.
• Expression of ECM components and inflammatory markers was modulated differently by each macrophage subtype, consistent with their distinct roles in inflammation and tissue remodeling.

Interpretation and Significance

• MΦ1 secretome promotes fibroblast gene expression related to inflammation and connective tissue growth, implying a role in initiating repair and immune responses during endometrial injury.
• MΦ2a secretome influences genes linked to the organization and maintenance of tissue structure, consistent with resolving inflammation and supporting tissue repair and remodeling.
• The balance or sequential activation of MΦ1 and MΦ2a signaling pathways may be crucial for effective and regulated endometrial repair in horses, minimizing fibrosis development.
• Understanding these macrophage-fibroblast interactions could aid in designing therapeutic strategies to prevent or treat endometrial fibrosis and improve reproductive health in mares.

Conclusion

• The study provides novel insights into how macrophage subpopulations differentially impact endometrial fibroblast behavior at molecular and functional levels.
• Targeting macrophage-fibroblast communication pathways may offer promising avenues for managing equine endometrial fibrosis and its associated reproductive problems.