Menstrual blood-derived stromal cells (MenSCs), a type of mesenchymal stromal cell (MSC) originating from the endometrium, are attracting attention for their potential in cell-based therapies due to their immunomodulatory capabilities. Their ease of non-invasive collection and ethical advantages over other MSC sources like bone marrow and adipose tissue make them particularly attractive. Previous research has demonstrated the therapeutic potential of MSCs in various inflammatory conditions, primarily through paracrine mechanisms that include angiogenic, anti-apoptotic, and immunomodulatory effects. While MSC immunomodulation is known to involve cytokine release and cell-cell interactions, the specific impact of MenSCs on innate immune cells, especially macrophages—key players in inflammation—remains largely unexplored. Understanding MenSC-macrophage interactions is critical for optimizing their clinical use in inflammatory disorders. This study aimed to evaluate the immunomodulatory effects of MenSCs on macrophages during acute inflammation using two distinct mouse models: thioglycollate (TGC)-elicited peritonitis (a sterile inflammatory model) and *Salmonella Typhimurium*-induced monobacterial sepsis (an infectious model). The impact of MenSCs on macrophage recruitment and other innate immune cells (neutrophils, monocytes), along with in vitro studies on human macrophage functional properties (phagocytosis, bacterial clearance, reactive oxygen species production), were analyzed to fully characterize MenSC activity.

The literature extensively documents the therapeutic potential of mesenchymal stromal cells (MSCs) derived from various sources, including bone marrow and adipose tissue. These cells exert their effects primarily through paracrine mechanisms, including the secretion of a wide range of factors with angiogenic, anti-apoptotic, and immunomodulatory properties. Their immunomodulatory actions are particularly promising for treating inflammatory diseases. However, the tissue of origin significantly influences the functional properties and effects of MSCs, highlighting the need for source-specific investigations. MenSCs, first described by Meng et al. in 2007, offer advantages due to their non-invasive collection method and lack of ethical concerns, making them a valuable alternative to other MSC sources. While some studies have investigated MenSC effects in various inflammatory models (colitis, LPS-induced injury, polymicrobial sepsis), these studies primarily focused on systemic outcomes (survival rates, reduced histopathological damage) or the impact on T and B cells. The direct effect of MenSCs on macrophage function, a central component of innate immunity and inflammation, remained largely unknown, motivating the current study.

This study employed two mouse models of acute inflammation: thioglycollate (TGC)-elicited peritonitis and a *Salmonella Typhimurium* (ST)-induced monobacterial sepsis model, using BALB/c mice. MenSCs were isolated from menstrual blood samples collected from 15 healthy women (following ethical approval and informed consent) using density gradient centrifugation. The MenSC phenotype was confirmed via flow cytometry, demonstrating expression of typical MSC surface markers. In the TGC model, MenSCs were administered intraperitoneally (IP) 4 or 24 hours after TGC injection. Peritoneal lavages were collected 5 days later to assess cell numbers and phenotypes (macrophages, neutrophils, inflammatory monocytes) using flow cytometry. In the sepsis model, mice were IP-challenged with ST and an attenuated SseB- strain of *Salmonella*. MenSCs were administered 4 hours post-infection, and peritoneal lavages, spleen, and mesenteric lymph nodes were collected 24 hours later for cell analysis, bacterial burden determination (CFU), and quantitative PCR (mRNA expression of *Nos2*, *Tnf*, *Il10*, *Tgfb*). In vitro studies used THP-1 cells differentiated into macrophages with PMA. Co-culture experiments with MenSCs were performed using Transwell inserts to assess effects on macrophage phenotype (flow cytometry), phagocytosis (*Salmonella* SseB uptake), bacterial killing (gentamicin protection assay), and reactive oxygen species (ROS) production (DCFDA oxidation). Statistical analyses included one-way or two-way ANOVA with post-hoc tests (Tukey's or Sidak's) as appropriate.

The study revealed that MenSCs injection in the TGC peritonitis model reduced the number and percentage of macrophages recruited to the peritoneum, with the reduction being more pronounced when MenSCs were injected 24 hours post-TGC. This correlated with the formation of peritoneal aggregates containing MenSCs and macrophages, suggesting a localized immunomodulatory effect. While the macrophage percentage decreased, the percentage of neutrophils significantly increased, indicating a shift in the innate immune cell composition. In the *Salmonella* sepsis model, MenSC administration reduced the number of both macrophages and neutrophils recruited to the peritoneum, leading to higher bacterial loads in the peritoneum, spleen, and mesenteric lymph nodes, indicating impaired bacterial clearance. In vitro experiments using human THP-1-derived macrophages showed that MenSC co-culture increased bacterial internalization (phagocytosis) but significantly decreased bacterial killing and ROS production, indicating compromised microbicidal activity. Analysis of cytokine mRNA levels in the sepsis model showed increased expression of *Nos2* and *Il10*, suggesting a shift towards a regulatory macrophage phenotype characterized by impaired bactericidal activity and potential IL-10-mediated inhibition of neutrophil recruitment.

This study's findings highlight the complex immunomodulatory effects of MenSCs on macrophages during acute inflammation. While MenSCs have shown promise in preclinical models of inflammation, their effects on macrophages in the context of both sterile and infectious inflammation are multifaceted and require careful consideration for clinical translation. The results demonstrate that MenSCs can reduce macrophage recruitment and shift the immune response towards a regulatory phenotype, potentially limiting tissue damage but also potentially impairing bacterial clearance in the context of infection. The formation of peritoneal aggregates in the TGC model suggests MenSCs might act as a scaffold for immune cell modulation. The in vitro findings further support the hypothesis that MenSCs directly reprogram macrophages towards a regulatory phenotype, potentially through mechanisms involving IL-10 production and impaired ROS generation. The apparent contradictory effects on neutrophil recruitment in the two models underscore the dynamic nature of MenSC-mediated immune regulation, influenced by the specific inflammatory context (sterile vs. infectious).

This research provides novel insights into MenSC-macrophage interactions during acute inflammation. While MenSCs demonstrate immunomodulatory properties beneficial in reducing inflammation and tissue damage, their impact on bacterial clearance requires careful consideration, particularly in infectious settings. Future studies should focus on identifying the specific molecular mediators and signaling pathways mediating MenSC effects on macrophage polarization and function, as well as expanding studies to encompass other immune cell populations to refine strategies for clinical applications.

The study primarily focused on mouse models and in vitro studies using THP-1 cells, which may not fully capture the complexity of human immune responses. The use of BALB/c mice may limit the generalizability of the findings. Additionally, while the study identified key effects on macrophage function, the precise mechanisms underlying these effects require further investigation, particularly the role of specific cytokines and signaling pathways. The limited number of human donors for MenSC isolation should also be considered.