Abstract

Background: Renal ischemia-reperfusion (I/R) injury is a major cause of graft dysfunction and failure, driving inflammation and tissue damage. Mesenchymal stem cells (MSCs) possess therapeutic potential due to their immunomodulatory properties. Notably, neutrophils express the inhibitory receptor CD32b, which is a specific target of the immunosuppressive molecule soluble fibrinogen-like protein 2 (sFgl2).

Aims: This study aimed to investigate the therapeutic efficacy and underlying mechanisms of genetically engineered MSCs expressing sFgl2 (sFgl2-MSCs) in treating renal I/R injury, with a focus on neutrophil regulation.

Study Design: An in vivo renal I/R injury mouse model.

Methods: Following imaging to localize MSCs, mice were randomly allocated into four treatment groups. Treatments were administered according to group assignments. Renal function was assessed using serum creatinine and blood urea nitrogen levels, while systemic inflammation was evaluated by measuring serum interleukin-1 beta (IL-1β), interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and IL-10 via enzyme-linked immunosorbent assay (ELISA). Neutrophil proportions in the blood and kidney were analyzed by flow cytometry. At 24 h, surface expression of CD95 and CD206 was assessed; CD206 was used to define neutrophils with N2-like (CD206+) and N1-like (CD95+) phenotypic features. Histopathological scoring of renal tissue was performed at 24 h. Infiltration of Ly6G+ neutrophils and citrullinated histone H3 (CitH3) as well as myeloperoxidase/CitH3 co-localization (an indicator of neutrophil extracellular traps, NETs) were detected by immunohistochemistry and immunofluorescence. Circulating free DNA (cf-DNA) in plasma was quantified using PicoGreen dye. Furthermore, the impact of sFgl2-MSCs on bone marrow-derived neutrophil polarization and function was evaluated in vitro using flow cytometry, ELISA, and a co-culture system.

Results: While unmodified MSCs exhibited a moderate therapeutic effect, sFgl2-MSCs treatment was significantly more effective. sFgl2-MSCs markedly improved renal function, reduced histopathological damage (e.g., tubular necrosis), and modulated systemic cytokine levels by decreasing pro-inflammatory (IL-1β, IL-6, TNF-α) and increasing anti-inflammatory (IL-10) cytokines. Crucially, sFgl2-MSCs regulated neutrophil responses in the kidney: they increased the proportion of N2-like neutrophils and decreased N1-like neutrophils, concurrently reducing NET-related markers, as evidenced by decreased CitH3 and cf-DNA. Mechanistically, sFgl2-MSCs enhanced neutrophil immunoregulatory function via the TGFβ-Smad2/3 signaling pathway.

Conclusion: Genetically modified sFgl2-MSCs alleviate renal I/R injury. This protective effect is associated with engagement of neutrophil CD32b receptors, activation of the TGFβ-Smad2/3 pathway, promotion of a protective N2-like neutrophil phenotype, and suppression of N1-like and NET-related markers.