(W1222) Altered matrix environments enhance the Immunomodulatory Activity of Mesenchymal Stromal/Stem Cells through TNFα/NF-kappa B and TNFα/JNK/AP1 signalling pathways

Abstract: Mesenchymal stromal/stem cells (MSCs) are recognised for their role in modulating immune cells during tissue repair. Given the dynamic nature of the extracellular matrix (ECM) in such situations, this study aimed to identify the signalling pathways in MSCs that are influenced by the ECM and how this impacts their immunomodulatory activity.
Human MSCs were cultured in a monolayer on tissue culture plastic (TCP) or encapsulated in three 3D ECM matrices (Fibrin, Collagen Type 1 and Collagen Type 1-Fibrin). After 24 hours of subjecting them to a low-dose pro-inflammatory environment (2ng/µl TNFα and 5ng/µl IFNγ), transcriptional profiles were analysed using bulk RNA sequencing (RNAseq). The significant transcriptional changes were validated using RTqPCR, ELISA and Western blot. RNA silencing and pharmacological inhibition were employed to investigate key signalling pathways.
Previously, we showed that human MSC expression of TNFAIP6 and CXCL10 in 3D environments is significantly upregulated in response to pro-inflammatory stimuli. Here, RNA-seq analysis identified 2,085 genes that were significantly upregulated in 3D matrices compared to TCP, with over 90% of the highly expressed genes (including FOSB, FOS, and TNFAIP6) being consistently shared across all hydrogel scaffolds. In response to cytokine treatment, subsequent gene ontology analysis highlighted the TNF signalling pathway as one of the most enriched. Protein-protein interaction predictions revealed that the hydrogel environment differentially influenced the TNF-alpha/NF-kappa B and AP1 pathways. Pathway inhibition studies showed that 3D environments regulate the immunomodulatory activity of MSCs treated with TNFα and IFNɣ through the TNFα/NF-kappa B and TNFα/JNK/AP1 signalling pathways.
These findings highlight that MSCs exhibit distinct immunomodulatory responses in 3D environments compared to traditional monolayer cultures. This underscores the importance of 3D models in more accurately mimicking (patho)physiological conditions for studying MSCs and their therapeutic potential.

Funding Source: King Abdulaziz City for Science and technology

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