Regulation of inflammatory responses in Toll-like receptor-stimulated human mesenchymal stem cells

Abstract

Various stem cell types are being studied for their potential therapeutic benefits, which may derive from their differentiation potentials or immunomodulatory functions. For example, human mesenchymal stem cells (hMSCs), the subject of this Thesis, are widely studied for the regeneration and treatment of inflammatory diseases. MSCs can interact with immune cells and modulate a variety of effector functions. MSCs can be recruited into sites of tissue injury and participate in repair. MSCs express Toll-like receptors (TLRs) that can modulate immune and migration responses. Specially, stimulation of TLR3 and TLR4 mediates immunomodulation and cell migration in MSCs. Activation of TLR3 and TLR4 induces polarization of MSCs and regulates immune responses. TLR3 and TLR4 stimulation play an important role in MSCs. However, the mechanisms by which TLRs regulate hMSCs migration are unknown. Therefore, the present study determined and interpreted the transcriptional profiles of TLR3- and TLR4-stimulated hMSCs. In addition, the transcriptional regulation of LPS-inducible enhancer was studied in TLR4-stimulated hMSCs. Firstly, hMSCs were TLR4-stimulated by adding lipopolysaccharide (LPS), and the resulting transcriptional profiles determined. LPS induced the expression of interferon-stimulated genes, cytokines, and chemokines known to be involved in the chemotaxis response. In their -950 to +50 bp regions, the LPS-induced genes were enriched with binding motifs for the transcription factors interferon regulatory factor 1 (IRF1) and nuclear factor kappa B 1 (NF-κB1). Gene ontology analysis revealed that the target genes of IRF1 were associated with immune responses. Moreover, ChIP experiments demonstrated direct binding of IRF1 to the promoter regions of its target genes, and RNA interference directed against IRF1 inhibited their expression. As for NF-κB1, its target genes were related to the response to wounding and chemotaxis, and inhibition of NF-κB reduced the LPS-induced cell hMSC migration. Taken together, these results indicate that NF-κB and IRF1 orchestrate the TLR4-stimulated immunomodulatory response of hMSCs. Secondly, the transcriptional profiles of TLR3-stimulated hMSCs were studied after the addition of polyinosinic:polycytidylic acid (poly(I:C)). Activation of TLR3 increased the expression of inflammation-related genes such as interferon stimulating genes, cytokines, and chemokines, as well as of cell migration-related genes. The latter were enriched within the +1000 bp regions with putative binding motifs for NF-κB1, RELA, forkhead box protein O1 (FOXO1) and FOXO3. The potential NF-κB1 and FOXO1 target were related to the cellular response to interleukin-1 and to the immune response. Pharmacological inhibition of FOXO1 in the TLR3-stimulated hMSCs led to a significant down-regulation of cell migration-related genes and an inhibition of poly(I:C)-induced cell migration. Taken together, these data indicate that in TLR3-stimulated hMSCs, FOXO1 is important for the expression of inflammation- and cell migration-related genes as well as for cell migration. Thirdly, a functional study of transcriptional regulation via LPS-inducible enhancers was performed. For this study, we selected the gene that encodes the interferon-inducible transmembrane protein 1 (IFITM1). In the earlier part of this study, we had found that IFITM1 is one of the genes that are controlled by LPS, NF-κB, and IRF1 (see above); in line with this, IFITM1 has previously been shown to be related to immunity, cell migration, and adhesion. Moreover, IFITM1 appears to play a role in migration since we also found that RNAi directed against IFITM1 reduced the cell migration response in TLR4-stimulated hMSCs. With respect to the enhancer regulation, we find that upon induction with LPS, the IFITM1 gene-associated chromatin was highly enriched for histone H3 acetylation on lysine 27 (H3K27ac). Upon induction with LPS, the R5 and especially the R2 enhancer regions showed a significant induction of enhancer RNA (eRNA) expression and enrichment of H3K27ac. In line with this, both R2 and R5 showed enhancer activity in luciferase reporter gene assays. Finally, pharmacological inhibition of NF-κB decreased its binding to the IFITM1 enhancer regions and the expression of the respective eRNAs. These results indicate that the IFITM1 gene enhancers, R2 and (to a lesser degree) R5, are activated by the LPS-controlled binding of NF-κB. In summary, we investigated transcriptional profiles and functional gene regulation in TLR3- or TLR4-stimulated hMSCs. Activation of TLR3 and TLR4 induced inflammation- and cell migration-related genes and mediated cell migration responses regulated by the transcription factors NF-κB and IRF1. TLR3-stimulated hMSCs were induced to migrate via activation of FOXO1. In TLR4-stimulated hMSCs, IRF1 directly regulated LPS-induced genes while NF-κB signaling regulated cell migration responses. Furthermore, IFITM1 was a key molecule of the cell migration response. IFITM1 enhancer activity was modulated by NF-κB. We investigated the regulation of cell migration and gene regulation of enhancer activity in TLR3- or TLR4-stimulated hMSCs. These findings contribute to our understanding of the molecular mechanisms underlying cell migration and immunomodulation in TLR-stimulated hMSCs.