Project description:Cardiovascular diseases (CVDs) include atherosclerosis, which is an inflammatory disease of large and medium vessels that leads to atherosclerotic plaque formation. Key factors contributing to onset and progression of atherosclerosis include the pro-inflammatory cytokines Interferon (IFN)α and IFNγ and the Pattern Recognition Receptor (PRR) Toll-like receptor 4 (TLR4). Together, they trigger activation of IFN Regulatory Factors (IRFs) as well as Signal Transducer and Activator of Transcription (STAT)s. Based on their promoting role in atherosclerosis, we hypothesized that combined inhibition of IRFs and STATs blocks pro-inflammatory target gene expression and may be a promising strategy to treat CVDs. Using comparative in silico docking of multiple IRF-DBD models on a multi-million natural compound library, we identified the novel multi-IRF inhibitor, ALEKSIN. This compound targets the DBD domain of IRF1, IRF2, and IRF8 with the same affinity and simultaneously inhibits expression of multiple IRF-target genes in HMECs in response to IFNa and IFNg. Under the same conditions, ALEKSIN was also shown to inhibit phosphorylation of STATs, potentially through low-affinity STAT-SH2 binding, however with lower potency as the known multi-STAT inhibitor STATTIC. This was in line with the common inhibition of ALEKSIN and STATTIC observed on genome-wide expression of pro-inflammatory IRF/STAT/NF-κB target genes, as well as on migration of HMECs. Finally, we identified a novel signature of 46 ALEKSIN and STATTIC commonly inhibited pro-atherogenic target genes, that was upregulated in atherosclerotic plaques in aorta's of high-fat diet fed APOE-/- mice and associated with inflammation, proliferation, adhesion, chemotaxis and response to lipid. Together, this suggests that ALEKSIN represents a novel class of multi-IRF inhibitors, which inhibit IRF- , STAT- and NF-κB-mediated transcription and could offer great promise for the treatment of CVDs. Also, the ALEKSIN and STATTIC commonly inhibited pro-inflammatory gene signature could serve to monitor plaque progression during experimental atherosclerosis.

Project description:Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T-cells into regulatory T-cells in vitro. The marker CD69 is a target of canonical NF-κB signaling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3+ T-cells were activated and cultured in the presence or absence of MSCs. CD4+ cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 and a siRNA against RELB were used to explore the differential roles of canonical and non-canonical NF-κB signaling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69+ cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69+ cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signaling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signaling on the 3rd day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signaling. In order to study the molecular basis by which Multipotent Mesenchymal Stromal/Stem Cells (MSC) exert their immune regulatory function, immunomagnetically purified CD3+ T-cells from the peripheral blood of 3 individuals were activated and cultured in the presence or absence of MSCs. Following 5 days, CD4+ and CD8+ T-cells were further immunomagnetically selected and their gene expression profiles were obtained by microarrays and compared. Paired samples from 3 individuals were used for this analysis.

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Inhibition of Mesenchymal Phenotypes in Glioblastoma by Combined Targeting of NF-κB and STAT3 Pathways

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