In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter

Abstract

Background Mesenchymal stromal cells (MSCs) are being investigated as therapies for inflammatory diseases due to their immunosuppressive capacity. IFN-γ treatment primes MSC immunosuppression partially through induction of Indoleamine 2,3-dioxygenase (IDO1), which depletes tryptophan necessary to support proliferation of activated T-cells. We investigated the role of histone modifications in the timing and maintenance of induced IDO1 expression in MSCs under clinical manufacturing conditions, such as cryopreservation. Methods We used chromatin immunoprecipitation and quantitative polymerase chain reaction (PCR) to assay levels of transcriptionally permissive acetylated H3K9 and repressive trimethylated H3K9 histone modifications surrounding the transcriptional start site for IDO1, and reverse transcriptase PCR and immunoblotting to detect mRNA and protein. Results MSCs derived from three donors approached maximum IDO1 mRNA levels following 24 hours of in vitro cytokine treatment. Induction of IDO1 expression correlated with increased acetylation of H3K9 concomitant with reduction of trimethylated H3K9 modifications at the promoter. Examination of two additional donors confirmed this result. While induced IDO1 levels declined within two days after cytokine removal and freeze thawing, the activated chromatin state was maintained. Upon re-exposure to cytokines, previously primed MSCs accumulated near-maximum IDO1 mRNA levels within four to eight hours. Discussion Our data indicate that in vitro priming of MSCs causes chromatin remodeling at the IDO1 promoter, that this alteration is maintained during processing commonly used to prepare MSCs for clinical use, and that once primed, MSCs are poised for IDO1 expression even in the absence of cytokines.

Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies

“…The expression of IDO decreases after medium removal and freeze-thawing the MSC products [ 33 ]. However, preconditioning of MSC with IFN-γ and TNF-α induces chromatin remodeling in the IDO1 promoter, which correlates with increased H3K9 (histone H3 at lysine 9) acetylation concomitantly with a reduction in trimethylated H3K9.…”

“…Curiously, these chromatin alterations are maintained even after the cryopreservation process. Upon re-exposure to cytokines, previously IFN-γ-treated MSC are able to quickly accumulate high IDO1 mRNA levels [ 33 ].…”

The Role of Indoleamine-2,3-Dioxygenase in Cancer Development, Diagnostics, and Therapy

“…Indoleamine-2,3-dioxygenase1 production is elevated upon (i) IFN-γ production of effector T cells ( 2 ), (ii) inflammatory cytokine production of innate immune cells ( 6 , 38 ), (iii) IL-10 and IL-27 stimulation ( 39 ), (iv) CTLA4 expression on Treg cells causing increased IDO1 secretion by dendritic cells (DCs) ( 40 ), and (v) TGF-β, IL-10 and adenosine production of Treg and other immunosuppressive cells ( 40 – 42 ), (vi) cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) stimuli that are mediated through the PKC, PI3K, and MAPK pathways (several types of tumors carry PI3K or MAPK oncogenic mutations leading to constitutive IDO1 expression.) ( 43 ).…”

Umbilical Cord-Derived Mesenchymal Stem Cells Are Able to Use bFGF Treatment and Represent a Superb Tool for Immunosuppressive Clinical Applications

“…In immunosuppressive applications the treatment with pro-inflammatory cytokines and growth factors are used. Interferon gamma (IFN-y) or the combination of IFN-y and tumor necrosis factor alpha (TNF-α) are the most often used according to the ISCT recommendations [ 26 ] to increase the immunosuppressive effects of MSCs [ 27 , 28 , 29 , 30 ]. Extended combinatory strategies of other cytokines include interleukin-1 beta, interleukin-6 (IL-6), interleukin-17, interleukin-23, basic fibroblast growth factor (bFGF) or transforming growth factor beta (TGFβ).…”