Der Einfluss der kurzkettigen Fettsäure Valproat auf die T-Zell-vermittelte Immunantwort

Valproinsäure, beziehungsweise ihr Salz Valproat, wird seit mehr als 50 Jahren als Antikonvulsivum eingesetzt und ist das am häufigsten verschriebene antiepileptische Pharmakon in Deutschland. In den letzten Jahren ist diese Substanz aufgrund der Entdeckung ihrer antineoplastischen Wirkungen, wie zu...

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Bibliographic Details
Main Author: Breidenbend, Carina
Contributors: Visekruna, Alexander (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2021
Online Access:PDF Full Text
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Valproic acid, or its salt valproate, has been used as an anticonvulsant for more than 50 years and is the most commonly prescribed antiepileptic pharmaceutical in Germany. In recent years, this substance has increasingly become the focus of research due to the discovery of its antineoplastic effects, such as the induction of apoptosis in tumor cells. In this context, it was discovered that valproate is a potent histone deacetylase (HDAC) inhibitor. HDACs are enzymes that can modulate various cellular processes such as apoptosis, differentiation and proliferation through epigenetic changes. Inhibition of these thus leads to widespread changes, particularly in tumor cells where HDACs are often dysregulated. Structurally, valproic acid is a synthetically produced branched short-chain fatty acid (SCFA). SCFAs are physiologically produced via anaerobic fermentation of undigested food components in the colon and are considered major metabolites of the intestinal microbiome. Our research group has intensively studied the effect of various SCFAs such as butyrate and valerate on inflammation and carcinogenesis. This has revealed a wide-ranging modulatory influence on the immune system, for which it is known that dysregulation can lead to the development of autoimmune diseases and tumors. A special role is played by T-lymphocytes, which are form a central column of the adaptive immune response and are crucially involved in anti-tumor responses. Based on these findings, new therapeutic approaches have been designed in terms of modulating the immune system using various techniques and substances, for example in the context of immunotherapy using adoptive cell transfer of genetically engineered chimeric antigen receptor (CAR)-coupled T cells. This led us to ask whether valproate could also have a similar immunomodulatory effect and thus potentially be used in the context of such cellular immunotherapy to combat tumors. This should be systematically elucidated in this dissertation, since the influence of valproate on different subtypes of T lymphocytes has not yet been fully clarified. To this end, an HDAC activity assay validated that valproate can significantly suppress HDAC activity in CD4+ and CD8+ T cells. Cell biological and protein biochemical methods such as flow cytometry and enzyme-linked immunosorbent assays (ELISAs) were then used to further characterize the influence of the substance on the different T cell subtypes. For this purpose, T cells were isolated from murine lymph nodes and spleens, differentiated to the corresponding T cell subtypes in cell cultures and treated with valproate as well as valerate. We demonstrated that in all lines considered expression of the immunostimulatory cytokine IFN-γ was induced, which could provide conclusions about a possible phenotype switch toward TH1 cells or cytotoxic T cells (CTLs). Moreover, in TH17 and TC17 cells, the cytokine IL-17A involved in tumor progression was almost completely suppressed by valproate, as was the transcription factor Foxp3 characteristic of regulatory T cells (Tregs). At the same time, our data illustrated that CTLs can upregulate key effector molecules to combat tumors in the presence of valproate. These included the cytokine IFN-γ as well as granzyme B, which is essential for apoptosis initiation in tumor cells. We identified the HDAC inhibitory effect of valproate as a basic mechanistic explanation. A combination of these effects could mediate optimization of the effector functions of CTLs, allowing even more potent tumor targeting. In summary, our data shed light on the immunomodulatory influences of valproate and identify it as a compound with potential therapeutic utility in the context of cellular immunotherapy. Currently, such an adoptive cell transfer has been established in a murine in vivo melanoma model with valproate's "sister substance", valerate, by our research group in cooperation with the University Hospital of Würzburg. Successful tumor elimination has already been demonstrated, raising hopes that valproate may also be used successfully in such a context. This approach should be further investigated in future studies.