Understanding the chemotherapy-induced bystander effect; an evaluation of the role of cytokine storm in an in-vitro model of the human bone marrow

Asurappulige, Harshini

Abstract

The phenomenon of donor cell leukaemia (DCL) occurs following haematopoietic stem cell transplantation (HSCT), but its aetiology is currently unknown. Previous work within the research group identified a chemotherapy-induced bystander effect (CIBE) in an in vitro bone marrow (BM) model, which has been suggested to play a role in DCL. As DCL predominantly occurs as myeloid lineages, this research study investigated the role of cytokines in CIBE. Utilising the same in vitro model, the induction of cytokine ‘storm’ was measured in the human bone marrow mesenchymal stem cell line HS-5. Five candidate cytokines (TNFα, IL-6, TGF-β1, G-CSF and GM-CSF) were selected and tested, either alone or in combination, for their ability to directly induce genotoxicity in the form of micronuclei in TK6 cells. IL-6 was focussed on for mechanisms of action; IL-6 production was knocked down in HS-5 cells, and IL-6 signalling pathways were inhibited in TK6 cells to ascertain the role of IL-6 in genotoxicity.
The chemotherapeutic agents chlorambucil and mitoxantrone, induced an overall increase in cytokine secretion, with all 80 cytokines detected in either treated or untreated cells. This heightened cytokine response, resembled a cytokine storm, and for the first time, provided a comprehensive profiling of cytokine secretion in the HS-5 cell line. ELISA results confirmed storm levels of expression of the five selected cytokines, by HS-5 cells in-vitro. Additionally, the study revealed the direct genotoxic effects of these five cytokines on the TK6 cell line, with combination treatments exacerbating genotoxicity.
IL-6 emerged as a central candidate, with the significant baseline expression and sustained elevated secretion post-chemotherapy, as well as a notable contribution to genotoxicity induction in bystander cells. The central role of IL-6 in CIBE was shown for the first-time using IL-6 gene knockdown in HS-5 cells, resulting in reduction of micronuclei in bystander TK6 cells to levels similar to untreated cells; this proved significant for chlorambucil (p<0.05). This novel finding underscores IL-6 as a central contributor to the induction of genotoxic effects in studies of bystander models.
Efforts to mitigate IL-6-induced genotoxicity using JAK/STAT and RAS/MAPK pathway inhibitors to TK6 yielded unexpected results, in that significant micronuclei increased in the presence of the pathway inhibitors FLLL-32 and BAY-293 with IL-6 treatment. These data suggest that alternative pathways, perhaps more relevant to genotoxicity, may be promoted when using these inhibitors in the presence of IL-6, highlighting the complexity of IL-6 signalling pathways.
In conclusion, this study illuminates the intricate dynamics of cytokines in the context of CIBE and their possible contribution to DCL, offering valuable insights for further investigations and potential therapeutic interventions. The complexities unveiled underscore the need for continued exploration into the multifaceted mechanisms governing HSCT outcomes and associated complications.

• Thesis Type: Thesis
• Deposit Date: Jan 3, 2024
• Publicly Available Date: Jul 25, 2024
• Public URL: https://uwe-repository.worktribe.com/output/11548758
• Award Date: Jan 3, 2024