Abstract
Cytokines are critical for initiating, mediating and resolving the immune response. Macrophages are key cytokine-secreting cells that can express both pro- and anti-inflammatory cytokines. When the production of pro-inflammatory cytokines becomes dysregulated, cytokine storm can occur. Infection, autoimmune diseases, and treatment with cancer immunotherapies can trigger this, and multiple-organ failure, seizures and death can result. There are several methods for managing cytokine storm, however, there is a need for safe therapies that successfully alter the cytokine profile to become more anti-inflammatory.
Ascorbate has many functions in the cell, including a role as an essential cofactor for TET2. TET2 is a DNA demethylase, and DNA demethylation is typically associated with an increase in gene expression. Ascorbate can support immune cell function and influence the production of cytokines. The ability of ascorbate to regulate cytokine production via TET2 cofactor activity has not been sufficiently researched. Therefore, the aim of this project was to determine the effect of ascorbate on the epigenetic control of human macrophage cytokine production, focusing on whether ascorbate encourages a pro- or anti-inflammatory phenotype by modulating TET2 activity. The research question was investigated using primary human macrophages.
Intracellular ascorbate levels increased dramatically when macrophages were cultured with 100 μM ascorbate, meaning the macrophages were successfully loaded. Addition of ascorbate into the cell culture media did not significantly alter cell viability. PBMCs do not survive in culture, and by driving differentiation of monocytes into macrophages, monocyte colony stimulating factor allows cells to survive in vitro.
ii
Macrophages stimulated with LPS for 24 hours secreted significantly more IFN-γ, TNF-α, MCP-1, IL-6, and IL-12p70 when treated with ascorbate during differentiation and stimulation. These are all pro-inflammatory cytokines, excepting IL-6, which is both pro- and anti- inflammatory. This suggests that ascorbate drives a predominantly pro-inflammatory cytokine response 24 hours after stimulation with LPS. More research is recommended to determine whether ascorbate and time after stimulation affects the macrophage cytokine profile. Testing other stimuli, such as the anti-inflammatory stimulus IL-4/IL-13, is also suggested.
Global levels of 5-hydroxymethylcytidine, the product of TET2 activity, did not increase when human macrophages were treated with ascorbate. However, the ability of ascorbate to drive TET2 activity in vitro is well established. Ascorbate may have promoted TET2-catalysed demethylation at specific gene loci, such as those encoding cytokines, but further investigation was outside the scope of this study. Overall, this research demonstrates that ascorbate alters the cytokine profile of stimulated macrophages, and this could be via epigenetic regulation.