title: Effects of Chromatin Remodelling on Neutrophil Gene Expression
Creators: name: Makki, FA
Publisher: name: University of Liverpool Repository
Year: 2525

description: Neutrophils have traditionally been studied in the context of acute infection, forming the first line of defence against invading pathogens, but with a restricted set of proinflammatory functions. More recently, it has become appreciated that neutrophils are, in fact, more complex cells that exhibit heterogeneity and functional plasticity, and capable of a wide array of specialised functions that contribute to adaptive immune responses and pathology of chronic inflammatory conditions. These later roles of neutrophils arise largely as a result of changes in their transcriptional activity in response to changes in the microenvironment in vivo and in vitro. However, relatively little is known of the mechanisms regulating this gene expression. Recent work has shown that, in response to long-term exposure to the TLR8 agonist R848, human neutrophils undergo chromatin remodelling and de novo synthesis of TNFa to express the cytokine, IL-6, expression of which by human neutrophils was previously controversial. In this thesis, the effect of R848 on global gene expression and on changes in the chromatin architecture that may control gene expression of neutrophils, alone or in combination with the proinflammatory factors TNFa or GM-CSF was investigated. Neutrophils were freshly-isolated from healthy individuals and treated with R848, either alone or in combination with TNFa or GM-CSF for different time periods. Histone post- translational modifications (e.g. pan and residue-specific acetylation and citrullination) were investigated by western blotting. RNA-seq was also performed on ultrapure neutrophils (&gt;99%) stimulated with the above agents for 6 h, followed by computational and bioinformatics analysis to predict activation mechanisms. All major findings were validated by qRT-PCR at the mRNA level, and, in some cases, at the protein level. Further functional assays, such as apoptosis, ELISA and measurement of ROS, were performed as appropriate. R848, alone or in combination with the cytokines, induced acetylation of H3 after 6 h of treatment while it decreased H3 citrullination. These effects were more pronounced after 18 h of stimulation. Acetylation and transamination correlated with activation of gene expression in response to R848, but the effects of R848 used together with the cytokines were not simply additive. Unlike the cytokines, R848 induced the expression of interferon- response genes (IRGs) in an interferon-independent, but endogenous TNFa-dependent, manner. Exogenous TNFa failed to have any further effect on R848-induced expression of IRGs, while GM-CSF inhibited their induction. In contrast, R848 plus TNFa synergistically increased the expression of members of the IL-12 family cytokines. IL-23 was found, for the first time, to be expressed and secreted by neutrophils under such co-stimulation. On the other hand, R848 plus GM-CSF synergistically induced the expression of chemokines of the CC family and this effect was entirely dependent on endogenous TNFa. Finally, R848 and the cytokines had a pro-survival effect on neutrophils, but this effect was the outcome of different and complementary patterns of regulation of the anti-apoptotic proteins Mcl-1 and Bfl-1. The absence of an additive effect in apoptosis delay in response to the combined treatments excluded the possibility that TNFa or GM-CSF further increased gene expression in R848-treated neutrophils, simply because they further enhance neutrophil survival. In conclusion, these data show, for the first time, that chromatin remodelling agents, such as R848, can induce substantial changes in the patterns of gene expression of human neutrophils, when used alone or in combination with pro-inflammatory cytokines. The data predict that such chromatin re-modelling may occur in vivo in certain inflammatory conditions, substantially changing how neutrophils can respond to local cytokines. The data also show the importance of endogenous TNF expression and how this may result in autocrine signalling to regulate neutrophil function in human disease.