The role of EZH2 in myeloid driven inflammation

UoM administered thesis: Phd

  • Authors:
  • Thomas Hopwood

Abstract

The aberrant activities of the histone methyl transferase EZH2 have long been implicated in cancer development and progression. However, EZH2 has recently emerged as a coordinator of multiple physiological mechanisms including the circadian clock and both adaptive and innate arms of the immune response. Recent evidence has emphasised the importance of EZH2 in cell plasticity and the response to changing environmental stimuli. In this thesis, I examined the role of EZH2 in myeloid cells utilising in vivo genetic targeting in conjunction with Streptococcus pneumoniae infection and exposure to lipopolysaccharide (LPS), ex vivo investigations and in vitro immune challenges. My first studies of Ezh2 knock-out myeloid cells revealed an increased output of inflammatory cytokine mRNA after an LPS challenge in vitro. Moreover, in vivo experiments showed that the inhibition of EZH2 using UNC1999, an S-adenosyl-L-methionine competitive inhibitor, alongside genetically targeted myeloid cells revealed enhanced systemic inflammation after an intraperitoneal injection of LPS. I observed that Ezh2 deletion in pulmonary macrophages had limited effect on the response to nebulised LPS. However, the clearance of S. pneumoniae bacteria in the lung and the restriction of bacterial entry into the blood stream was significantly greater in the absence of Ezh2 in alveolar and interstitial macrophages. I then isolated genetically targeted macrophages and, utilising Nanostring and RNA-sequencing, identified the TLR-4:NF-kB signalling cascade as a potentially perturbed circuit in the absence of Ezh2. Transcription factor activation assays in conjunction with a TLR-agonist panel showed that the loss of Ezh2 selectively targeted the TLR-4 receptor cascade resulting in higher Rel-A activation. The loss of EZH2 in neutrophils has previously been shown to alter migration efficiency. I showed that this resulted in a reduced response to nebulised LPS and a loss of ability to control intranasal infection with S. pneumoniae, a phenotype which could be rescued by tail vein injection of Ezh2 intact neutrophils. Overall, these results suggested a far more dynamic role for EZH2 than has previously been observed. This revealed EZH2 to be vitally important in producing a co-ordinated and appropriate immune response to an LPS challenge in vitro and in vivo and in response to bacterial infection. This evidence provided a significant warning to the administration of EZH2 inhibitors as a treatment for cancer progression.

Details

Original languageEnglish
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Supervisors/Advisors
Award date31 Dec 2018