Abstract |
Macrophages are the central mediators of inflammatory responses, they constitute
the basis of innate immunity, while, at the same time, exhibit an important role in several
functions of homeostasis1,2
. Macrophage activation requires strict and multiple layers of gene
regulation, which may lead in a vast spectrum of pro- or anti- inflammaroty phenotypes,
termed as “M1” or “M2”, respectively2,3
. A growing body of evidence indicates that this
enormous macrophage plasticity is excessively regulated at the epigenetic level and that
different metabolic adaptations influence the outcome of the immune response.
In the present postgraduate thesis, the role of histone demethylases “PHF8” (KDM7B)
and “PHF2” (KDM7C) in macrophage activation was investigated, including the expression of
genes and secretion of cytokines that are implicated in TLR4-induced inflammation, the
impact on immunometabolic regulation, as well as macrophage phagocytosis capacity. Finally,
a possible interaction of PHF8 with AKT kinases, which have a crusial role in immune response,
was tested.
For that purpose, stable macrophage cell-lines which under- or over- express the
aforementioned proteins were created with CRISPR. Several molecular- and cell- biology
techniques were used, such as si RNA on primary macrophages, FACS, RT-qPCR, ELISA, and
WB.
It was exhibited that PHF8 plays both a pro- and an anti- inflammatory role in
macrophage activation. Furthermore, it was demonstrated that PHF8 downregulates
mitochondrial biogenesis, and, by extension, oxidative phosphorylation. Overexpression of
PHF8 led to elevated phatocytosis capacity. Initial indications suggested that AKT1 and AKT2
kinases control PHF8 expression in an opposite manner and that they may affect its total and
phosphorylated protein levels.
Histone demethylase PHF2 was found to exhibit a strong pro-inflammatory activity. It
was displayed that it regulates a number of immune regulation genes in an opposite way than
PHF8, suggesting a potential competitive relashionship between the two proteins. Moreover,
in some cases similar gene regulation was also observed, indicating a synergistic mechanism
of action. Finally, it was displayed that PHF2 is necessary for effective phagocytosis.
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