Abstract |
Inflammation is the first barrier against external and internal stimuli. It’s a multistep
process including several complex mechanisms and driven by a great number of mediators
and immune cells. Macrophages are effector cells of the immune system involved both in
the initiation and resolution of inflammation that derive from monocyte precursors. They
acquire an array of phenotypes broadly characterized as M1 and M2-like phenotypes. M1
macrophages express pro-inflammatory cytokines such as TNFa, IL-6 and IL-12 and secrete
nitric oxide. On the other hand, M2 macrophages are involved in the resolution of
inflammation and wound healing by expressing anti-inflammatory mediators such as
Arginase1, MRC1, Fizz1 and Ym1. A broad spectrum of transcriptional regulatory programs
such as the NF-kB and the MAPK/AKT signaling cascades ensure a well-coordinated
inflammatory response. Tight coordination of the inflammatory response is pivotal for
maintaining homeostasis as inflammatory dysregulation potentially results in the initiation
of inflammation-related diseases such as autoimmunity, atherosclerosis and cancer.
In the first part of this study, we screened for new chemical compounds with antiinflammatory
properties deriving from the marine algae Laurencia glandulifera and
Dictyopteris membranaceae. Having screened several compounds we showed that diterpenes
1-3 exhibited significant anti-inflammatory properties in RAW 264.7 macrophages inducing
anti-inflammatory mediators’ production in naïve macrophages whereas reduced the
production of pro-inflammatory cytokines in LPS activated macrophages. Diterpenes 1 and
3 exhibited the most potent anti-inflammatory properties with IC50 of 2.32μM and 2.92μM,
respectively. Interestingly, diterpenes 2 and 3 managed to alleviate intestinal inflammation
in a model of DSS induced colitis, reducing pro-inflammatory cytokine production and
maintaining intestinal tissue integrity. Moreover, we showed that disulfides 4-6 deriving
from D. membranaceae suppress macrophage activation in response to LPS stimulation by
reducing pro-inflammatory cytokine production and this is mediated through the
MAPK/AKT pathway exhibiting elevated AKT phosphorylation and diminished ERK1/2
phosphorylation. Then, we aimed to investigate the potential use of total dried algae
Laurencia and Dictyopteris as dietary supplements and their potential role in alleviating
inflammation related diseases. Indeed, we showed that diet supplementation with Laurencia
or Dictyopteris reduced insulin resistance in high-fat induced obese mice and modulated the intestinal microbiome. Specifically, it not only maintained colonization of rare microbial
taxa but also induced colonisation of beneficial bacteria in a sex-dependent manner. Using
also a DSS-induced colitis model we showed that diet supplementation with Dictyopteris
ameliorated the colitis phenotype in mice by reducing colon length shortening and proinflammatory
cytokine and chemokine secretion.
Although there is plenty of information on macrophage activation and polarization,
little is known about the epigenetic regulation of the same processes. In the second part of
the present study, we focus on investigating the role of histone demethylases PHF2 and
PHF8 in macrophage activation and metabolism. First, we showed that PHF2 and PHF8 are
differentially expressed upon TLR4-mediated activation indicating differential roles in
macrophage activation. PHF2 expressed early (2h) upon LPS stimulation, whereas PHF8
expressed late (12h) following LPS stimulation, indicating a potential role in endotoxin
tolerance. Then, we generated stable overexpressing and knock-out RAW 264.7
macrophages using a CRISPR-Cas9 lenti-viral system and studied their activation profile in
response to LPS stimulation. We showed that PHF2 positively regulated pro-inflammatory
gene production by actively binding on inflammatory gene promoters as well as it was
necessary for the phagocytic capacity macrophages. On the contrary, PHF8 is a negative
regulator of macrophage activation reducing pro-inflammatory gene expression in the
context of endotoxin tolerance by actively binding on inflammatory gene promoters and
positively regulates the phagocytic capacity of macrophages. In addition, PHF8 is a major
regulator of metabolism during macrophage activation reducing major glycolytic genes and
negatively regulating mitochondrial biogenesis and function. Specifically, PHF8 over
expressing macrophages possess reduced mitochondrial mass and impaired mitochondrial
function, exhibiting reduced ATP production and basal respiration capacity.
Overall, this study adds to the current knowledge on macrophage activation and
metabolism and proposes new chemical compounds as anti-inflammatory drugs with
potential pharmaceutical interest in inflammatory responses. Moreover, it shades light into
the field of the epigenetic regulation of innate immunity unravelling the major functions of
histone demethylases PHF2 and PHF8 in macrophage activation and metabolism.
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