| Abstract |
Summary
Endotoxin
tolerance
occurs
to
protect
the
organism
from
hyperactivation
of
innate
immune
responses,
primarily
mediated
by
macrophages.
Regulation
of
endotoxin
tolerance
occurs
at
multiple
levels
of
cell
responses
and
requires
significant
changes
in
gene
expression.
During
macrophage
activation,
induced
expression
of
miR-‐155
and
miR-‐146a
contributes
to
the
regulation
of
the
inflammatory
response
and
endotoxin
tolerance.
Herein,
we
demonstrate
that
expression
of
both
miRNAs
is
co-‐
ordinately
regulated
during
endotoxin
tolerance
by
a
complex
mechanism
involving
mono-‐allelic
inter-‐chromosomal
association,
alterations
in
histone
methyl
marks
and
transcription
factor
binding.
Upon
activation
of
naïve
macrophages,
Histone3
was
tri-‐methylated
at
lysine4
(H3K4me3)
and
NFkBp65
was
bound
on
both
miR-‐155
and
miR-‐146a
gene
loci.
However,
at
the
stage
of
endotoxin
tolerance
both
miR
gene
loci
were
occupied
by
C/EBPβ,
NFkBp50
and
the
repressive
Histone3
marks
H3K9me3.
DNA
fluorescence
in
situ
hybridization
(DNA-‐FISH)
experiments
revealed
mono-‐allelic
inter-‐chromosomal
co-‐localization
of
miR-‐155
and
miR-‐146a
gene
loci
at
the
stage
of
endotoxin
tolerance,
while
RNA-‐DNA-‐FISH
experiments
showed
that
the
co-‐localized
alleles
were
silenced,
suggesting
a
common
repressive
mechanism.
Genetic
ablation
of
Akt1,
which
is
known
to
abrogate
endotoxin
tolerance,
abolished
induction
of
loci
co-‐localization
and
C/EBPβ
binding,
further
supporting
that
this
mechanism
occurs
specifically
in
endotoxin
tolerance.
This
thesis
demonstrates
that
two
miRNAs
are
co-‐ordinately
regulated
via
gene
co-‐localization
at
the
three
dimensional
chromatin
space,
similar
transcriptional
machinery
and
Histone3
methylation
profile,
contributing
to
the
development
of
endotoxin
tolerance.
Further
insight
into
the
role
of
AKT
in
regulation
of
M1/M2
polarization,
revealed
the
essential
role
of
these
microRNAs
in
macrophage
phenotype.
Akt1
ablation
promotes
miR-‐155
expression
in
LPS-‐stimulated
macrophage.
Measuring
miR-‐155
in
Akt2-‐depleted
macrophages
revealed
that
Akt2
ablation
had
the
opposite
effect,
reducing
miR-‐155
expression
in
both
resting
and
LPS-‐activated
macrophages.
Therefore,
down-‐regulation
of
miR-‐155
in
Akt2-‐defiecient
macrophages
results
in
up-‐regulation
of
its
target
C/EBPβ
and,
consequently,
in
the
induction
of
Arg1,
a
hallmark
of
M2
macrophage
polarization.
Akt2
deficiency
9
resulted,
however,
in
a
significant
upregulation
of
miR-‐146a,
which
mediates
M1
phenotype
suppression
and
assure
endotoxin
tolerance.
miR-‐146a
transfection
in
WT
macrophages
was
able
to
inhibit
iNOS
induction
while
miR-‐146a
suppression
in
Akt2-‐depleted
mice
resulted
in
upregulation
of
iNOS
expression.
The
physiological
and
clinical
significance
of
these
miRs
in
sepsis
was
supported
by
further
data
in
humans.
Critically
ill
patients
with
impaired
immune
responses
(CARS
syndrome)
are
associated
with
increased
miR-‐155
and
miR-‐146
expression.
In
vivo
transferring
of
these
miRs
by
using
amphoteric
liposomes
seems
to
be
highly
promising,
underlining
miR-‐155
and
miR-‐146
as
potential
novel
molecular
biomarkers
of
macrophage
sensitivity
and
CARS
syndrome
and
tools
for
therapeutic
purposes.
|
Regulation of microRNAs during activation of macrophages / Christina Doxaki.
