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BACKGROUND
Macrophage activation due to TLR4 and TLR7/8 agonists is
limited by NRF2/HO-1 stimulation
Jasmin Ortolan*1, Anna Lisa Furfaro1, Caterina Ivaldo2, Cristina d’Abramo3,4, Luca Giliberto3,4,5, Mariapaola Nitti1
1Department of Experimental Medicine, University of Genova, Italy.
2Department of Surgical and Integrated Diagnostic Sciences, University of Genoa, Italy
3Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Northwell Health, Manhasset, New York, USA
4The Litwin-Zucker Center for Alzheimer's Disease & Memory Disorders, Institute of Molecular Medicine,Feinstein Institutes for Medical Research, Northwell Health System, Manhasset, New York, USA
5Institute for Neurology and Neurosurgery, Northwell Health System, Manhasset, New York, USA
IRF5 dysregulation plays a crucial role in the development of autoimmune
diseases, cancer, neuroinflammation and cardiovascular diseases. Our work
aim at understanding the role played by NRF2/HO-1 in the regulation of IRF5
downstream TLR4 and TLR7/8 stimulation, which has never been
investigated.
•Mouse RAW246.7 and PMA-differentiated human THP-1 cells were cultured under standard conditions.
•To detect dimeric IRF5, a Tris-glycine 10% Acryl/bis non-denaturating gel was used, followed by standard
blotting conditions
•For mouse TNF- detection in cell culture media a mouse TNF alpha SimpleStep ELISA Kit from AbCam
was used.
Protein Ab Brand Dilution
Mouse IRF5
Rabbit anti
-IRF5
Abcam
1:1000
Mouse HO
-1
Rabbit anti HO
-1
OriGene
1:1000
Mouse p
-IkB-α
Mouse anti p
-IkB-α
Santa Cruz Biotech
1:1000
Mouse Tubulin
Mouse anti
-Tubulin
Santa Cruz Biotech
1:10000
Gene FW primer Rv primer
mTNFαFw 5’ ATG AGC ACA GAA AGC ATG A 3’ Rv 5’ AGT AGA CAG AAG AGC GTG GT 3’
mHO-1 Fw 5’ GTA AAG CGT CTC CAC GAG GT 3’ Rv 5’ GGC GGT CTT ACG CTC TTC TG 3’
mACT Fw 5’ TTC TAC AAT GAG CTG CGT GTG 3’ Rv 5’ GGG GTG TTG AAG GTC TCA AA 3’
hIFNα1 Fw 5’ AGA AAT ACA GCC CTT GTG CCT 3’ Rv 5’ AGC AGG GGT GAG AGT CTT TGA 3’
hHPRT1 Fw 5’ CCT GGC GTC GTG ATT AGT GA 3’ Rv 5’ CGA GCA AGA CGT TCA GTC CT 3’
•Intracellular bilirubin generation was analyzed by transfecting
cells with a plasmid coding for UnaG protein (RIKEN BioResource
Center) that becomes fluorescent by reaction with bilirubin. Thus,
transfected cells were treated as needed and, at the end of
treatments, fluorescence was recorded in a multiwell-plate reader
(ex 485-em 520)and normalized on protein cellular content.
RAW264.7 cell exposure to 100ng/ml LPS induced TNF-mRNA expression at 6and 24h
(Fig.1A) confirming the pro-inflammatory activation of cells.
After 24hof LPS treatment, was observed a significant upregulation of HO-1 protein expression
(Fig.1B).
HO-1 was inhibited with 10M SnMP able to further increase TNF-mRNA expression
compared to LPS treatment alone (Fig.1C).
The analysis of IkB-phosphorylation also revealed that SnMP+LPS increased p-IKB at 24h
compared to LPS alone (Fig.1D), pointing out the involvement of HO-1 in the regulation of IKB-
activation.
By fluorimetric detection of bilirubin we tested the efficacy of SnMP that was confirmed on cells
transfected with UnaG plasmid (Fig.1E) that showed the reduction of bilirubin in cells exposed to
SnMP. Thus, HO-1 induction downstream TLR4 stimulation seems to counteract prolonged pro-
inflammatory activation, halting the transcription of TNF-.
The transcription factor NRF2 plays a crucial role in the regulation of antioxidant cell
responses to stressors. NRF2 controls a plethora of genes with pro-surviving activity,
among which heme oxygenase 1 (HO-1) which exerts a potent anti-inflammatory activity
through its metabolites carbon monoxide (CO) and bilirubin. Macrophages trigger
inflammation by recognizing pathogens and tissue damage using different toll like
receptors (TLRs) that, through the activation of complex signalling pathways, lead to
specific cell responses. In particular, TLR4 and TLR7/8 have been proved to have
downstream signal cascades highly interrelated, involving both NF-kB and interferon
regulatory factor 5 (IRF5) transcription factors, that drive pro-inflammatory cytokine
transcription.
•For WB protein analysis abs used are listed below
•qPCR was performed using LightCycler © 96 SW Roche and primers listed below. The gene expression
was calculated with 2-ΔΔCT analysis using mouse actin or human HPRT1 as housekeeping genes.
AB
To better understand if NRF2/HO-1 pathway is able to limit IRF5 activation we treated cells
with sulphorofane (SFN), to activate NRF2 and induce HO-1, and with Resiquimod (R848),
TLR7/8 agonist, positive control of IRF5 activation. 20 nM R848 treatment did not modify HO-1
expression (Fig.3A) but increased TNF-mRNA expression (Fig.3B). HO-1 induction obtained
by using 10M SFN (Fig.3B) was able to strongly reduce TNF-mRNA expression due to
R848 treatment (Fig.3B). Similar results were obtained by analyzing TNF- secreted in
culture media (Fig.3C) To prove the involvement of IRF5, a native page electrophoresis on
cells treated with R848 in combination with SFN has been performed. Indeed, TLR7/8
stimulation induces dimerization of IRF5 that is needed for transcriptional activity. Our results
proved that SFN was able to counteract IRF5 dimerization induced by R848 (Fig.3D). Similar
results were obtained analyzing TNF-αmRNA expression THP-1 cells exposed to R848 and
SFN (Fig.3E).
N T
25M S FN
2 0 n M R 84 8
R8 4 8 + S F N
0.00
0.01
0.02
0.03
0.04
0.05
0.10
0.15
0.20
0.25
0.30
H O - 1 /-a ct in m R N A e x p re s s io n
**
*p< 0.0 1 v s N T
n = 3
p g/m l/n g R N A
NT
2 0 n M R 848
2 5 M S FN
R84 8 + S FN
-0 . 1
0 .0
0 .1
0 .2
0 .3
(IRF5)2
IRF5
D im e ric IR F 5
NT
1M R 848
2 5 M S F N
R 8 48+S F N
0
200
400
600
800
N T
R8 4 8
S F N
S F N + R 8 4 8
0 .0
0 .2
0 .4
0 .6
0 .8
T N F -a /H P R T 1
m R NA e xp re ssio n
*
#
*p < 0 .0 5 v s N T
# p < 0 .0 01 vs R 84 8
n= 3
BA C
DE
N T
2 0 n M R 84 8
25M S FN
R8 4 8 + S F N
0.00
0.01
0.02
0.03
0.04
0.05
0.12
0.14
0.16
0.18
0.20
T N F /- ac tin m R N A e x p re s s io n
***
***p< 0.0 001 vs N T,
S FN , R 84 8+S FN
n = 3
DE
p-IKB-α
Tubulin
LPS SnMP SnMP+
LPS
NT
HO-1
Tubulin
NT 100ng/ml
LPS
H O - 1 /T u b u l in
NT
100ng/m L LPS
0
200
400
600
800
1000 ** p< 0 .0 1
A U F/m g p ro t
1
0
200
400
600
**
***
# #
NT
L P S 1 00
S n M P 1 0 uM
S n M P 1 0 u M + LP S 1 0 0
H e m in 4 0u M
# # p < 0.0 5 vs L PS 10 0
* * p< 0. 01 v s NT
** * p <0.0 01 v s N T
n = 3
FIGURE 1
ABC
N T
10 0 n g /ml LP S
1 0 M S n M P
Sn M P + L P S
0.00
0.05
0.10
0.15
0.20
0.25
T N F /- ac ti n m R N A e xp re ss io n
*
#
*p <0 .0 01 v s N T
# p< 0.0 5 v s L P S 1 00
n = 3
6h 24h
0 .0
0 .1
0 .2
0 .3
N T
1 0 0 ng /m l L P S
T N F /- ac tin m R N A e x p re ss io n
*
*p < 0 .0 1 v s N T
§
§ p < 0 .0 5 v s N T
n = 2 6 h
n = 5 2 4 h
AIM
METHODS
RESULTS
CONCLUSIONS
HO-1 is involved in a homeostatic mechanism downstream TLR4
stimulation that limits IRF5 dependent pro-inflammatory activation
Our data could open a new scenario in understandig
inflammatory disorders related to IRF5 dysregulation and
prevent or treat them by modulating NRF2/HO-1
Exogenous induction of NRF2/HO-1 prevents IRF5
activation downstream TLR7/8
RAW246.7 cells exposed to 100ng/ml LPS for 24hdidn’t modify significantly IRF5 expression
but co-treatment with 10µM SnMP (HO-1 inhibitor) was able to increase IRF5 level (Fig.2A),
suggesting that HO-1 could also control IRF5-dependent pathways.
To prove that, we also analysed THP-1 human monocytes, evaluating interferon Ia (IFN-Ia),
another downstream target of IRF5 activation.
As shown in Fig.2B, the HO-1 inhibition increased INF-Ia mRNA expression even though
without a statistical significance (n=2).
N T
10 0 n g /m l L PS
1 0 M S n M P
S n M P + L P S
0.000
0.002
0.004
0.006
0.008
IF N a 1 /H P R T 1 m R N A e x p r es s io n
FIGURE 2
FIGURE 3
1
3
2
📧jasminortolan92@gmail.com
📧Mariapaola.Nitti@unige.it
IR F 5 /T u b u lin
N T
10 0 ng/m L L PS
1 0 M S n M P
S n M P + L P S
0
100
200
300
* *
**p <0 .0 0 1 v s
1 0 0 n g /m l LP S
p IK B -/ T u b u li n
NT
10 0 ng/m l L P S
1 0 u M S n M P
S n M P + L P S
0
100
200
300
p < 0 .0 5