Heme oxygenase-1—Dependent anti-inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice
Heme oxygenase-1-Dependent anti- inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice
Ghewa A. El-Achkar 0 1 3
May F. Mrad 0 3
Charbel A. Mouawad 0 3
Bassam Badran 2 3
Ayad A. Jaffa 0 3
Roberto Motterlini 1 3
Eva Hamade 2 3
Aida HabibID 0 3
0 Department of Biochemistry and Molecular Genetics, American University of Beirut , Beirut , Lebanon
1 INSERM U955 , Equipe 12 , University Paris-Est, Faculty of Medicine, Cr e ?teil, France, 3 Nehme and Therese Tohme Multiple Sclerosis Center, American University of Beirut Medical Center , Beirut , Lebanon
2 Laboratory of Cancer Biology and Molecular Immunology, Faculty of Sciences I, Lebanese University , Hadath, Beirut , Lebanon , 5 INSERM-U1149, CNRS-ERL8252, Centre de Recherche sur l'Inflammation, Sorbonne Paris Cit e ?, Laboratoire d'Excellence Inflamex, Faculte ? de M e ?decine, Site Xavier Bichat, Universit e ? de Paris , Paris , France
3 Editor: William Durante, University of Missouri Health Care , UNITED STATES
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This study has been funded with
supports from the National Council for Scientific
Research in Lebanon (to AH and EH, 02-05-16),
the Medical Practice Plan (MPP to AH), the Faculty
of Medicine of the American University of Beirut (to
AH), the Ecole Doctorale Des Sciences et
Technologie of the Lebanese University (to E.H.),
Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have
previously reported that macrophages treated with statins increased the expression of
heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress
protein, responsible for the degradation of heme. In the present study, we investigated the
effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo.
Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.
p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were
administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae,
was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory
markers were determined in exudates collected from the pouch 24 hours post zymosan
injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with
atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin
domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the
air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells
expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice
treated with atorvastatin showed a significant reduction in cell influx in response to zymosan,
and in the expression of proinflammatory cytokines and chemokines such as interleukin-1?,
monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with
atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the
inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective
role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of
neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of
atorvastatin treatment on the modification of their relative proportion. These findings identify
INSERM (To RM), the Universite? Paris-Est Cre?teil
(to RM). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as
an in vivo anti-inflammatory agent.
Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase and inhibit cholesterol synthesis and low-density lipoprotein cholesterol (LDL-C).
Satins have been shown to have many beneficial pleiotropic effects beyond their ability to lower LDL-cholesterol, that include anti-inflammatory, antioxidant, anti-proliferative, and anti-thrombotic actions [1, 2].
Heme oxygenase (HO)-1 is the inducible isoform of heme oxygenase responsible for the
oxidative degradation of heme. Its products contribute to the antioxidant, anti-inflammatory
and anti-apoptotic actions of HO-1 [
]. HO-1 is induced by pro and anti-inflammatory
], lipopolysaccharide (LPS) [
] and nitric oxide (NO) [
]. HO-1 has been described
in vivo as a downstream effector of interleukin (IL)-10  and to play a role in the resolution
of inflammation [
As part of the feedback mechanisms, macrophages with anti-inflammatory activities are
activated. Subsets of anti-inflammatory macrophages are characterized with the expression
of arginase-1, mannose receptor-1 or the lectin C-type lectin domain family 7 member A
(CLEC7A) and are referred to as Th2 ?driven macrophage or M2 macrophages [
important in the tissue repair and the resolution of inflammation. Multiple studies suggested a role
of HO-1 induction in the polarization of macrophages into an anti-inflammatory M2
]. Zhang et al have shown that deletion of HO-1 in the myeloid lineage exacerbates
the pro-inflammatory phenotype of bone marrow-derived macrophages in response to
lipopolysaccharide and limits the anti-inflammatory phenotype in response to interleukin-4 [
Recent studies have shown that statin induces HO-1 in murine macrophage cell lines RAW
264.7 and J774A.1, in NIH 3T3 fibroblasts and in primary murine peritoneal macrophages
]. On the other hand, statins reduced the LPS-induced prostaglandin E2 synthesis, and
cyclooxygenase-2 (COX-2) expression in monocytes . However, little is known about the
effect of statins in vivo and the mechanisms underlying its beneficial effects in inflammation
]. Statin administration to mice was shown to increase the expression of HO-1 in heart
and lung tissue . Few studies investigated the mechanisms involved in the role of statins
in inflammation in vivo but did not assess the role of HO-1 [
]. HO-1 has been shown to
play a role in the anti-inflammatory effects of some drugs including the cannabinoid receptor
2 agonist JWH-133 [
In the present study, we employed the air pouch model in C57BL/6 mice to assess the
effect of atorvastatin on inflammation. We first determined the expression of the
anti-inflammatory genes in response to atorvastatin alone and characterized the subtypes of immune cells
recruited in response to zymosan and /or atorvastatin. We next demonstrated that the effect of
atorvastatin on zymosan-induced leukocytes recruitment and inflammation involves HO-1 as
a potential anti-inflammatory player.
Materials and methods
BSA, DMSO and zymosan A from Saccharomyces cerevisiae (Z4250) were from Sigma
Aldrich (St Louis, MO, USA). Tin protoporphyrin IX (SnPPIX) (Sn749-9) was obtained from
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Frontier Scientific (Logan, UT, USA). Atorvastatin (10493) and prostaglandin (PG) E2 EIA
measurement reagents were from Cayman Chemicals Company (Ann Arbor, MI, USA). Kits
for ELISA for mouse IL-1? (88-5019-77) and monocyte chemoattractant protein-1 (MCP-1)
(88-7391-86) were purchased from Thermo Fisher Scientific (Waltham, MA USA). Antibodies
for flow cytometry were from BioLegend (San Francisco, CA, USA).
Subcutaneous dorsal air pouch model. C57BL/6J female mice (20?25 g, 8 week-old)
were obtained from Charles River (Ecully, France) and the animal facility of the American
University of Beirut. They were housed 5 per cage with cotton cocoon as enrichment environ
ment in temperature- and humidity-controlled rooms, kept on a 12-hr light-dark cycle, and
provided with food and water ad lib in the animal facility of the American University of Beirut.
Body weight and food intake were monitored three times a week throughout the study period.
Approval for use of animals was obtained from the Institutional Animal Care and Use Com
mittee of the American University of Beirut (IACUC # 16-11-393).
Atorvastatin (5 mg/kg, i.p.) was diluted in DMSO: saline, 1:49 (v:v), and SnPPIX (12 mg/kg,
i.p.) in saline [
] and mice were injected every day for 10 days (Figs 1A and 2A). Air
pouches were established in mice as described previously . Briefly, mice were anesthetized
using isoflurane inhalation and air pouches were produced on day 5 by subcutaneously
injecting 5 ml of sterile air into the back of the mice. On day 8, pouches were maintained by
reinflation with 2.5 ml of sterile air. On day 10, 0.5 ml of sterile saline solution or 0.5 ml of 1%
zymosan in saline (w:v) was injected in the air pouch. 24 hours after the injection of zymosan,
mice were sacrificed by CO2 inhalation and the exudates were collected in 1 ml of Hanks buffer
containing 0.32% trisodium citrate to prevent cell aggregation. The number of cells in exudates
was counted using improved Neubauer hemocytometer. Supernatants were kept at -80?C for
the measurement of PGE2, mouse IL-1? and MCP-1. Total RNA was extracted from cell pellets
for real time RT-PCR. For vehicle and atorvastatin?treated alone, twelve mice were injected
and cells were pooled from 3 different mice. For zymosan, zymosan + atorvastatin and
zymosan + atorvastatin + SnPPIX, eight mice were used in each experimental group.
RT-PCR analysis. Cell pellets were suspended in QIAzol (QIAGEN, 79306) and extracted
as previously described [
]. 1 ?g of total RNA was reversed transcribed using High-Capacity
cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368813). RT-PCR was carried
out on CFX384 cycler using ABsolute Blue QPCR Mix, SYBR Green (Thermo Fisher Scientific,
AB4166B) and the primers obtained from TIB Molbiol (Berlin, Germany). Oligonucleotide
sequences were according to the references [
] and , except for Hmox1, Ptgs2, Pges and
Nos2, which were as follow: Hmox1 (F): GGCTAAGACCGCCTTCCTGCTC; Hmox1 (R):
GCAGGGGCAGTATCTTGCACCAG; Ptgs2 (F): AGACAGATTGCTGGCCGGGTTGCT; Ptgs2 (R):
TCAATGGAGGCCTTTGCCACTGCT; Pges (F): GATGGAGAGCGGCCAGGTGC; Pges (R):
GGCAAAAGCCTTCTTCCGCAGC; Nos2 (F): CCCTTGTGCTGTTCTCAGCCCAAC; Nos2 (R):
GGACGGGTCGATGTCACAT GCA. Gene expression was normalized to the housekeeping gene
Flow cytometry analysis. Flow cytometry analysis of cells collected from the air pouch
was performed. To characterize the inflammatory subsets in the pouch, multi-color
fluorescence cell staining was conducted using the combination of the following antibodies as
indicated in S1 Table: CD45 (PerCP-Cy5.5), TCR ? (FITC), CD11b (BV450/50), and Ly-6G (PE).
Dead cells were excluded using zombie yellow viability kit (BioLegend 423104) or Live/Dead
Fixable Blue dead stain kit (Thermo Fisher Scientific, L23105). For HO-1 and CD206 detection, air pouch cells were fixed with the Fixation Buffer (BioLegend 420801) and treated with
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Fig 1. Atorvastatin induces the expression of anti-inflammatory genes in air pouch of C57BL/6J mice. A) Outline of the air pouch
model. Atorvastatin (5 mg/kg, i.p.) or vehicle was injected every day for 10 days in C57BL/6J mice and cell were harvested as described in
the method section, B) Gene expression of Hmox1, Arg1, Clec7a, and Mrc1. C) Representative gating strategy for the quantification of
the proportion of cells expressing HO-1 cells in air pouches of vehicle- or atorvastatin-treated mice. Viable CD45+ cells were gated in the
total exudate cells. Neutrophils were identified as viable CD45+CD11b+Ly-6G+ cells and were excluded from subsequent monocyte/
macrophage gating. Monocyte/macrophage were selected as viable CD45+CD11b+Ly-6G- cells, D) Representative flow cytometry dot
plots of HO-1 expression and summary data. Mean ? SEM (n = 6). P<0.05, P<0.01.
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Fig 2. Atorvastatin inhibition of zymosan-induced cell recruitment to the air pouch is HO-1 dependent and does not involve
modification of leukocyte subsets. A) Outline of the air pouch model of inflammation induced by zymosan. Atorvastatin (5 mg/kg, i.p.) and/
or SnPPIX (12 mg/kg, i.p.) were injected daily for 10 days. On day 10, air pouches of mice were injected with 0.5 ml of saline and/or 1% (w/v)
zymosan in saline. The exudates were collected after 24 hours. B) Number of cells the air pouches. C) Representative gating strategy for the
characterization of the exudate of air pouches injected with zymosan in mice treated with vehicle or atorvastatin. Viable CD45+ cells were
gated in the total exudate cells. T-cells were identified as viable CD45+TCR?+ cells and were excluded form subsequent gating. Neutrophils
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were identified as viable CD45+TCR?-CD11b+Ly-6G+ cells and monocyte/macrophage as viable CD45+TCR?-CD11b+Ly-6G- cells. D)
Summary data. Mean ? SEM (n = 8?9). p<0.01, p <0.001.
the intracellular staining permeabilization Wash Buffer (BioLegend 421002) according the
manufacturer?s instructions. Rabbit polyclonal anti-HO-1 1/200 [
], rat anti-rabbit IgG?
FITC (Thermo Fisher Scientific F-2765), and anti-CD206 (for MRC1, BioLegend 141705)
were used. Isotype controls and a control without the primary anti-HO-1 antibody were run.
Three mice were pooled for the treatment with vehicle or atorvastatin alone. Analysis was per
formed at the faculty of medicine core facility at the AUB using FACS Aria SORP (BD
Biosciences). Data were analyzed using FlowJo (TreeStar, Ashland, Or). Neutrophils were defined as
living (live/dead cell stain negative) CD45+TCR?-CD11b+Ly-6G+. T cells were defined as
living CD45+TCR?-. Infiltrating monocytes/macrophages are defined as viable
TGCTGGCAGGGTTGCTGGTGGTA; COX-2 R: GGGCTTCAGCATAAAGCGT
TTGCGG; mPGES-1 F: CCTGGTGATGAGCAGCCCGG; mPGES-1 R: GGCAAAGGCCTTCTTCCGCAG; COX-2 F:
TGCTGGCAGGGTTGCTGGTGGTA; CO2 R: GGGCTTCAGCATAAAGCGT TTGCGG; mPGES-1 F: CCTGGTGATGAGCAGCCCGG; mPGES-1 R: GGCAAAGGCCTTCTTCCGCAG;
Statistical analysis. Statistical analysis was performed using GraphPad Prism 5 (La Jolla,
CA 92037 USA). Results are presented as the mean ? SEM. The level of statistical significance
was determined by Mann-Whitney and p<0.05 was considered statistically significant.
Atorvastatin induces the expression of anti-inflammatory markers in cells
isolated from the sterile dorsal air pouch
We first investigated the effect of atorvastatin alone in mice. We assessed the levels of gene
expression in cells isolated from the sterile cavity of the air pouch after 10 days treatment with
atorvastatin (5 mg/kg, i.p.) (Fig 1A). HO-1 was significantly increased in atorvastatin-treated
mice compared to untreated mice (p<0.05) (Fig 1B). We also checked the expression of some
anti-inflammatory genes. Atorvastatin significantly increased the expression of arginase-1
(Arg-1) (p<0.01), C-type lectin domain family 7 member A (CLEC7A), and Mannose receptor
C-type 1 (MRC1) (p<0.05). To determine the cell types that express HO-1 following atorva
statin treatment, we analyzed their phenotype in the air pouch of mice injected with
atorvastatin alone (Fig 1C and S1 Fig). Leukocyte expressing HO-1 and the anti-inflammatory
marker, MRC1, (also as CD206) were increased in mice treated with atorvastatin compared to
vehicle. Moreover, monocyte/macrophage gated on leukocytes (CD45+) and expressing HO-1
were increased compared to mice treated with vehicle alone (Fig 1D).
Thus, atorvastatin significantly induced the expression of HO-1 and other anti-inflammatory markers in resident cells of the air pouch.
HO-1 mediates the inhibitory effect of atorvastatin on zymosan-dependent
We next investigated whether HO-1 is involved in the inhibitory effect of atorvastatin on the recruitment of cells in the air pouch. Mice were treated with atorvastatin and/or SnPPIX, an
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inhibitor of heme oxygenase, daily for 10 days prior to inducing inflammation in the air pouch
with zymosan (Fig 2A). Fig 2B shows that zymosan injection in the air pouch increased
significantly the number of recruited cells compared to vehicle. Atorvastatin administration
significantly reduced zymosan-induced cell recruitment by 61% (p<0.001, atorvastatin+zymosan vs
zymosan) in response to zymosan alone. Co-treatment with SnPPIX abolished the inhibitory
effect of atorvastatin on cell recruitment (p<0.01, atorvastatin+zymosan+SnPPIX vs
atorvastatin+zymosan) indicating a role for HO-1 in the anti-chemotactic effect of atorvastatin
We further characterized the inflammatory subsets in the pouch using flow cytometry anal
ysis (Fig 2C). Zymosan-recruited leukocytes (CD45+) were 96% of total viable cells in the air
pouch, and consisted mainly of neutrophils (75% of CD45+), monocytes/macrophages (11% of
CD45), and T cells (2.9% of CD45+). The proportions of zymosan-recruited leukocytes sub
populations were not modified by atorvastatin (Fig 2D).
HO-1 is involved in the effect of atorvastatin on zymosan-induced
expression of proinflammatory genes
Next, we analyzed the expression of some proinflammatory cytokines and chemokines. Zymo
san-injected air pouches showed a significant increase in gene expression of Il1a, Il1b, Il6, and
Tnfa. Atorvastatin significantly decreased the gene expression of Il1a by 82% (p<0.001), 73%
for Il1b by 73% (p<0.05), Il6 by 81% (p<0.001), and Tnfa by 67% (p<0.05). Mice co-treated
with SnPPIX reversed the effect of atorvastatin (Fig 3A). Fig 3B illustrates the gene expression
of chemokines under the same experimental conditions. Atorvastatin also reduced the
expression of Ccl3 by 68% (p<0.05), Ccl4 by 68% (p<0.05) and chemoattractant chemokine Cxcl1 by
This inhibitory effect of atorvastatin on zymosan was reduced by SnPPIX treatment. Since
both COX-2/mPGES-1 and NOS-II are responsible for the synthesis of proinflammatory
mediators such as PGE2 and nitric oxide, respectively, and are important players in the
inflammatory response and cytokine synthesis, and in agreement with in vitro statin-mediated
modulation of their expression in leukocytes, we analyzed their expression in response to zymosan
in vivo. Fig 4A shows a strong increase in gene expression of Ptgs2, Pges and Nos2 by zymosan.
Atorvastatin significantly inhibited Ptgs2 by 64% (p<0.01), Pges by 83% (p<0.05) and Nos2 by
75% (p<0.01). SnPPIX reversed this inhibitory effect of atorvastatin.
We finally assessed the effect of atorvastatin and SnPPIX on the protein synthesis of some
inflammatory mediators. Zymosan-injected air pouches showed an increased secretion of
cytokine IL-1? and MCP-1 (Fig 4B). In atorvastatin-treated group, IL-1? was inhibited by
44% (p = 0.06) and MCP-1 by 71% (p<0.05) (zymosan+atorvastatin vs zymosan). Similarly to
gene expression, SnPPIX attenuated atorvastatin inhibitory effect on IL-1? and MCP-1. PGE2
formation was also significantly decreased by 53% in the atorvastatin-treated group compared
to zymosan (p<0.001). However, SnPPIX did not show any significant reversal effect on PGE2
inhibition by atorvastatin, suggesting either a HO-1-independent mechanism or a direct
inhibition of the cyclooxygenase by SnPPIX since cyclooxygenase is a heme binding protein and
that different protoporphyrin can compete with its heme [
It has been reported that statins have many beneficial protective effects including improvement of endothelial dysfunction, antioxidant, and anti-inflammatory effects. Statins were first shown to enhance NO production in aortic endothelial cells by activating endothelial nitric oxide synthase  (NOS-III) and to possess an antioxidant activity by scavenging hydroxyl
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Fig 3. HO-1 -dependent suppression of proinflammatory cytokines and chemokines by atorvastatin. Mice were treated as described in the legend for Fig 2.
Gene expression of A) Cytokines, B) Chemokines. Mean ? SEM (n = 8); p<0.05; p<0.01; p <0.001.
and peroxyl radicals in vitro [
]. Moreover, statins inhibited IL-6 and IL-8 mRNA and
protein expressions in LPS-stimulated human bronchoepithelial cells [
]. We have previously
shown that statins inhibits COX-2, a proinflammatory enzyme in monocytes in a Rac and
NF-?B?dependent manner . In addition statins have been shown to induce HO-1 expres
sion and to inhibit the production of IL-6 and TNF-? in macrophages stimulated with LPS
16, 18, 37
Few studies have attempted to address the mechanisms of the beneficial effects of statins in
vivo. Studies have shown an improvement of endothelial dysfunction by enhancing NOS-III
expression in a rat model of pulmonary hypertension and in apolipoprotein E
(ApoE)?deficient mice [
]. Treatment of mice with atorvastatin or rosuvastatin had an antioxidant
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Fig 4. Atorvastatin- mediated inhibition of Ptgs2, Pges and Nos2 gene expression is HO-1 dependent. Mice were treated as described in the legend for Fig 2. A)
Gene expression of Ptgs2, Pges, and Nos2, B) IL-1?, MCP-1 and PGE2 concentration. Mean ? SEM (n = 8).
effect in the heart through the induction of HO-1 and the production of its products, carbon
monoxide (CO) and bilirubin . In the present study, we provide the in vivo evidence
for the protective anti-inflammatory effects of atorvastatin. Our findings demonstrate that
daily administration of atorvastatin for 10 days increased the gene expression of
anti-inflammatory markers such as CLEC7A, Arg-1, MRC1, and HO-1 in the cells isolated from the
exudate of air pouch. A significant population of the leukocyte CD45+cells of the cell exudate was
CD45+CD11b+Ly-6G, representing mainly monocyte/macrophage/dendritic populations and
expressed HO-1 in mice treated with atorvastatin.
We also demonstrated that the anti-inflammatory effect of atorvastatin involves the reduction in cell influx in the air pouch in response to zymosan injection, and this effect was abolished by treatment with the selective HO inhibitor SnPPIX. HO-1 was expressed in the leukocytes migrating into the exudates of zymosan-induced mouse air pouch in a time-dependent increase,
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reaching maximal expression at 24?48 h [
]. Analysis of the composition of the cells in the air
pouch by flow cytometry showed a high percentage of CD45+ leukocytes with a predominance
of neutrophils CD11b+Ly6G+ and monocytes/macrophages CD11b+Ly6G- in response to
zymosan. However, pre-treatment of air pouch with atorvastatin did not result in the modification of
the percentage of any leukocyte subsets.
Interleukins and chemokines have an important role in cellular trafficking of leukocytes,
and in enhancing and maintaining inflammation [
]. IL-6 production in air pouch model in
mice is strongly associated with inflammation, where cellular infiltration was strongly reduced
in IL-6 knockout mice [
]. In our experimental model, inhibition of the expression of these
proinflammatory markers by atorvastatin was mediated via HO-1. The decrease in the
inflammatory cell recruitment observed in response to atorvastatin was accompanied by a reduction
in the levels of mediators measured in the air pouch. At the same time, our data showed that
the modulation of the expression of the proinflammatory cytokines, chemokines and enzymes,
performed on the remaining inflammatory cells in the air pouch was also significantly
attenuated. These findings support an inhibitory role of atorvastatin on both the recruitment of cells
in the air pouch and the regulation of gene expression. It was demonstrated that HO-1
induction resulted in reducing COX-2 and NOS-II expression and PGE2, nitrite, LTB4, IL-1? and
TNF-? synthesis [
]. The role of HO-1 was further reinforced using myeloid-restricted
deletion of HO-1 that revealed an increase in neutrophil infiltration and enhancement of the
inflammatory mediators IL-1?, TNF-?, MMP-3, and PGE2, highlighting an important
antiinflammatory role of HO-1 in the zymosan-induced air pouch model [
]. Importantly, CO
and biliverdin/bilirubin, the products of HO reaction, exhibit anti-inflammatory effects with a
reduction of proinflammatory cytokine expression [
] and leukocyte?endothelial
interactions, supporting a role in cell recruitments [
]. Moreover, CORM-3 and CORM-A-1,
compounds that deliver CO and mimic the effect of HO-1-derived CO, have been reported to exert
significant anti-inflammatory effects in addition to their cardioprotective and anti-atherogenic
In line with our finding on isolated macrophages, we showed that statins inhibited the gene
expression of inflammatory enzymes COX-2, NOS-II and mPGES-1 in a HO-1 dependent
manner. The statin-dependent inhibition of PGE2 in the air pouch in mice confirmed our
previous results in cultured human monocytes [
]. SnPPIX has been widely used as an HO-1
inhibitor with success [
] despite few HO-1 independent reports [
Our study unravels in vivo HO-1 as an anti-inflammatory player important in the protective
effects of statins and supports both statins and HO-1 induction as promising and useful
antiinflammatory strategy in vivo.
S1 Fig. Comparison of HO-1 and MRC1 expression in CD45+ cells from air pouch of
C57BL6/mice treated with vehicle or atorvastatin.
S1 Table. Antibody references for flow cytometry.
S1 File. Arrive guideline checklist.
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We thank Miss Maria Esmerian (Molecular and Cellular Biology Unit, Faculty of Medicine,
American University of Beirut) for her help in performing and analyzing the flow cytometry analyses.
Conceptualization: Roberto Motterlini, Eva Hamade, Aida Habib.
Formal analysis: Aida Habib.
Funding acquisition: Roberto Motterlini, Eva Hamade, Aida Habib.
Investigation: Ghewa A. El-Achkar, Aida Habib.
Methodology: Ghewa A. El-Achkar.
Project administration: Aida Habib.
Supervision: Eva Hamade, Aida Habib.
Writing ? original draft: Ghewa A. El-Achkar, Eva Hamade, Aida Habib.
Writing ? review & editing: Ghewa A. El-Achkar, May F. Mrad, Charbel A. Mouawad, Bassam Badran, Ayad A. Jaffa, Roberto Motterlini, Eva Hamade, Aida Habib.
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