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of June 14, 2017.
Infiltrating Monocyte-Derived Macrophages
and Resident Kupffer Cells Display Different
Ontogeny and Functions in Acute Liver
Injury
Ehud Zigmond, Shany Samia-Grinberg, Metsada
Pasmanik-Chor, Eli Brazowski, Oren Shibolet, Zamir
Halpern and Chen Varol
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
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Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 193:344-353; Prepublished online 2 June
2014;
doi: 10.4049/jimmunol.1400574
http://www.jimmunol.org/content/193/1/344
The Journal of Immunology
Infiltrating Monocyte-Derived Macrophages and Resident
Kupffer Cells Display Different Ontogeny and Functions in
Acute Liver Injury
Ehud Zigmond,*,†,1 Shany Samia-Grinberg,*,†,1 Metsada Pasmanik-Chor,‡
Eli Brazowski,*,† Oren Shibolet,*,† Zamir Halpern,*,† and Chen Varol*,†
M
acrophages (MF) are a heterogeneous population of
immune cells that are strategically positioned at distinct anatomic locations, where they are transcriptionally
programmed to perform tissue-specific functions (1). A foundational dogma has been that homeostasis of tissue-resident MF
relies on the constant recruitment of blood monocytes (2). However, several recent pioneering studies revealed two parallel developmental pathways of mammalian MF (3–9). Accordingly,
most tissue-resident MF, including liver Kupffer cells (KC), are
derived from progenitor cells generated during development in
the yolk sac and/or fetal liver and maintain themselves through
adulthood by self-renewal independent from blood-borne mono-
*The Research Center for Digestive Tract and Liver Diseases, Sourasky Medical
Center, Tel Aviv 64239, Israel; †Sackler Faculty of Medicine, Tel Aviv University,
Tel Aviv 69978, Israel; and ‡Bioinformatics Unit, G.S. Wise Faculty of Life Science,
Tel Aviv University, Tel Aviv 69978, Israel
1
E.Z. and S.S.-G. contributed equally to this work.
Received for publication March 6, 2014. Accepted for publication April 25, 2014.
This work was supported by an internal grant of Tel Aviv Sourasky Medical Center
(to C.V.).
The microarray data presented in this article have been submitted to the National
Center for Biotechnology Information Gene Expression Omnibus public database
(http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE55606.
Address correspondence and reprint requests to Dr. Chen Varol, The Research Center
for Digestive Tract and Liver Diseases, Sourasky Medical Center, Weizmann 6st, TelAviv 64239, Israel. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ADAM, a disintegrin and metalloprotease domain;
AILI, acetaminophen-induced liver injury; Anxa1, Annexin 1; APAP, N-acetyl-paminophenol; BM, bone marrow; COX, cyclooxygenase; DC, dendritic cell; ECM,
extracellular matrix; KC, Kupffer cell; Lcn2, lipocalin-2; MF, macrophage; MHC II,
MHC class II; MMP, matrix metalloproteinase; MoMF, monocyte-derived MF;
qPCR, quantitative PCR; Thbs1, thrombospondin-1; VEGF, vascular endothelial
growth factor.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400574
cytes. A second MF system originates from hematopoietic stem
cells and is based on definitive hematopoiesis. Short-lived circulating Ly6Chi monocytes generated in this pathway are recruited to
tissues in homeostasis and injury-associated inflammation. Ly6Chi
monocytes and their MF descendants are plastic and can either
promote inflammation or assist the healing process depending on
the type of injury and the tissue milieu (1, 10, 11).
The role of MF in liver injury is controversial with plethora of
studies reporting on both deleterious and hepatoprotective functions of these cells (12–23). This discrepancy could arise from the
heterogeneity of liver-MF and the difficulty in distinguishing between distinct subpopulations of MF, especially under inflammatory conditions. Studies in human and murine models of acute and
chronic liver injury attempted to discriminate between resident
KC and infiltrating Ly6Chi monocytes recruited to the inflamed liver
(12, 13, 17, 18, 20, 22, 24, 25). Yet, the recent progress in multiparameter flow cytometry and transcriptome analyses allowed more
accurate distinguishing between MF subsets unraveling higher
complexity in MF compartment at other organs (5, 11, 26–28).
Moreover, the question whether monocytes can contribute to the
maintenance of KC following inflammatory insults or, alternatively,
give rise into a functionally distinct MF entity remained elusive.
Using these techniques, we wished in this study to perform indepth phenotypic, ontogenic and functional characterization of
liver-MF compartment at steady state and following acute injury.
To do that, we took advantage of the acetaminophen (N-acetyl-paminophenol [APAP]; paracetamol)–induced liver injury (AILI)
model. APAP, a widely used analgesic/antipyretic, is a type A
hepatotoxin causing dose-dependent severe hepatic necrosis that
results in innate immune activation (29, 30). We show that resident
KC constituted the main MF population in the steady-state liver,
but were significantly reduced 24 h following AILI and started
recovering 72 h later by self-renewal of residual cells. In contrast,
circulating Ly6Chi monocytes, recruited in a CCR2-dependent
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The liver has a remarkable capacity to regenerate after injury; yet, the role of macrophages (MF) in this process remains controversial mainly due to difficulties in distinguishing between different MF subsets. In this study, we used a murine model of acute
liver injury induced by overdose of N-acetyl-p-aminophenol (APAP) and defined three distinct MF subsets that populate the liver
following injury. Accordingly, resident Kupffer cells (KC) were significantly reduced upon APAP challenge and started recovering
by self-renewal at resolution phase without contribution of circulating Ly6Chi monocytes. The latter were recruited in a CCR2and M-CSF–mediated pathway at the necroinflammatory phase and differentiated into ephemeral Ly6Clo MF subset at resolution
phase. Moreover, their inducible ablation resulted in impaired recovery. Microarray-based molecular profiling uncovered high
similarity between steady-state KC and those recovered at the resolution phase. In contrast, KC and monocyte-derived MF
displayed distinct prorestorative genetic signature at the resolution phase. Finally, we show that infiltrating monocytes acquire
a prorestorative polarization manifested by unique expression of proangiogenesis mediators and genes involved with inhibition of
neutrophil activity and recruitment and promotion of their clearance. Collectively, our results present a novel phenotypic,
ontogenic, and molecular definition of liver-MF compartment following acute injury. The Journal of Immunology, 2014, 193:
344–353.
The Journal of Immunology
manner and controlled by M-CSF, massively infiltrated the injured
liver, becoming the dominant liver-MF subset at both necroinflammatory and early resolution phases and had no contribution
to the recovery of resident KC. Selective ablation of monocytederived MF (MoMF) resulted in impaired recovery manifested
by greater hepatotoxic damage. Finally, microarray-based molecular
profiling of MF subsets uncovered that resident KC and infiltrating
MoMF acquire a distinct gene-expression signature, suggesting
different prorestorative functions of these cells.
Materials and Methods
Mice
The following 8–12-wk-old male mouse strains were used: C57BL/6 mice
were purchased from Harlan Laboratories and Zbtb46gfp/+ mice from The
Jackson Laboratory (B6; 129P2-Zbtb7btm2Litt/JAX). CD45.1 and CD45.2
Cx3cr1gfp/+ and Ccr22/2 Cx3cr1gfp/+ mice were bred at the Sourasky
Medical Center animal facility. All murine experiments were approved by
the Sourasky Medical Center Animal Care Committee.
AILI
Isolation of hepatic nonparenchymal cells
Perfused livers were cut into small fragments and incubated (37˚C, 250 rpm
for 40 min) with 5 ml digestion buffer (5% FBS, 0.5 mg/ml collagenase
VIII [Sigma-Aldrich, Rehovot, Israel], and 0.1 mg/ml DNase I [Roche] in
PBS+/+). This was followed by three cycles of washings with PBS2/2 at
400 rpm from which the supernatant was taken, omitting the parenchymal
cell pellet. The supernatant cell pellet was then lysed for erythrocytes by
2-min incubation with ACK buffer (0.15 M NH4C and 0.01 M KHCO3).
Flow cytometry analysis, sorting of liver-MF subsets, and
ELISA
Abs used for liver-MF characterization included: CD45 (30-F11), CD45.2
(104), CD45.1 (A20), Ly6C (HK1.4), CD64 (X54-5/7.1), CD11c (N418),
CD11b (M1/70), IAb (AF6-120.1), CD19 (HIB19), TCRb (H57-597), B220
(RA3-6B2), and MPDCA1 (927) (all from BioLegend, San Diego, CA);
NK1.1 (PK136) (BD Biosciences, San Jose, CA); F4/80 (CI: A3-1) (AbD
Serotec); and Ki67 (SolA15; eBioscience, San Diego, CA). Abs used for
the sorting of bone marrow (BM) monocytes used in the adoptive transfer
experiments included: CD45.1 (A20), CD11b (M1/70), CD115 (AFS98),
Ly6C (HK1.4), and c-Kit (2B8). Cells were analyzed with LSR Fortessa
or FACSCanto II flow cytometers (BD Biosciences) and sorted with an
FACSAria machine (BD Biosciences). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR). PGE2 levels were
examined in supernatants isolated from overnight incubated sorted Ly6Chi
monocytes from APAP 24 h–challenged livers using the PGE2 ELISA KitMonoclonal (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.
BrdU pulsing
Mice received three i.p. injections of 2 mg BrdU (BD Biosciences) 3 h apart.
To assess BrdU incorporation, isolated nonparenchymal cells were stained,
fixed, and permeabilized using the Cytofix/Cytoperm and Perm/Wash buffer
(BD Biosciences). Cells were incubated at 37˚C for 60 min in 30 mg
DNase1 and subsequently stained with anti–BrdU-allophycocyanin.
MC21 and M-CSF treatment
Mice were i.p injected with 150 ml anti-CCR2 mAb (clone MC21)–conditioned
media (29 mg Ab/ml), starting at 12 h prior to APAP challenge and every day,
with the last treatment at 48 h post–APAP treatment. rM-CSF (PeproTech,
Rocky Hill, NJ) was given by i.p. injection (25 mg/mouse) for 3 d, everyday
from the beginning of APAP treatment until 24 h before sacrificing.
Real-time quantitative PCR
Total RNA was extracted from murine liver tissue with the Perfect Pure
RNA Tissue Kit (5 PRIME, Hilden, Germany) and from sorted MF subsets
with the microRNeasy Kit (Qiagen, Venlo, Limburg, The Netherlands).
RNA was reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). PCRs were performed
with the SYBR Green PCR Master Mix Kit (Applied Biosystems). Quantification of the PCR signals of each sample was performed by comparing the
cycle threshold values, in duplicate, of the gene of interest with the cycle
threshold values of the TBP housekeeping gene. The genes and primers were
as follows: TBP, 59-GAAGCTGCGGTACAATTCCAG-39 (forward) and 59CCCCTTGTACCCTTCACCAAT-39 (reverse); Ptgs2, 59-TGAGCAACTATTCCAAACCAGC-39 (forward) and 59-GCACGTAGTCTTCGATCACTATC-39 (reverse); Annexin 1 (Anxa1), 59-ATGTATCCTCGGATGTTGCTGC-39 (forward) and 59-’TGAGCATTGGTCCTCTTGGTA-39 (reverse);
TNFaip6, 59-GGGATTCAAGAACGGGATCTTT-39 (forward) and 59-TCAAATTCACATACGGCCTTGG-39 (reverse); lipocalin-2 (Lcn2), 59-TGGCCCTGAGTGTCATGTG-39 (forward) and 59-CTCTTGTAGCTCATAGATGGTGC-39 (reverse); M-CSF, 59-GACAGCCAGCTACTACCAGACATACT-39 (forward) and 59-CGCATAGGTGGTAACTTGTGTTTC-39
(reverse); GM-CSF, 59-GGCGCTCAATGCTGGCTTCA-39 (forward)
and 59-TCTGCCTCCAGCCTCAGGTT-39 (reverse); IL34, 59-CTTTGGGAAACGAGAATTTGGAGA-39 (forward) and 59-GCAATCCTGTAGTTGATGGGGAAG-39 (reverse); and IL4, 59-CCAGCTAGTTGTCATCCTGCTCTTCTTTCTCG-39 (forward) and 59-CAGTGATGTGGACTTGGACTCATTCATGGTGC-39 (reverse).
Microarray performance
Total RNA was extracted from freshly flow cytometry–sorted liver-MF
subsets with the microRNeasy Kit (Qiagen, Venlo, Limburg, Netherlands). Infiltrating Ly6Chi monocytes were sorted from a pool of five mice
at the inflammatory peak of the necrotic phase, 24 h post-AILI (33 biological repeats). Their Ly6Clo MoMF descendants were sorted at the recovery phase, 72 h post-AILI from pool of seven mice (33 biological
repeats). Finally, resident KC were sorted from both pool of seven steadystate mice (23 biological repeats) and a pool of seven mice at 72 h postAILI (33 biological repeats), for a total of 11 arrays. RNA purity was
assessed with BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA).
The cDNA was hybridized to GeneChip Mouse Gene 1.0 ST arrays
(Affymetrix, Santa Clara, CA). Hybridized chips were stained, washed,
and scanned with the Affymetrix GeneChip 3000 7G plus scanner (Affymetrix). Microarray analysis was performed using Partek Genomics Suite
version 6.6 (Partek, St. Louis, MO). Data were normalized and summarized
with the robust multiaverage method (31). Heat maps and Venn diagrams
were performed using Partek Genomics Suite software with Pearson’s dissimilarity correlation and average linkage methods. Functional enrichment
analysis was performed using DAVID and GOEAST tools.
All microarray data have been deposited at the National Center for
Biotechnology Information Gene Expression Omnibus public database
(http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE55606.
Histopatological quantification of hepatic damage
Liver samples were obtained at steady state and 24 and 48 h after AILI,
fixed (4% paraformaldehyde), paraffin embedded, sectioned, and stained
with H&E, and pathologic evaluation was performed by a pathologist
(E.B.). Necrosis was scored as 0 (no necrosis), 1 (spotty necrosis), 2 (confluent, zone 3 necrosis), 3 (confluent, zone 2 plus 3 necrosis), or 4 (panlobular necrosis). Bridging necrosis was scored as 0 (absent) or 1 (present)
and ballooning of hepatocytes as 0 (absent), 1 (mild), 2 (moderate), or 3
(severe).
Statistical analysis
Data were analyzed either by ANOVA followed by Bonferroni’s multiple
comparison test or by unpaired, two-tailed t test with GraphPad Prism 4
(GraphPad, San Diego, CA). Data are presented as mean 6 SEM; p values ,
0.05 were considered statistically significant.
Results
Phenotypic characterization of liver-MF compartment in
steady state and following AILI
Given the broad expression of the fractalkine receptor Cx3cr1 gene
within the mononuclear phagocyte system, we characterized the
composition of the liver-MF compartment using the Cx3cr1gfp/+
reporter mice (32) and multiparameter flow cytometry analysis.
Liver-MF were defined as cells mutually positive for CD45,
CD11b, MHC class II (MHC II), F4/80, and the FcgR1 (CD64)
markers. Using this gating strategy, we show that CX3CR1-GFP
expression could discriminate under homeostatic conditions between a predominant population of CX3CR1neg/lo KC and a sig-
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Mice were fasted overnight for 12 h prior to administration of vehicle or
300 mg/kg APAP (Tiptipot Novimol; CST, Kiryat Malakhi, Israel; 100 mg
paracetamol/ml) by oral gavage. Water was returned with APAP administration and the food 2 h later.
345
346
nificantly smaller population of CX3CR1hi MF (Fig. 1A, 1B). At
24 h following AILI, there was a massive infiltration of circulating monocytes characterized as Ly6ChiCD11bhiMHC IInegCX3CR1GFP+ cells concomitantly with marked reduction in the frequency
of resident KC. Notably, at 72 h following AILI, the liver-MF
compartment was dominated by CX3CR1-GFPhiLy6Clo–infiltrating
MF, whereas KC did not yet fully recover (Fig. 1A, 1B). At 120 h
following AILI, the KC returned to be the predominant liver-MF
subset, similar to pre-APAP administration, whereas the CX3CR1+
Ly6Clo–infiltrating MF were hardly detectable (Fig. 1A, 1B).
Importantly, flow cytometry analysis of APAP 72-h livers of
Zbtb46gfp/+ transgenic mice, in which cells positive for the dendritic cell (DC)–associated transcription factor Zbtb46 are labeled
with GFP (33), confirmed that GFP+ liver-DC are excluded from
our gating strategy for liver-MF (Supplemental Fig. 1A). Moreover, staining for TCRb, NK1.1, plasmacytoid DC Ag-1, SiglecF,
and Ly6G confirmed that our gating strategy excludes contamination with T cells, NK cells, plasmacytoid DC, eosinophils, and
neutrophils, respectively (Supplemental Fig. 1B).
To investigate the contribution of circulating Ly6Chi monocytes to
the ontogeny of liver-MF subsets following AILI, we performed
adoptive transfer experiments of purified BM-derived CD45.1
Cx3cr1gfp/+ Ly6Chi monocytes into APAP-challenged wild-type
FIGURE 1. Phenotypic characterization of
mouse liver-MF compartment in health and
following AILI. (A) Flow cytometry analysis of
nonparenchymal Cx3cr1gfp/+ liver cells isolated
from steady state or at 24, 72, and 120 h following AILI. (B) Dynamics of liver-MF subsets
in steady state and at different time points following AILI: resident CX3CR1-GFPneg/lo KC
(circle), infiltrating Ly6Chi CX3CR1-GFP+ monocytes (square), and Ly6CloCX3CR1-GFPhi MF
(triangle), presented as percentage out of CD45+
living cells (mean [SEM]; n $ 5 for any time
point).
recipients. The monocyte graft was administered by i.v. injection at 12 h following AILI, and recipient mice were sacrificed
at following consecutive time points (Fig. 2A). The engrafted
Ly6Chi monocytes infiltrated the injured liver already by 24 h
following APAP challenge and, subsequently, started differentiating toward the Ly6CloF4/80hiCX3CR1-GFP+ MF population we named MoMF. At 96 h post–APAP administration, the
MoMF could hardly be detected, suggesting that these cells are
ephemeral (Fig. 2B). Importantly, at all examined time points,
the engrafted Ly6Chi monocytes did not differentiate to CX3CR1GFPneg cells.
The chemokine receptor CCR2 is required for emigration of
Ly6Chi monocytes from the BM (34). Flow cytometry analysis of
Ccr22/2Cx3cr1gfp/+ mice subjected to APAP overdose revealed
a failure of these animals to accumulate Ly6Chi monocytes (24 h)
and Ly6CloCX3CR1-GFP+ MoMF (72 h) in their livers (Fig. 2C,
2D). Importantly, maintenance and recovery of KC were not affected by CCR2 deficiency (Fig. 2C, 2D). M-CSF controls the
differentiation, proliferation, and survival of certain MF subsets
(35). Quantitative PCR (qPCR) analysis of M-CSF expression in
APAP-challenged livers revealed a 4-fold increase at 24 h that
remained above baseline level during the recovery phase (72–
120 h) (Fig. 3A). Interestingly, we could not detect any significant
change in the expression of other factors associated with MF
differentiation including IL-4, IL-34, and GM-CSF during the
course of AILI (Fig. 3B). Injection of recombinant mouse M-CSF
resulted in a significant increase in the cell number of liver
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Ly6Chi monocytes are recruited in a CCR2- and
M-CSF–dependent manner and give rise to the CX3CR1+Ly6Clo
liver-MF subset
MF HETEROGENEITY IN ACUTE LIVER INJURY
The Journal of Immunology
347
Ly6Chi monocytes and MoMF, but had no effect on KC levels
(Fig. 3C, 3D). Collectively, these results show that the recovery
of KC does not relay on CCR2+Ly6Chi monocytes or M-CSF.
Liver-KC recover by self-renewal following AILI
To investigate whether KC can self-renew by local proliferation,
we resorted to BrdU pulsing regimen as it is empirically incorporated into dividing cell. BrdU labeling was performed at 48 h
following AILI with the initiation of KC recovery (Fig. 1). Flow
cytometry analysis 24 h later revealed incorporation of BrdU in
.5% of KC and ,2% of MoMF (Fig. 4A). To delineate whether
BrDU+ KC were recently derived from proliferating precursor or,
alternatively, are capable of self-renewal, we stained for the Ki67
proliferation marker present only during active phases of cell
cycle. Flow cytometry analysis revealed a 7-fold increase in the
fraction of Ki67+ KC at 72 h following AILI (Fig. 4B, 4C). In
contrast, we could not observe any increase in the fraction of
proliferating cells within the MoMF compartment. Furthermore,
introduction of rM-CSF had no effect on the frequency of Ki67+proliferating KC or MoMF (Fig. 4B, 4C).
Ablation of Ly6Chi monocytes and their MoMF descendants
impairs the recovery from AILI
To investigate the contribution of Ly6Chi monocytes and MoMF
in AILI, we took advantage of the anti-CCR2 MC21-depleting Ab
(11, 24, 36). MC21 treatment specifically eliminated the Ly6Chi
monocytes and their MoMF progenies, but had no effect on resident KC (Fig. 5A, 5B). Histopathological analysis revealed more
extensive damage, with bridging necrosis and ballooning degeneration in livers of MC21-treated mice at both 24 and 48 h following APAP administration (Fig. 5C). Pathological scoring confirmed a significant prolonged hepatic damage (Fig. 5D). Thus,
MoMF contribute to the recovery from liver injury.
Molecular characterization of resident KC, infiltrating liver
monocytes and their MoMF descendants
We next sought to molecularly define the distinct liver-MF subsets during the inflammatory and resolution phases of liver injury.
To do that, we performed a transcriptome microarray analysis of
highly purified cell populations freshly sorted from the livers of
APAP-challenged Cx3cr1gfp/+ mice (Supplemental Fig. 2). Specifically, Ly6Chi-infiltrating liver monocytes were sorted from APAP
24-h livers, whereas KC and MoMF were sorted from APAP 72-h
livers. These were compared with resident liver KC from unchallenged mice. Microarray analysis performed with a stringent statistical significance cutoff of p , 0.05 (with false discovery rate
correction) revealed very high resemblance between steady-state
KC and recovered KC, with a Pearson correlation coefficient of
0.975 in support of their self-renewal (Fig. 6A, 6B). Nevertheless,
there were 2196 genes that were differentially expressed between
the distinct liver-MF subsets with at least 2-fold change. In particular, there was high variability between steady-state KC and both
Ly6Chi monocytes and their MoMF descendants, with a Pearson
correlation coefficient of 0.38 and 0.73, respectively (Fig. 6B).
Indeed, we identified 2059 genes that exhibited at least a 2-fold
change in expression between Ly6Chi monocytes and steady-state
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FIGURE 2. CX3CR1-GFP+ infiltrating liver-MF, but not KC, arise from Ly6Chi monocytes following AILI in a CCR2-dependent pathway. (A and B)
Flow cytometry analysis of recipient wild-type mice receiving adoptive transfer of 1.5 3 106 purified BM-derived CD45.1-Cx3cr1gfp/+ Ly6Chi monocytes.
Analysis was performed at 24, 48, and 72 h following APAP administration (n = 3 for any time point). (C) Flow cytometry analysis of Ccr22/2Cx3cr1gfp/+
mice versus Cx3cr1gfp/+ mice comparing levels of CD45+CD11b+MHC II+CD64+F4/80+ liver MF at steady state or after AILI (24 and 72 h). (D) Graphical
summery showing the percentage of indicated liver-MF subsets out of CD45+ living cells (mean [SEM]; n $ 5 for any time point). *p , 0.05.
348
MF HETEROGENEITY IN ACUTE LIVER INJURY
shift upon their differentiation into MoMF manifested by a Pearson
correlation coefficient of 0.76 and 1267 genes with at least 2-fold
change in their expression (Fig. 6B). Importantly, our microarray
results show that the expression of CD169 (Siglec1), F4/80 (Emr1),
CD64 (Fcgr1), MER receptor tyrosine kinase (Mertk), macrosialin
(CD68), the AXL receptor tyrosine kinase, and MHC II, all extensively used in the literature as definitive markers of resident
tissue MF, were similarly expressed in KC and infiltrating MoMF.
Instead, the molecular profiling revealed 603 genes that were differentially expressed by at least a 2-fold change between these
subsets. Among them, there were various cell-surface markers that
can be used for discrimination (Fig. 6D).
Infiltrating liver Ly6Chi monocytes regulate neutrophil
recruitment, activity, and removal
KC and 802 genes between MoMF and steady-state KC (Fig. 6C).
In addition, the Ly6Chi monocytes induced a major gene expression
FIGURE 4. KC recovery by local proliferation following AILI. (A) Representative flow cytometry analysis images of nonparenchymal cells isolated from
perfused livers of Cx3cr1gfp/+ mice at 72 h post-AILI
and 24 h post–33 consecutive pulses with BrdU.
Bottom panel: graphical summary indicating the percentage of BrdU+ cells out of indicated cell subsets
(mean [SEM]; n = 3). (B) Graphical summary showing
percentage of Ki67+ cells out of KC or MoMF at
steady state or AILI 72 h, with or without concomitant
treatment with M-CSF (mean (SEM); n = 5). (C) Flow
cytometry analysis of Ki67 proliferation marker in KC
and MoMF in steady-state Cx3cr1gfp/+ mouse livers
and at 24, 48, and 72 h following AILI. *p , 0.05.
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FIGURE 3. M-CSF specifically promotes the differentiation of MoMF.
(A and B) Graphical summary of qPCR analysis showing mRNA expression
of M-CSF, GM-CSF, IL-34, and IL-4 in livers isolated from steady-state
mice and at 24, 48, 72, and 120 h following AILI (mean [SEM]; n = 5).
(C) Flow cytometry analysis of Cx3cr1gfp/+ mouse livers at 72 h following
AILI gated on CD45+CD11b+MHC II+CD64+F4/80+ liver-MF subsequent
to treatment with rM-CSF or saline. (D) Graphical summary showing the
cell number of indicated liver-MF subsets normalized for liver tissue weight
(mean [SEM]; n = 5). *p , 0.05.
Corroborating previous results (13), we show in this study a significant increase in the frequency of neutrophils in the livers of
Ccr22/2 mice at 24 h following APAP administration (Fig. 7A).
Supportively, the inducible ablation of Ly6C hi monocytes and
their MoMF descendants resulted in a profound increase in neutrophil levels (Fig. 7B), suggesting that the former negatively
regulate the latter. PGE2 has been recently established as a potent
inhibitor of neutrophil activation (10). In this relation, the Ly6Chi
monocytes expressed uniquely high levels of the Ptgs2 gene,
which encodes for cyclooxygenase-2 (COX2), and of the microsomal PGE synthase-1 (Ptges), both of which constitute key
enzymes in PGE2 synthesis (37, 38), as well as lower levels of the
Hpgd gene encoding for the PG-degrading enzyme hydroxyl-PG
dehydrogenase 15-(NAD), overall resulting in higher PGE2 levels.
Moreover, both Ly6Chi monocytes and MoMF produced Anxa1
(Fig. 7C), a positive regulator of PGE2 synthesis, a potent antiinflammatory mediator in MF and an inhibitor of neutrophil migration (39). Conversely, KC expressed higher levels of Ptgs1 gene
The Journal of Immunology
349
encoding for COX1 and of the Hpgds gene encoding for hematopoietic PGD synthase that catalyzes the conversion of PGH2 to
PGD2 (Fig. 7C). qPCR and ELISA analyses further confirmed
the unique activation of the PGE2 metabolic pathway in Ly6Chi
monocytes (Fig. 7D, 7E).
The Ly6Chi monocytes also expressed the product of TNFstimulated gene-6 (Tnfaip6), a potent inhibitor of neutrophil migration (40) and a positive regulator of COX2 expression in MF
(41). Moreover, they expressed Lcn2 that induces cell death in
neutrophils (42) and plays a significant hepatoprotective role in
liver injury (43) (Fig. 7C). Validating qPCR analysis confirmed
the expression of Lcn2 and TNFaip6 specifically in Ly6Chi monocytes (Fig. 7D). The Ly6Chi monocytes and MoMF also expressed
genes involved in the removal of apoptotic neutrophils such as
thrombospondin-1 (Thbs1) and its receptor CD36 (44) and the
aL (Itgal) and b2 (Itgb2) integrin components of the lymphocyte function-associated Ag 1 that is involved with engulfment
of ICAM-3+ apoptotic neutrophils (45). In this relation, they
also expressed the guanine-nucleotide exchange factor Vav3,
which is essential for the b2 integrin–dependent MF phagocytosis of apoptotic neutrophils in wound healing (46) (Fig. 7C).
Collectively, these results suggest a hepatoprotective role for infiltrating liver monocytes in mediating neutrophil recruitment,
activity, and clearance.
Hepatoprotective polarization of Ly6Chi monocytes and MoMF
Shaping of MF function is an essential component of tissue
damage and repair (47, 48). The Ly6Chi monocytes expressed
exclusively proinflammatory genes associated with classical activation M1 phenotype including triggering receptor expressed on
myeloid cells-1 (Trem1), NO synthase 2 (Nos2), COX2 (Ptgs2),
and the chemokines Ccl2 and Ccl7. Nevertheless, they expressed
a wider panel of genes associated with both alternatively activated regulatory M2 and prorestorative wound-healing MF phenotypes (48), including arginase I (Arg1), Lcn2, chitinase-3-like
protein 3 (Chil3l3, also known as Ym1), IL-4R subunit a (Il4ra),
TNF ligand superfamily member 14 (Tnfsf14), TNF, TNFAIP3interacting protein 3 (Tnip3, also named Abin3), and the a-induced
protein 3 (Tnfaip3, also named A20). They also uniquely expressed
oncostatin M (Osm) reported to be indispensable for liver regeneration following injury (49, 50) (Supplemental Fig. 3A, 3B).
Upon their differentiation into MoMF, the Ly6Chi monocytes
downregulated their expression of the proinflammatory mediators
mentioned above while upregulating the expression of other prorestorative wound healing genes including Trem2, Gpnmb, insulinlike growth factor 1 (Igf1), mannose receptor 1 (Mrc1), the resistinlike molecule a (Retnla, also known as Fizz-1), Scarf1, and Cd163
(Supplemental Fig. 3).
Interestingly, Ly6Chi monocytes expressed high levels of key
proangiogenesis mediators including vascular endothelial growth
factor-A (VEGF-A; Vegfa), semaphorin 4A and D (Sema4a and
Sema4d), hypoxia-inducible factor-1a (Hif1a), the plasminogen
activator urokinase receptor gene (Plaur), Thbs1, Vav3, Anxa2,
ephrin type A receptor 2 (Epha2), fibronectin 1 (Fn1), the cytokines TGF-b1 (Tgfb1), ILe-6 (Il6), and IL-1b (Il1b) as well as the
extracellular matrix (ECM) remodeling enzymes matrix metalloproteinase (MMP) 18, a disintegrin and metalloprotease domain (ADAM) 8, and heparanase (Hpse) (Supplemental Fig. 3A).
Collectively, these results establish that following AILI, monocytes undergo genetic reprogramming, acquiring alternatively
activated and proresolution phenotypes important for wound
healing.
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FIGURE 5. Inducible ablation of Ly6Chi monocytes and their MoMF descendants impairs recovery from AILI. (A) Flow cytometry analysis images of
Cx3cr1gfp/+ mouse livers demonstrating the inducible ablation of Ly6Chi monocytes and their MoMF descendants following treatment with MC21. (B)
Graphical summary presenting cell number normalized for liver tissue weight of indicated subsets (mean [SEM]; n = 10). (C) Representative images of
H&E-stained liver sections at 24 and 48 h following AILI. Original magnification 310. (D) Histopathological score at 24 and 48 h post-AILI in mice
treated simultaneously with saline or MC21 (mean [SEM]; n = 10). *p , 0.05.
350
MF HETEROGENEITY IN ACUTE LIVER INJURY
KC and MoMF display distinct prorestorative molecular
signature
The microarray results revealed major gene expression differences
that strongly imply task division between KC and MoMF in the
resolution from liver injury. In this relation, functional enrichment
analysis based on the DAVID and GOEAST bioinformatics tools
revealed variable expression of wound healing associated genes
between the recruited MoMF and liver-resident KC (Supplemental
Table I). Deeper overview revealed that both liver-MF subsets
displayed variable expression of pattern recognition receptors including scavenger receptors and C-type lectins important for the
clearance of apoptotic cells and cellular debris (Supplemental
Fig. 3C). Specifically, KC displayed exclusive expression of the
T cell Ig mucin 4 (gene Timd4) and stabilin 2 (Stab2) receptors,
both of which mediate the engulfment of phosphatidylserineexpressing apoptotic cells (51, 52). They also expressed higher
levels of the scavenger receptors Marco, CD163, Colec12, and
mannose receptor (Mrc1) as well as of the C-type lectin genes
Clec1b, Clec4f, and Clec9a and DC-specific ICAM3-grabbing
nonintegrin isoform Cd209f. In contrast, Ly6Chi monocytes and
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FIGURE 6. Molecular characterization
of liver-MF subsets. (A) Affymetrix microarray hierarchical clustering performed on
mRNA of sorted liver-MF subsets. A colored bar indicating the standardized log2
intensities accompanies the expression profile. (B) Correlation matrix with Pearson
correlation coefficient performed for all
genes above background and above 2-fold
change. (C) Venn diagram of liver-MF subsets showing the distribution of the 2196
genes with above 2-fold expression difference in comparison with steady-state KC.
(D) Heat map analysis showing the differential raw expression level of MF-associated
genes.
MoMF expressed uniquely high levels of T cell Ig mucin 3 (Havcr2),
TLR2 gene (Tlr2), the C-type lectin genes Clec4d, Cle4e, and
Clec5a, and DC-specific ICAM3-grabbing nonintegrin isoform
Cd209a. They also expressed uniquely the C1q receptor CD93,
shown to be important for the removal of apoptotic cells (53).
Extracellular matrix (ECM) remodeling is of major importance
in resolution from liver injury. The microarray results revealed
variable expression of ECM-remodeling enzymes. Accordingly,
KC expressed higher levels of genes encoding for matrix metalloproteinases-12 and 13 (Mmp12 and -13) and ADAM-23 as well as
Adamdec1, Tgm1, and Timp2 and -3. In contrast, Ly6Chi monocytes and MoMF expressed higher levels of MMP-8, -14, and -19,
ADAM-8, -15, and -19, and heparanase (Hpse). With respect to
ECM structural components, they also expressed higher levels of
the thrombospondin-1 (Thbs1), fibronectin-1 (Fn1), versican (Vcan),
emilin-2, and embigin (Emb) genes, whereas KC expressed higher
levels of nidogen-1, 2 (Nid1 and -2), biglycan (Bgn), and osteonectin (Sparc) (Supplemental Fig. 3D). Finally, the activation of
coagulation and complement cascades are also important for resolution. The microarray results revealed variable expression of
The Journal of Immunology
351
coagulation mediators and complement system factors (Supplemental
Fig. 3E).
Discussion
Recent studies have highlighted marked heterogeneity in the origins of tissue MF. Fate-mapping approaches revealed that most
tissue-resident MF have a prenatal origin and do not rely on blood
monocytes for their renewal under steady-state conditions (3–9).
We have shown that intestinal lamina propria MF are exceptional
and maintained by Ly6Chi monocytes (11, 54). In respect to KC,
their ontogeny and maintenance is disputed. Thymidine incorporation experiments revealed that KC are in mitosis at any given
time in the healthy liver (21, 55). Yet, other studies suggested that
KC are generated by BM-derived cells or by local proliferation,
depending on the nature of stimulation (56–59). Nevertheless, emerging studies have shown that liver-KC are established prenatally
by yolk sac–derived precursors and can persist in adult mice independently of hematopoietic stem cells (3, 4). Yet the question
whether circulating monocytes can contribute to the renewal of KC
following inflammatory insults remained elusive. In this study, using
several complementary approaches, including adoptive transfer, selective ablation, and molecular profiling, we report that following
liver injury, Ly6Chi monocytes infiltrate the liver in a CCR2- and
M-CSF–dependent manner and give rise to a phenotypically and
transcriptionally distinct MF population and do not contribute to the
replenishment of resident KC. Instead, the latter are replenished
following liver injury, at least partially, by self-renewal independently of CCR2 and M-CSF. Confirmatively, our microarray results
revealed very high similarity between steady state and recovered KC.
Ly6Chi monocytes display high plasticity and are directly involved in both the establishment and resolution of inflammatory
reactions. Previous studies based on the use of Ccr22/2 and Ccl22/2
transgenic mice revealed an impaired recovery from AILI, strongly
suggesting that infiltrating MF play a pivotal hepatoprotective
role in the resolution phase (13, 25, 60). In this study, we further
substantiated these findings, demonstrating that inducible and selective ablation of Ly6Chi monocytes and MoMF results in impaired
recovery manifested by greater hepatotoxic damage. Indeed, our
microarray profiling provides compelling evidence that Ly6Chi
monocytes and MoMF express a genetic signature associated with
alternatively activated and prorestorative phenotypes. Worth mentioning is the unique expression of key angiogenesis mediators by
the Ly6Chi monocytes. These include VEGF-A, which is critically
important to the process of hepatocyte regeneration and restoration of liver microvasculature following AILI (61). Moreover,
VEGF-A induces the paracrine release of hepatocyte growth factor,
IL-6, and other hepatotrophic molecules by liver sinusoidal endothelial cells and the protection from hepatotoxic damage (62).
Moreover, they expressed the urokinase plasminogen activator and
its receptor, which are central players in wound healing and angiogenesis and in liver repair (63, 64).
Neutrophils recruited to inflammatory sites release a diverse set
of deleterious substances and take substantial part in the collateral
damage to the tissue. Thus, opportune termination of neutrophil
activity is essential for resolution from liver injury. Holt et al. (13)
were the first to make the association between absence of infiltrating MF and increased frequency of neutrophils following AILI.
Substantiating these results, we show in this study increased accumulation of neutrophils at 24 h following APAP administration
in Ccr22/2 mice and in mice subjected to inducible ablation of the
Ly6Chi monocytes. Our results revealed several hepatoprotective
mechanisms used by these cells to regulate neutrophil activity,
apoptosis, and removal. In particular, we show their unique activation of the PGE2 synthesis pathway recently shown in a murine
model of acute mucosal infection with Toxoplasma gondii to inhibit neutrophil activation (10).
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FIGURE 7. Infiltrating liver Ly6Chi monocytes
regulate neutrophil recruitment, activity, and removal. (A) Graphical summary showing neutrophil
percentage out of total CD45+ liver hematopoietic
cells. (B) Graphical summary presenting neutrophil
cell number normalized for liver tissue weight (mean
[SEM]; n = 10). (C) Heat map analysis showing the
differential raw expression level of selected genes
associated with regulation of neutrophil activity by
liver-MF. (D) Graphical summary of qPCR analysis
showing the mRNA expression level ratio of Anxa1,
Ptgs2, Lcn2, and Tnfaip6 genes in distinct liver-MF
subsets versus steady-state KC. (E) Graphical summary showing ELISA readout for the production of
PGE2 in Ly6Chi monocytes with or without simultaneous LPS activation. *p , 0.05, ***p = 0.001.
352
Acknowledgments
We thank Prof. Matthias Mack from the Department of Internal Medicine
at Regensburg University (Regensburg, Germany) for the generous gift of
the anti-CCR2 MC21 Ab. We also thank M. Kaplan (Sourasky Medical
Center) for excellent animal care.
Disclosures
The authors have no financial conflicts of interest.
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Impaired recovery from AILI has been demonstrated in mice
lacking KC (65) or MoMF (13) and was greater in mice deficient
for both MF subsets (23), suggesting that both play critical role in
this process. Nevertheless, the question of whether MoMF compensate functionally for the reduced presences of KC at the initial phase of recovery, or, oppositely, these MF subsets are functionally distinct has not been addressed. In an effort to unravel
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