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CCR2+ monocytes aggravate the early phase of acetaminophen induced acute liver injury

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Conclusion: Infiltrating monocyte-derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of APAP-induced liver injury. This article is protected by copyright. All rights reserved.
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CCR2+ monocytes aggravate the early phase of acetaminophen
induced acute liver injury
Jana C. Mossanen*,1,2, Oliver Krenkel*,1, Can Ergen1, Olivier Govaere3, Anke
Liepelt1, Tobias Puengel1, Felix Heymann1, Sandra Kalthoff4, Eric Lefebvre5, Dirk
Eulberg6, Tom Luedde1, Gernot Marx2, Christian P. Strassburg4, Tania Roskams3,
Christian Trautwein1, Frank Tacke1
1Department of Medicine III, University Hospital Aachen, Aachen, Germany,
2Department of Intensive and Intermediate Care, University Hospital Aachen,
Aachen, Germany, 3Department of Imaging & Pathology, University of Leuven,
Leuven, Belgium, 4Department of Medicine I, University Hospital Bonn, Bonn,
Germany, 5Tobira Therapeutics, Inc., South San Francisco, USA, 6NOXXON Pharma
AG, Berlin, Germany
Total word count (incl. references): 5543 words
Abstract word count: 269 words
Keywords: macrophages, acute liver failure, APAP, chemokine, therapy
Corresponding author: Frank Tacke, M.D., PhD
Department of Medicine III, University Hospital Aachen
Pauwelsstrasse 30, 52074 Aachen, Germany
Phone: -49-241-80-35848, Fax: -49-241-80-82455
Email: frank.tacke@gmx.net
Abbreviations: Acetaminophen (APAP); acute liver failure (ALF); APAP-induced
acute liver failure (AALF); Cenicriviroc (CVC); chemokine (C-C motif) receptor (CCR);
chemokine (C-C motif) ligand (CCL); chemokine (C-X-C motif) receptor (CXCR);
chemokine (C-X-C motif) ligand (CXCL); damage-associated molecular patterns
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/hep.28682
This article is protected by copyright. All rights reserved.
(DAMPs); glutathione (GSH); green fluorescent protein (Gfp); Kupffer cells (KC);
monocyte-derived macrophages (MoMF); N-acetyl-p-benzoquinonimine (NAPQI);
two-photon laser scanning microscopy (TPLSM).
Conflicts of interest: E.L. is an employee of Tobira Therapeutics Inc. (San Francisco,
CA), D.E. is an employee of NOXXON Pharma AG (Berlin, Germany). The anti-
murine MCP-1 inhibitor mNOX-E36 was kindly provided by NOXXON Pharma AG,
the CCR2/CCR5 inhibitor cenicriviroc by Tobira Therapeutics, Inc.
Financial support: This work was supported by the German Research Foundation
(DFG; Ta434/5-1 and SFB/TRR57), the Interdisciplinary Center for Clinical Research
(IZKF) Aachen, and the B. Braun Foundation.
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Abstract
Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver
failure (ALF) in humans and induces hepatocyte necrosis, followed by the activation
of the innate immune system further aggravating liver injury. The role of infiltrating
monocytes during the early phase of ALF is still ambiguous. Upon experimental
APAP overdose in mice, monocyte-derived macrophages (MoMF) massively
accumulate in injured liver within 12-24h, while the number of tissue-resident
macrophages (Kupffer cells) decreases. The influx of MoMF is dependent on the
chemokine receptor CCR2, since Ccr2-/- mice display reduced infiltration of
monocytes and attenuated liver injury after APAP overdose at early time-points. As
evidenced by intravital multiphoton microscopy of Ccr2-reporter mice, CCR2+
monocytes infiltrate liver as early as 8-12h after APAP overdose and form dense
cellular clusters around necrotic areas. The CCR2+ MoMF express a distinct pattern
of inflammatory but also repair-associated genes in injured livers. Adoptive transfer
experiments revealed that MoMF primarily exert pro-inflammatory functions early
after APAP, thereby aggravating liver injury. Consequently, early pharmacological
inhibition of either CCL2 (by the inhibitor mNOX-E36) or CCR2 (by the orally
available dual CCR2/CCR5 inhibitor cenicriviroc, CVC) reduces monocyte infiltration
and APAP-induced liver injury in mice. Importantly, neither the early nor continuous
inhibition of CCR2 impair repair processes during resolution from injury. In line,
human livers of ALF patients requiring liver transplantation reveal increased CD68+
hepatic macrophage numbers with massive infiltrates of periportal CCR2+
macrophages that display a pro-inflammatory polarization. Conclusion: Infiltrating
monocyte-derived macrophages aggravate APAP hepatotoxicity, and the
pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential
by reducing the inflammatory reaction during the early phase of APAP-induced liver
injury.
Abstract word count: 269 words
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Introduction
Acetaminophen (N-acetyl-p-aminophenol, APAP, or paracetamol) is a commonly
used analgesic and antipyretic drug, which is considered to be safe at therapeutic
concentrations. When taken as an overdose APAP can cause severe liver injury (1),
which is the most frequent reason for acute liver failure (ALF) in some Western
countries (2). APAP is rapidly taken up via the intestine and processed by
hepatocytes into the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI)
causing oxidative stress and hepatocyte necrosis (3). The degree of hepatocyte
necrosis, as reflected by circulating cytokeratin-18 fragments (M65), is a strong
predictor of an unfavorable prognosis in human ALF (4). Hepatocyte necrosis leads
to the activation of innate immune cells, e.g. resident hepatic macrophages (Kupffer
cells, KC). Activated KC secrete a variety of pro-inflammatory cytokines and
chemokines leading to sterile inflammation and leukocyte infiltration (3,5). The
secretion of the chemokine (C-C motif) ligand 2 (CCL2) provokes the recruitment of
chemokine receptor (C-C motif) 2 expressing (CCR2+) monocytes towards areas of
necrosis in the liver (6). In experimental sterile liver injury, monocyte-derived
macrophages (MoMF) can undergo a maturation process from a pro-inflammatory
towards a restorative phenotype (7–9), characterized by the downregulation of the
surface marker Ly-6C (Gr1) in mice (7).
Due to the lack of treatment options in ALF, understanding the contribution of
inflammatory reactions to the outcome of liver injury has gained increasing attention.
Several investigators have unambiguously demonstrated the massive accumulation
of monocytes in APAP-induced liver injury (6,7,10,11), and MoMF appear to be
functionally important during the late repair phase from injury in mice (10,11).
However, as MoMF express pro-inflammatory markers in mouse models (7) and are
largely found in human patients with severe APAP-induced ALF that required liver
transplantation (12), we aimed at characterizing their functional role during the early
phase of APAP-induced liver injury.
In this study, we demonstrate that monocytes are massively recruited into
APAP poisoned livers in a CCR2-dependent fashion, forming large cellular clusters
adjacent to necrotic areas. Although CCR2+ MoMF express a mixed profile of pro-
inflammatory and tissue-repairing genes, MoMF aggravate inflammation and injury
during the early phase of ALF. Consequently, pharmacological inhibition of monocyte
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infiltration by blocking either CCL2 or CCR2/CCR5 attenuated liver damage without
affecting repair processes at later stages, demonstrating the diverse roles of
infiltrating monocyte-derived macrophages in APAP-induced liver failure and the
feasibility of monocyte-targeted interventions in this disease setting.
Methods
Mice
C57BL6/J wildtype (WT) and Ccr2-/- mice were housed under specific pathogen free
conditions. Mice for pharmacological treatment experiments were purchased from an
external animal supplier (Janvier Labs, France). For ex vivo and intravital two-photon
laser scanning microscopy we used transgenic CCR2.gfp reporter mice, which
express GFP under the CCR2 promoter (Ccr2+/eGfp), as well as transgenic CCR2.gfp
knock-in mice lacking functional CCR2 (Ccr2eGfp/eGfp) (13). Experiments were
performed with male 9-13 weeks old mice and have been approved by the
appropriate authorities according to German legal requirements.
Induction of acute liver injury and pharmacological inhibitors
Acute liver injury was induced in mice as described before (1). Briefly, after fasting for
12h, mice received 250mg/kg APAP (Actavis, Germany) by a single IV injection. The
CCR2/CCR5 inhibitor cenicriviroc (CVC) was applied by oral gavage (100mg/kg) and
solved in sterile water containing 0.5% methylcellulose (400cps) and 1% Tween-80.
The L-RNA “Spiegelmer” mNOX-E36 was applied as described before (14). Alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) activity (UV test at
37°C) were measured in serum (Roche Modular preanalytics system, Rotkreuz,
Switzerland). Conventional H&E stainings were performed according to standard
protocols (15), and necrotic areas were quantified by area fraction analysis (ImageJ).
Analysis of blood and intrahepatic leukocytes
Livers were digested by collagenase type IV (Worthington, USA), and intrahepatic
leukocytes were isolated by multiple differential centrifugation steps as detailed
earlier (16). Whole blood was obtained by heart puncture. All cells were subjected to
red cell lysis by Pharmlyse (BD) and stained with fluorochrome-conjugated antibodies
for multi-color fluorescent-activated cell sorting (FACS) analysis.
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Intravital multiphoton microscopy
Long term intravital two-photon laser scanning microscopy (TPLSM) was performed
as described before (17), using transgenic CCR2.gfp reporter mice (Ccr2+/eGfp) that
received APAP 6h before imaging. KC were labelled by IV injection of 0.5-µm red
fluorescent carboxylated latex (Lx) particles (Life Technologies, Carlsbad, CA) with a
concentration of 0.04% (v/v) (18). Before imaging, mice were anesthetized by an IP
injection of ketamine/xylazine (100 and 10 mg/kg), followed by tracheotomy and
controlled respiration with 2.5% isoflurane in 100% O2 (17). Several viewfields per
mouse were imaged over a time-period of at least 2-3h and analyzed for their track
length, track speed, and mobility by Imaris (Bitplane, Zurich, Switzerland).
nCounter gene expression analysis
Gene expression analysis was performed using NanoString assays (nCounter Mouse
Immunology Kit) with FACS-separated cells. Differential gene expression was
calculated by the R package “DESeq2” (19). Differentially expressed genes were
subjected to gene set enrichment analysis (GO biological process) by using the
Cytoscape (20) plug-in BinGO (21). For network visualization, the Cytoscape plugins
Enrichment Map (22) and Word Cloud (23) were used.
Adoptive cell transfer
CD115+ monocytes were isolated by using biotinylated CD115 (clone AFS98,
eBioscience, Germany) and streptavidin microbeads (Miltenyi Biotec), B cells were
isolated by CD45R0 (B220) microbeads (Miltenyi Biotec). The isolated cells were
resuspended in sterile 0.9% sodium chloride solution (B. Braun, Germany) and 5x105
cells were injected IV.
Immunohistochemistry of human samples
The study was approved by the ethical committee of the University Hospitals Leuven,
Belgium. Formalin-Fixed Paraffin Embedded human tissue samples (n=15) were
obtained from patients diagnosed and treated at the University Hospitals in Leuven.
Acetaminophen-induced acute liver failure samples were obtained from explanted
livers (n=8), normal tissue was obtained adjacent to focal nodular hyperplasia or
colorectal cancer metastasis (n=7). Four µm thick tissue slides were stained using
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the BondTM Polymer Refine Detection kit on the Bond Max autostainer (Leica).
Primary antibodies were direct against CD68 (Ready-To-Use, Dako), CD163 (1/100,
Novocastra), S100A9 (1/1000, ABCAM) and CCR2 (1/100, ABCAM). CCR2-,
S100A9-, CD68- and CD163-positive cells were quantified in five high power fields in
the portal tract area and/or areas of remaining parenchyma. Necrotic areas were not
quantified.
Statistical analysis
All data are represented as mean ± SEM. Statistical significance between groups
was calculated by two-tailed unpaired Student t-test (GraphPad Prism, USA).
Additional details on the methodology are reported as Supplementary Material.
Results
Ccr2-/- mice display attenuated liver injury and reduced infiltration of
monocytes after APAP overdose. As a result of APAP-induced liver injury in mice,
Ly-6Chigh monocytes strongly accumulate in the liver (6,7,10). There is compelling
evidence that macrophages derived from these infiltrating monocytes (MoMF) play an
essential role during the restoration phase, because the lack of MoMF delays liver
repair (7,10). Until now the contribution of infiltrating monocytes to the inflammatory
reaction during the initial phase of progressing APAP-induced liver injury has
remained elusive (12). Because monocytes are predominantly recruited via the
CCR2-CCL2 axis into injured liver (6,7), we subjected wildtype (WT) and Ccr2-/- mice
to a sublethal dose of APAP and evaluated the early course of liver injury. Both
genotypes showed a clear increase in necrotic area fraction and serum transaminase
levels (ALT) after 6h. However, while liver damage continued to progress in WT mice
at 12h after APAP, hepatic injury was significantly attenuated in Ccr2-/- mice at this
time-point (Fig.1A).
Infiltrating monocytes give rise to the population of MoMF, which are separate
from resident KC (3,7,24). In WT mice, flow cytometric analysis revealed a distinct
kinetics of different hepatic macrophage populations in APAP injury. Between 6h and
12h after APAP application, monocytes massively infiltrate the liver in WT mice,
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shown by an increased population of MoMF at the 12h time-point, while the KC
decrease within the first 24h after injury induction (Fig.1B). It is known that KC are
reduced upon APAP-induced liver injury (7,25), possibly due to direct toxic effects of
APAP (26). Of note, neutrophils precede the monocyte influx (Fig.1C) (27). Total
myeloid cell numbers in the liver (Suppl.Fig.1) also reflected these data, while
lymphocyte counts did not vary significantly between WT and Ccr2-/- mice
(Suppl.Fig.2).
In line with the hepatic monocyte infiltration, serum levels of CCL2 significantly
increased in WT mice challenged with an APAP overdose (Fig.1D). In contrast, mice
lacking CCR2 are unable to recruit monocytes into the inflamed liver between 6h and
24h after APAP (Fig.1B). In addition, hepatic monocytes in Ccr2-/- mice also show a
significantly reduced expression of the surface marker Ly-6C (Fig.1E), with a high
expression of Ly-6C being characteristic for freshly infiltrated MoMF (7,8).
CCR2+ monocytes infiltrate the liver at 8h after APAP overdose and form dense
cellular clusters around necrotic areas. In order to better apprehend the dynamics
and spatial distribution of monocyte recruitment, we evaluated the CCR2-dependent
infiltration of monocytes during APAP-induced liver injury ex vivo and in vivo by two-
photon laser scanning microscopy (TPLSM), using CCR2-eGFP reporter (Ccr2+/eGFP)
and knock-out (Ccr2eGFP/eGFP) mice. Ex vivo TPLSM showed the influx of CCR2+
monocytes between 6h and 12h after APAP application (Fig.2A-B), starting from
periportal fields and forming large clusters in areas of hepatic necrosis. This
accumulation of CCR2+ monocytes is absent in livers of CCR2-deficient mice
(Fig.2A-B). We next investigated Ccr2+/eGFP reporter mice by intravital TPLSM,
covering the period of 8h to 12h after APAP overdose (Fig.2C, Suppl.Movies 1-2).
The migratory behavior of CCR2+ monocytes was determined by speed (mean speed
of individual cells), displacement (distance between the first and the last spot of each
track irrespective of its directionality) and straightness (directionality of the movement
of the cell, with a high rank indicating a straight movement along vessels) (17). Upon
APAP administration, infiltrating CCR2+ monocytes show reduced speed,
displacement and straightness in an injured liver compared to untreated control mice
(Fig.2D). The reduced speed is linked to the extravasation of monocytes into the liver
tissue and their migration towards areas of necrosis forming dense CCR2+ cell
aggregates (Suppl.Movies 1-2). While cells moving within vessels show a high
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straightness as they are following the structure of the vessel, CCR2+ monocytes in
APAP-injured livers clearly demonstrate a slower and more focalized movement of
the cells around centrolobular areas, which become necrotic after APAP
administration (Fig.2D). The establishment of dense infiltrates containing GFP+
monocytes in areas of necrosis is also clearly visible in time-lapse videos of APAP
treated compared to control mice (Suppl.Movies 1-2). Taken together, CCR2+
monocytes infiltrate into the liver as early as 8h after APAP application in mice, show
a reduced mobility and form cell clusters associated with centrolobular necrotic
areas.
Gene expression analyses indicate a dual role of Ly-6Chigh MoMF in the
pathogenesis of APAP-induced liver injury. To better characterize the different
populations of hepatic macrophages, we isolated Ly-6Chigh MoMF, Ly-6Clow MoMF
and KC from livers 12h after APAP-induced liver injury by FACS sorting (Fig.3A) and
subjected these cells to a quantitative gene expression array of 561
inflammation/immunity-related genes (NanoString® analysis). The CCR2-dependent
Ly-6Chigh MoMF show a significant upregulation of genes associated with
inflammation and tissue infiltration, as well as genes responsible for resolution of
injury and angiogenesis, compared to KC or Ly-6Clow MoMF (Fig.3B-C). Pattern-
recognition receptors, such as toll-like receptors (Tlr2, Tlr4, Tlr5, Tlr8, Tlr9) or the
LPS-receptor (Cd14), are upregulated in Ly-6Chigh compared to Ly-6Clow MoMF. The
mRNA expression of the chemokines Ccl2, Ccl3 and Ccl6 and the chemokine
receptor Ccr2 is also upregulated in Ly-6Chigh MoMF, whereas the expression of the
chemokine Cx3cl1 is higher in Ly-6Clow MoMF and KC. Most of the analyzed
cytokines (e.g., Il2, Il4, Il6, Il10, Il17a, Il23a) were poorly expressed in the three
populations (data not shown), whereas variant cytokine receptors, like the interferon
γ receptor 2 (IFNγR2; Ifngr2), the IL-4Rα (Il4ra), or the IL10Rβ (Il10rb) were highly
expressed in Ly-6Chigh MoMF. The pro-inflammatory S100 calcium binding protein A8
and A9 (S100A8/9, also calgranulin A/B; S100a8, S100a9), the integral matrix protein
fibronectin 1 (Fn1), and the cell adhesion molecule L-selectin (Sell) are also strongly
expressed by Ly-6Chigh MoMF compared to Ly-6Clow MoMF and KC (Fig.3B-C).
Gene enrichment mapping revealed significantly upregulated (p<0.05) gene
ontology (GO) pathways when comparing Ly-6Chigh MoMF to KC. The GO pathway
analysis revealed that upregulated genes from Ly-6Chigh MoMF are involved in the
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inflammatory response, chemotaxis and complement activation. In addition, also
genes linked to wound healing and extracellular matrix remodeling were upregulated
in Ly-6Chigh MoMF (Fig.3D). However, comparing Ly-6Chigh MoMF 12h after APAP
and the corresponding control treated mice, only a few genes, like Ccl2, Msr1, Cd14,
and Mif are significantly upregulated (data not shown), indicating that infiltrating Ly-
6Chigh MoMF do not undergo major changes in their gene expression pattern during
the first 12h of APAP-induced liver injury. In addition to the gene expression analysis,
the altered phenotype of the macrophages was confirmed by FACS analysis for
surface markers (Suppl.Fig.1D). While only MoMF up-regulate pro-inflammatory
proteins like Ly-6C and MHC-II 12h after APAP, both KC and MoMF down-regulate
anti-inflammatory markers such as CD206 and CD301 in injured livers
(Suppl.Fig.1D). Thus, our data indicate that Ly-6Chigh MoMF are clearly distinct from
Ly-6Clow MoMF and KC and characterized by expression of pro-inflammatory genes
including chemokines, pattern-recognition receptors, S100A8/9, and Trem1, but also
genes involved in liver repair processes like Il4rα, Trem2 and fibronectin 1 (Fn1).
Early infiltration of Ly-6Chigh monocytes aggravates liver injury. Even though Ly-
6Chigh MoMF expressed not only many inflammatory factors but also genes related to
liver repair, we hypothesized that the accumulation of Ly-6Chigh MoMF in APAP-
treated livers aggravates hepatic injury in the early phase. To explore this hypothesis,
we performed adoptive transfer experiments of Ly-6Chigh monocytes isolated from
bone marrow of CD45.1+ WT mice into CD45.2+ WT or Ccr2-/- mice 3h after induction
of APAP injury; the transfer of splenic B cells served as a control condition (Fig.4A).
Indeed, adoptive transfer of bone marrow monocytes, but not of control B cells, gave
rise to CD45.1+ Ly-6Chigh MoMF (CD11b+ F4/80+ Ly-6G-) in the liver of recipient mice
(Fig.4B). Strikingly, the adoptive transfer of monocytes led to a significant increase in
the extent of liver injury in both, WT and Ccr2-/- mice, in contrast to the adoptive
transfer of B cells, which does not affect APAP-induced liver injury (Fig.4D).
Interestingly, the adoptive transfer of the monocytes early during APAP damage fully
abolishes the differences in the extent of liver injury between WT and Ccr2-/- mice
(Fig.4D).
Pharmacological inhibition of CCR2 blocks monocyte infiltration and
attenuates liver injury. The pharmacological targeting of monocytes by inhibiting
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either the chemokine CCL2 or the receptor CCR2 has been proposed to reduce
inflammation and fibrosis in chronic liver diseases (28). We hypothesized that
inhibiting monocyte infiltration early in the course of APAP-induced liver injury may
hold therapeutic potential as well. Thus, we tested the CCL2 inhibitor mNOX-E36
(Suppl.Fig.3) and the dual CCR2/CCR5 inhibitor cenicriviroc (CVC, formerly TBR-652
(29)) in the mouse model (Fig.5). Both inhibitors effectively block the CCL2-
dependent chemotaxis of primary mouse bone marrow monocytes in transmigration
assays in vitro (14) (data not shown). Pretreatment of mice with subcutaneous
injections of mNOX-E36 (Suppl.Fig.3A), the murine-specific surrogate of the human
CCL2 inhibitor NOX-E36 (tested in a phase IIa study in patients suffering type 2
diabetes, http://clinicaltrials.gov, NCT01547897), significantly reduces liver injury
(Suppl.Fig.3B) and inhibits the infiltration of bone-marrow derived monocytes into the
liver without affecting KC (Suppl.Fig.3C).
The oral CCR2/CCR5 inhibitor CVC is currently being evaluated in a phase IIb
study in patients with NASH and fibrosis (NCT02217475) (30). Early treatment of
mice with CVC after APAP administration significantly reduced liver injury, as
determined by necrotic area fraction and serum transaminase levels (Fig.5B). Mice
that received CVC display a strong inhibition of the accumulation of MoMF in livers
12h and 24h after APAP (Fig.5C). At 48h after APAP, no differences in hepatic
MoMF between CVC and vehicle treated mice can be observed, indicating that
monocyte infiltration may have ceased at this time-point (Fig.5C). Liver neutrophil
levels are not affected by CVC treatment, corroborating the specificity of this
approach (Fig.5C). More importantly, the CVC administration early after APAP injury
significantly reduces liver damage at 12h, but does not influence damage at later
stages (Fig.5B), indicating that repair processes during injury resolution are not
impaired by the inhibition of early monocyte recruitment.
As CVC is a dual inhibitor of CCR2 and CCR5, we next aimed at dissecting the
contribution of both chemokine receptors for hepatic monocyte recruitment. CVC
ameliorates liver damage and efficiently inhibits MoMF accumulation in APAP-
challenged Ccr5-/- but not Ccr2-/- mice (Suppl.Fig.4), demonstrating that CCR2 is the
main mechanism of monocyte recruitment in APAP injury. Furthermore, we could
exclude the possibility that CVC influences APAP hepatotoxicity, metabolism or
clearance. CVC does not affect APAP toxicity in isolated primary hepatocytes in vitro
(Suppl.Fig.5A) nor does it increase glutathione levels in the liver, the main route of
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APAP metabolism (Suppl.Fig.5B). Moreover, CVC does not influence UDP-
glucuronosyltransferase-1 polypeptide-A-cluster (UGT1A), the main pathway for
APAP glucuronidation (31), as UGT1A is not induced by CVC itself in a UGT1A-
reporter cell assay (Suppl.Fig.5C). Conclusively, pharmacological inhibition of the
CCL2-CCR2 axis successfully inhibits monocyte infiltration into the liver, which leads
to a reduced liver injury within the first 12h following an APAP overdose.
Early pharmacological inhibition of CCR2 reduces liver injury without impairing
repair processes. The mouse model of APAP IV administration is highly accelerated
compared to the course of human disease after enteral ingestion and resorption (1),
thereby allowing to assess the effects of early vs delayed pharmacological
intervention (Fig.6A). Early therapeutic treatment of mice with orally given CVC at 1h
and 2h after APAP administration demonstrates the same trend of reduced liver
injury as seen for simultaneous administration with APAP, while a late therapeutic
treatment does not protect from liver injury (Fig.6B). However, either early or late
CVC administration is able to efficiently block the accumulation of MoMF in livers 12h
after APAP, whereas KC and neutrophils are not influenced by CVC treatment
(Fig.6C).
Importantly, even the continuous therapeutic treatment of mice with CVC after
APAP, started at 2h after injury, does not influence liver repair processes, as
determined by equal necrotic areas and serum transaminase levels 24h and 48h
after APAP (Fig.6D-E). Continuous treatment with CVC was associated with
significantly reduced hepatic MoMF numbers at 48h after APAP, whereas liver KC
and neutrophil populations remained unaltered by CVC (Fig.6F). These data on the
one hand emphasize that pharmacological inhibition of monocyte infiltration likely
only translates into beneficial clinical outcomes if administrated very early after acute
liver injury, but on the other hand demonstrate that even a continuous blockade of
monocyte infiltration by CVC after APAP is a safe procedure, because it does not
impair liver repair processes.
CCR2+ infiltrating monocytes display a pro-inflammatory phenotype in patients
with ALF. In order to validate our findings from experimental setups of ALF, we
analyzed liver samples explanted from patients due to APAP-induced ALF (AALF).
Livers of AALF patients show a strong increase of CCR2+ MoMF, especially in the
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periportal region, as compared to control livers (Fig.7A) as well as increased overall
numbers of CD68+ hepatic macrophages (Fig.7B). On the contrary, CD68+
macrophages are significantly reduced in the remaining parenchyma of livers from
patients with AALF compared to normal controls (Fig.7B), suggesting a depletion of
KC during AALF, similar to observations in the mouse model. In order to further
characterize the infiltrating, CCR2+ MoMF, we stained for S100A9 as a pro-
inflammatory marker (32) and the secretory leukocyte protease inhibitor (SLPI,
CD163) as an anti-inflammatory macrophage marker in AALF (33). CCR2+ cells
mainly clustered around the portal tract, indicating that these cells have recently
infiltrated the liver, while CD68+ macrophages are equally distributed between the
portal tract and the remaining parenchyma (Fig.7C). Periportal CCR2+ MoMF strongly
express S100A9, whereas CD163+ macrophages can be found to equal amounts in
the remaining parenchyma as well as in the portal tract (Fig.7C). These findings
collectively demonstrate the massive accumulation of CCR2+ MoMF and their pro-
inflammatory polarization in human AALF.
Discussion
Macrophages are central regulators of homeostasis and inflammation in the liver
(34). The heterogeneity of liver macrophage populations, relating to the origin,
differentiation and function of these cells, however, poses a considerable challenge
to target macrophages in disease settings (8). In our study, we identified the specific
aggravating role of infiltrating monocytes during the onset of acute liver injury. We
could also demonstrate that the pharmacological inhibition of either CCL2 or CCR2
reduced the influx of pro-inflammatory monocytes into the liver, which significantly
attenuated the early phase of APAP-induced liver injury in mice.
In accordance with these experimental data from mouse models, we could
clearly demonstrate the abundant presence of CCR2+ and S100A9+ pro-inflammatory
MoMF in the portal tracts in livers of patients with AALF, whereas CD163+ anti-
inflammatory macrophages (33) as well as CD68+ resident macrophages are not
restricted to the portal tracts. The fact that these findings have been obtained from
AALF patients that required liver transplantation corroborates the suggested
contribution of MoMF-related pro-inflammatory responses to detrimental liver injury.
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In corroboration of our data, increased serum CCL2 levels in ALF patients have been
found to predict an unfavorable prognosis (death or need for emergency liver
transplantation) following APAP intoxication (12).
In the repair phase from APAP-induced liver injury, MoMF were previously
shown to phagocytize cell debris, promote angiogenesis and induce neutrophil
apoptosis, in mice (6,7,10,11) as well as in men (12). Their accumulation can be
augmented by the growth factor CSF1 (M-CSF), which significantly improved the
recovery from APAP-induced liver injury in mice (11). Our data suggest a dual
mechanism of action for MoMF in APAP-induced liver injury. On the one hand,
monocytes enhance hepatic inflammation after extravasation early after injury; on the
other hand, they can contribute to the resolution of inflammation and tissue repair at
later time-points, by maturation into ‘restorative’ Ly-6Clow MoMF (9,35). However, the
continuous pharmacological blockade of monocyte infiltration does not alter the
resolution of liver injury, most likely due to compensatory effects of other macrophage
populations, including KC and Ly-6Clow cells.
Neutrophils are among the first immune cells, which start to infiltrate into the
liver during APAP-induced liver injury (7,36). Huebener et al. have recently identified
a major role of neutrophils and HMGB1 in APAP-induced liver injury (27). As we
could not detect differences in the overall number of liver neutrophils between WT
and Ccr2-/- mice at any of the investigated time-points, the pathway of neutrophil
recruitment via the HMGB1-TLR4-IL23a-IL17a axis (37), seems to depend on KC
and not MoMF. We found that neutrophils infiltrate into the liver within the first 6h of
APAP-induced liver injury, whereas monocytes started to accumulate slightly later.
Thus, both neutrophils and monocytes are important key players in driving
inflammation during the early stages of APAP-induced liver injury, possibly opening
the opportunity for synergistic effects by therapeutically targeting both mechanisms.
Until now, N-acetylcysteine (NAC) administration is the only pharmacological
strategy for patients suffering from APAP-induced liver injury (2). NAC dampens the
initial metabolic liver injury but shows no benefit if applied at later stages, whereas
specifically modulating immune cells carries the potential risk of increased infectious
complications including sepsis. In line, HMGB1 antibody treatment in mice has been
shown to increase bacterial translocation after APAP application (38). Instead,
targeting monocytes might not result in severe immune suppression, as the
Page 14 of 48
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scavenging role of KC and the anti-bacterial functions of neutrophils would remain
unaltered (39).
In our study, we used for the first time two pharmacological inhibitors of either
CCL2 or CCR2/CCR5 in APAP-induced liver injury in mice. We could show that
pretreatment with the CCL2 inhibitor mNOX-E36 results in reduced liver injury after
an APAP overdose. The inhibitor mNOX-E36 has already been tested successfully in
models of chronic liver injury in our laboratory (14,40) as well as in human patients
with diabetic nephropathy (41). Even more striking, the application of the dual
CCR2/CCR5 inhibitor CVC, when given directly after APAP application, led to a solid
decrease of liver injury, alongside a significantly reduced population of freshly
infiltrated Ly-6Chigh MoMF in the liver. A similar beneficial trend is observed when
CVC is given very early after injury, but the therapeutic window apparently closes
after injury is further established, despite a successful inhibition of monocyte
accumulation. We did not observe effects of CVC on APAP hepatotoxicity,
metabolism or clearance, indicating that the ameliorated liver injury after CVC
treatment is caused by the reduced monocyte infiltration.
Promisingly, the continuous therapeutic application of CVC does not impair
resolution of liver injury. These data imply a very rapid involvement of CCR2+ cells in
the response to liver damage, but also emphasize the plasticity of hepatic
macrophage populations. Bone-marrow derived monocytes can repopulate the KC
niche in the liver after depletion (24). These findings indicate that KC, possibly
alongside other myeloid precursors, can independently of CCR2 exert functions
traditionally linked to MoMF like tissue remodeling during resolution from damage
(9,10). Nonetheless, our data demonstrate that CCR2+ pro-inflammatory monocytes
are important promoters of injury progression during the early phase of APAP-
induced liver failure, making them a potential novel target for therapeutic
interventions in acetaminophen poisoning. The transient inhibition of monocyte
recruitment via CCR2-CCL2, started as early as possible after APAP-induced liver
injury, might be both, sufficient with respect to efficacy and not harmful with respect
to resolution of injury.
Acknowledgements
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We thank Eric Pamer (Memorial Sloan Kettering Cancer Center, New York) for
providing the CCR2-egfp reporter mice and Julio Saez-Rodriguez (RWTH Aachen)
for helpful discussions. This work was supported by the German Research
Foundation (DFG; Ta434/5-1 and SFB/TRR57), the Interdisciplinary Center for
Clinical Research (IZKF) Aachen, and the B. Braun Foundation.
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Author names in bold designate shared co-first authorship
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Figures
Figure 1. Ccr2-/- mice display attenuated liver injury and reduced hepatic
monocyte-derived macrophages after APAP overdose. Acute liver injury was
induced by 250 mg/kg APAP IV in C57bl/6 wildtype (WT) and Ccr2-/- mice. Mice were
examined after 6h, 12h and 24h. (A) Liver histology (H&E staining) show necrotic
patches and infiltrating cells in areas of necrosis. Original magnification 10X, scale
bar 400µm. Quantification of necrotic area fraction and serum alanine transaminase
levels (ALT). (B-C) Representative flow cytometric plots from liver leukocytes
showing MoMF (red) and KC (green) populations. Quantification of hepatic KC,
MoMF (B) and neutrophils (C) as percentage of total liver leukocytes for WT (white
bars) and Ccr2-/- mice (grey bars). (D) Serum CCL2 concentrations in WT mice after
APAP challenge. (E) Percentage of Ly-6Chigh expressing MoMF of all hepatic MoMF
in WT and Ccr2-/- mice. All data are presented as mean ± SEM (n 8).*p<0.05,
**p<0.01 (unpaired Student t test).
Page 20 of 48
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Figure 2. CCR2+ monocytes infiltrate liver at 8-12h after APAP overdose and
form dense cellular clusters around necrotic areas. Ccr2+/eGFP reporter and Ccr2-
deficient (Ccr2eGFP/eGFP) mice were subjected to APAP injury and analyzed by two-
photon laser scanning microscopy (TPLSM). (A) Representative images from APAP
treated Ccr2+/eGfp and Ccr2eGfp/eGfp mice, obtained by ex vivo TPLSM. CCR2+ cells are
shown in green, collagen structures in blue, and autofluorescent hepatocytes in red.
(B) Quantification of CCR2-Gfp+ cells as detected by ex vivo TPLSM in APAP treated
Page 21 of 48
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Ccr2+/eGfp and Ccr2eGfp/eGfp mice. (C) Representative images from in vivo TPLSM of
APAP injured livers from Ccr2+/eGfp mice, showing the speed of single tracks with
slow (blue) and fast (violet) movement during whole imaging time-frame. CCR2+ cells
are shown in green, collagen structures in blue; KC had been labelled in red by prior
injection of fluorescent particles. (D) Quantification of average speed, maximum
speed, speed variation, displacement and straightness of Gfp+ cells between control
and APAP treated mice. Data are presented as mean ± SEM (n≥3). *p<0.05,
**p<0.01, ***p<0.001.
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Figure 3. Gene expression pattern of Ly-6Chigh MoMF after APAP overdose. (A)
MoMF (CD11bhigh and F4/80intermediate) and KC (CD11blow and F4/80high) were FACS-
sorted from liver leukocytes with a strategy displayed by the representative FACS
Page 23 of 48
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plots. (B-C) Highly pure (>95%) isolates of the different populations were subjected to
quantitative microarray gene expression analysis (Nanostring® immunology kit,
covering 561 genes). Log2-fold change gene expression of 50 chosen candidates
comparing Ly-6Chigh and Ly-6Clow MoMF (B), as well as for Ly-6Chigh MoMF versus
KC (C), 12h after APAP application. (D) Clustered gene enrichment map of
significantly upregulated (p<0.05) gene ontology pathways between Ly-6Chigh MoMF
and KC 12h after APAP application. n=2 Nanostring® assays per population and
condition (APAP vs control).
Page 24 of 48
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Figure 4. Adoptive transfer of monocytes aggravates liver injury after APAP. (A)
CD115+ monocytes or CD45R0+ B-cells were isolated by magnetic bead separation
from bone-marrow or spleen of CD45.1+ mice, respectively, and adoptively
transferred into WT or Ccr2-/- mice (both CD45.2) 3h after APAP application. (B)
Representative FACS plots of MACS isolated CD45.1+ leukocytes, revealing high
purity of cells used for transfer (upper panel). Corresponding representative FACS
plots of CD45.1+ cells isolated from CD45.2+ recipient mice, showing the
differentiation of transferred monocytes into hepatic macrophages (CD11b+, F4/80+)
(lower panel). (C-D) Liver histology (H&E staining) and corresponding serum ALT
levels from WT and Ccr2-/- mice that received either monocytes or B cells. Original
magnification 10X, scale bar 400µm. All data are presented as mean ± SEM
(n≥3).*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
Page 25 of 48
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Figure 5. Pharmacological inhibition of CCR2 blocks monocyte infiltration and
attenuates liver injury. (A) WT mice received the CCR2/CCR5 inhibitor cenicriviroc
(CVC) PO directly with APAP and another 3h later. Mice were analyzed after 12h,
24h and 48h. (B) Assessment of liver injury by histology, quantification of necrotic
area fraction and serum ALT levels. Original magnification 10X, scale bar 400µm. (C)
Quantification of liver MoMF (red gate) and KC (green gate) as percent of liver
Page 26 of 48
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leukocytes, determined by FACS analysis 12h, 24h and 48h after APAP.
Representative FACS plots are shown. All data are presented as mean ± SEM (n≥3
for control groups, n=8 for treated groups).*p<0.05, **p<0.01, ***p<0.001 (unpaired
Student t test).
Page 27 of 48
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Figure 6. Early pharmacological inhibition of CCR2 reduces liver injury, but
does not impair repair processes. (A) WT mice received the CCR2/CCR5 inhibitor
cenicriviroc (CVC) PO either 1h and 2h after APAP (250mg/kg IV) (early therapeutic
treatment) or 3h and 6h after APAP (late therapeutic treatment). Mice were analyzed
after 12h. (B) Assessment of liver injury by serum ALT levels. (C) Quantification of
liver MoMF and KC as percent of liver leukocytes, determined by FACS analysis 12h
after APAP. (D) WT mice received the CCR2/CCR5 inhibitor cenicriviroc (CVC) PO
either 2h after APAP and then every 12h for 48h. Serum was analyzed after 24h and
mice were sacrificed after 48h. (E) Assessment of liver injury by serum ALT levels.
(F) Quantification of liver MoMF, KC and neutrophils as percent of liver leukocytes,
determined by FACS analysis 48h after APAP. Original magnification 10X, scale bar
400µm. All data are presented as mean ± SEM (n≥4 for vehicle treatment, n≥7 for
cenicriviroc treatment).*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
Page 28 of 48
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Figure 7. CCR2+ infiltrating monocytes display a pro-inflammatory phenotype
in patients with ALF. Consecutive sections of liver samples from patients with
acetaminophen-induced acute liver failure (AALF, n=8) or controls (n=7) were stained
for CCR2 (A), CD68 (B), S100A9 and CD163 (C), and five high power fields were
Page 29 of 48
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counted for each individual. Data are presented as median (n≥7).*p<0.05, (Mann-
Whitney U test). After quantification of immunopositive cells, the ratio of CCR2,
CD68, S100A9 and CD163 positive cells found in the portal tract vs. the remaining
parenchyma of AALF patients was determined. Original magnification 100x.
Page 30 of 48
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Figure 1. Ccr2-/- mice display attenuated liver injury and reduced hepatic monocyte-derived macrophages
after APAP overdose. Acute liver injury was induced by 250 mg/kg APAP IV in C57bl/6 wildtype (WT) and
Ccr2-/- mice. Mice were examined after 6h, 12h and 24h. (A) Liver histology (H&E staining) show necrotic
patches and infiltrating cells in areas of necrosis. Original magnification 10X, scale bar 400µm.
Quantification of necrotic area fraction and serum alanine transaminase levels (ALT). (B-C) Representative
flow cytometric plots from liver leukocytes showing MoMF (red) and KC (green) populations. Quantification
of hepatic KC, MoMF (B) and neutrophils (C) as percentage of total liver leukocytes for WT (white bars) and
Ccr2-/- mice (grey bars). (D) Serum CCL2 concentrations in WT mice after APAP challenge. (E) Percentage
of Ly-6Chigh expressing MoMF of all hepatic MoMF in WT and Ccr2-/- mice. All data are presented as mean
± SEM (n ≥ 8).*p<0.05, **p<0.01 (unpaired Student t test).
190x207mm (299 x 299 DPI)
Page 32 of 48
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Figure 2. CCR2+ monocytes infiltrate liver at 8-12h after APAP overdose and form dense cellular clusters
around necrotic areas. Ccr2+/eGFP reporter and Ccr2-deficient (Ccr2eGFP/eGFP) mice were subjected to
APAP injury and analyzed by two-photon laser scanning microscopy (TPLSM). (A) Representative images
from APAP treated Ccr2+/eGfp and Ccr2eGfp/eGfp mice, obtained by ex vivo TPLSM. CCR2+ cells are shown
in green, collagen structures in blue, and autofluorescent hepatocytes in red. (B) Quantification of CCR2-
Gfp+ cells as detected by ex vivo TPLSM in APAP treated Ccr2+/eGfp and Ccr2eGfp/eGfp mice. (C)
Representative images from in vivo TPLSM of APAP injured livers from Ccr2+/eGfp mice, showing the speed
of single tracks with slow (blue) and fast (violet) movement during whole imaging time-frame. CCR2+ cells
are shown in green, collagen structures in blue; KC had been labelled in red by prior injection of fluorescent
particles. (D) Quantification of average speed, maximum speed, speed variation, displacement and
straightness of Gfp+ cells between control and APAP treated mice. Data are presented as mean ± SEM
(n≥3). *p<0.05, **p<0.01, ***p<0.001.
126x176mm (299 x 299 DPI)
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Figure 3. Gene expression pattern of Ly-6Chigh MoMF after APAP overdose. (A) MoMF (CD11bhigh and
F4/80intermediate) and KC (CD11blow and F4/80high) were FACS-sorted from liver leukocytes with a
strategy displayed by the representative FACS plots. (B-C) Highly pure (>95%) isolates of the different
populations were subjected to quantitative microarray gene expression analysis (Nanostring® immunology
kit, covering 561 genes). Log2-fold change gene expression of 50 chosen candidates comparing Ly-6Chigh
and Ly-6Clow MoMF (B), as well as for Ly-6Chigh MoMF versus KC (C), 12h after APAP application. (D)
Clustered gene enrichment map of significantly upregulated (p<0.05) gene ontology pathways between Ly-
6Chigh MoMF and KC 12h after APAP application. n=2 Nanostring® assays per population and condition
(APAP vs control).
210x298mm (299 x 299 DPI)
Page 35 of 48
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Figure 4. Adoptive transfer of monocytes aggravates liver injury after APAP. (A) CD115+ monocytes or
CD45R0+ B-cells were isolated by magnetic bead separation from bone-
marrow or spleen of CD45.1+ mice,
respectively, and adoptively transferred into WT or Ccr2 /- mice (both CD45.2) 3h after APAP application.
(B) Representative FACS plots of MACS isolated CD45.1+ leukocytes, revealing high purity of cells used for
transfer (upper panel). Corresponding representative FACS plots of CD45.1+ cells isolated from CD45.2+
recipient mice, showing the differentiation of transferred monocytes into hepatic macrophages (CD11b+,
F4/80+) (lower panel). (C-D) Liver histology (H&E staining) and corresponding serum ALT levels from WT
and Ccr2-/- mice that recei
ved either monocytes or B cells. Original magnification 10X, scale bar 400µm. All
data are presented as mean ± SEM (n≥3).*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
142x153mm (299 x 299 DPI)
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Figure 5. Pharmacological inhibition of CCR2 blocks monocyte infiltration and attenuates liver injury. (A) WT
mice received the CCR2/CCR5 inhibitor cenicriviroc (CVC) PO directly with APAP and another 3h later. Mice
were analyzed after 12h,
24h and 48h. (B) Assessment of liver injury by histology, quantification of necrotic
area fraction and serum ALT levels. Original magnification 10X, scale bar 400µm. (C) Quantification of liver
MoMF (red gate) and KC (green gate) as percent of liver leukocytes, determined by FACS analysis 12h, 24h
and 48h after APAP. Representative FACS plots are shown. All data are presented as mean ± SEM (n≥3 for
control groups, n=8 for treated groups).*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
210x262mm (299 x 299 DPI)
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Figure 6. Early pharmacological inhibition of CCR2 reduces liver injury, but does not impair repair processes.
(A) WT mice received the CCR2/CCR5 inhibitor cenicriviroc (CVC) PO either 1h and 2h after APAP (250mg/kg
IV) (early therapeutic treatment) or 3h and 6h after APAP (late therapeutic treatment). Mice were analyzed
after 12h. (B) Assessment of liver injury by serum ALT levels. (C) Quantification of liver MoMF and KC as
percent of liver leukocytes, determined by FACS analysis 12h after APAP. (D) WT mice received the
CCR2/CCR5 inhibitor cenicriviroc (CVC) PO either 2h after APAP and then every 12h for 48h. Serum was
analyzed after 24h and mice were sacrificed after 48h. (E) Assessment of liver injury by serum ALT levels.
(F) Quantification of liver MoMF, KC and neutrophils as percent of liver leukocytes, determined by FACS
analysis 48h after APAP. Original magnification 10X, scale bar 400µm. All data are presented as mean ±
SEM (n≥4 for vehicle treatment, n≥7 for cenicriviroc treatment).*p
<0.05, **p<0.01, ***p<0.001 (unpaired
Student t test).
180x216mm (300 x 300 DPI)
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Figure 7. CCR2+ infiltrating monocytes display a pro-inflammatory phenotype in patients with ALF.
Consecutive sections of liver samples from patients with acetaminophen-induced acute liver failure (AALF,
n=8) or controls (n=7) were stained for CCR2 (A), CD68 (B), S100A9 and CD163 (C), and five high power
fields were counted for each individual. Data are presented as median (n≥7).*p<0.05, (Mann-Whitney U
test). After quantification of immunopositive cells, the ratio of CCR2, CD68, S100A9 and
CD163 positive cells
found in the portal tract vs. the remaining parenchyma of AALF patients was determined. Original
magnification 100x.
153x213mm (300 x 300 DPI)
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Supplementary Methods
Data analysis from intravital multiphoton microscopy
Pre-analysis for improving the segmentation of GFP-positive cells was performed
using Ilastik (1). We calculated the tracks of individual cells by analyzing their
directed movement between consecutive frames of the video. Based on the tracks
we calculated the average speed, displacement, and straightness for each cell.
Average speed gives the mean speed over time for each track, track displacement is
the distance between the first and the last spot of each track irrespective of its
directionality, while straightness is a measurement of the directionality of the
movement of the cell, with a high rank indicating a straight movement. Straightness is
calculated as total cell displacement divided by the track displacement.
nCounter gene expression analysis
20,000 FACS-separated cells were lysed in RLT lysis buffer (Qiagen) and stored at -
80°C. The cell lysate was incubated with reporter and capture probe sets overnight in
a pre-heated thermocycler at 65°C and afterwards immobilized on a cartridge using
the nCounter Prep Station (NanoString, USA). Cartridges were afterwards scanned
at the nCounter Digital Analyzer at 555 fields of view. Differentially expressed genes
were selected by an adjusted p-value threshold of <0.001 and a log2 fold change >2.
To detect enriched GO pathways for gene enrichment map analysis, very permissive
criteria (p-value <0.05, FDR Q-value cutoff <0.25, similarity cutoff with Jaccard
coefficient <0.5) were chosen.
Primary mouse hepatocyte culture
Primary hepatocytes were isolated from mice by collagenase perfusion methodology
as detailed earlier (2). 400,000 hepatocytes per well were seeded in HepatoZYME-
SFM Medium (Thermo-Fisher Scientific, USA) on collagen-I pre-coated 6-well plates
for 24h at 37°C and 5 % CO2. After 24h the medium was renewed and 40 mM APAP
and 1 µM cenicriviroc were added. 1mM cenicriviroc stock solution was prepared by
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solving cenicriviroc in DMSO with 0.5 % acetic acid and 0.5 % BSA. The final
concentration of DMSO was less than 0.1 % in all experiments. All experiments were
performed in triplicate.
Hepatic glutathione concentrations
Mice were fasted for 12h while receiving cenicriviroc or the corresponding control
solution by oral gavage (100 mg/kg) directly with the removal of food and another 3h
later. After 12h mice were sacrificed and 50mg of liver tissue was snap frozen in
liquid nitrogen and stored at -80°C until measurement. For hepatic glutathione (GSH)
quantification the Glutathione Assay Kit (Abnova, USA) was used according to the
manufacturer’s protocol. All experiments were performed in triplicate.
UGT1a reporter assay
The potential induction of UDP-glucuronosyltransferase (UGT) by cenicriviroc was
tested in vitro using an established reporter cell assay, as previously described (3).
Briefly, cells were seeded in 12-well plates and transfected with respective reporter
gene constructs (800 ng /well) in addition to the pRL-TK plasmid using Lipofectin
Transfection Reagent (Fisher Scientific, Schwerte, Germany) to perform a dual
luciferase assay (Dual-Reporter Assay; Promega, Mannheim, Germany). On the next
day, cells were treated with cenicriviroc (1-10µM) or vehicle (DMSO) for 48h. Coffee,
known as a potent UGT1A inductor (3), was used as a positive control. All
experiments were performed in triplicate.
1. Sommer C, Straehle C, Köthe U, Hamprecht FA. ilastik: Interactive Learning and
Segmentation Toolkit. Proceedings. 2011;230–233.
2. Karlmark KR, Weiskirchen R, Zimmermann HW, Gassler N, Ginhoux F, Weber C,
et al. Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver
injury promotes hepatic fibrosis. Hepatology. 2009;50:261274.
3. Kalthoff S, Ehmer U, Freiberg N, Manns MP, Strassburg CP. Coffee induces
expression of glucuronosyltransferases by the aryl hydrocarbon receptor and Nrf2
in liver and stomach. Gastroenterology. 2010;139:16991710, 1710.e12.
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Supplementary Movies
Suppl. Movie 1: Monocyte migration in control mice
Ccr2+/eGFP reporter mice were analyzed by two-photon laser scanning microscopy
(TPLSM). CCR2+ cells are shown in green, collagen structures in blue; Kupffer
cells had been labelled in red by prior injection of fluorescent particles.
Suppl. Movie 2: Monocyte migration in APAP-injured mice
Ccr2+/eGFP reporter mice were subjected to APAP injury and analyzed by two-
photon laser scanning microscopy (TPLSM). The representative movie starts 8h
after APAP i.v. administration. CCR2+ cells are shown in green, collagen
structures in blue; Kupffer cells had been labelled in red by prior injection of
fluorescent particles.
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Supplementary Figures
Supplementary Figure 1. Myeloid leukocyte populations in liver and blood
after experimental acetaminophen injury in mice. (A) Distinct hepatic
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macrophage populations were assessed by FACS analysis. The gating strategy
to identify MoMF (CD11bhigh and F4/80intermediate) and KC (CD11blow and F4/80high)
is shown. (B) Absolute cell numbers per g liver tissue of hepatic MoMF, KC and
neutrophils. (C) Absolute and relative numbers of blood monocytes. (D) Surface
marker expression of MoMF and KC from representative APAP treated and
control mice. “Fluorescence minus one" (FMO) represents the cell-specific
autofluorescent background. All data are presented as mean ± SEM
(n≥3).*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
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Supplementary Figure 2. Lymphoid liver leukocyte populations after
experimental acetaminophen injury in mice. Distinct hepatic lymphocyte
populations were assessed by FACS analysis. Results are shown as absolute
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cell numbers per g liver tissue and relative numbers as percentage per total liver
leukocytes. (A) Liver CD8+ T cells are gated as living CD45+, NK1.1-, TCRβ+
and CD8+. (B) Liver CD4+ T cells are gated as living, CD45+, NK1.1-, TCRβ+
and CD4+. (C) Liver NK cells are gated as living, CD45+, NK1.1+, TCRβ- cells.
(D) Liver NKT cells are gated as living, CD45+, NK1.1+, TCRβ+ cells. All data
are presented as mean ± SEM (n≥3).*p<0.05, **p<0.01, ***p<0.001 (unpaired
Student t test).
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Supplementary Figure 3. Pharmacological inhibition of CCL2 blocks
monocyte infiltration and attenuates liver injury after APAP. (A) C57BL/6J
mice received the CCL2 inhibitor mNOX-E36 twice by subcutaneous injection
prior to APAP administration (250mg/kg IV) at the indicated time-points. (B)
Assessment of liver injury by histology, quantification of necrotic area fraction
and serum ALT levels. Original magnification 10X, scale bar 400µm. (C)
Quantification of liver MoMF (red gate) and KC (green gate) as percent of liver
leukocytes as determined by FACS analysis 12h after APAP (representative
FACS plots displayed for each condition). All data are presented as mean ± SEM
(n≥3).*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
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Supplementary Figure 4. Pharmacological inhibition of infiltrating
monocytes in Ccr2-/- and Ccr5-/- mice. (A) WT, Ccr2-/- and Ccr5-/- mice received
the CCR2/CCR5 inhibitor cenicriviroc (CVC) PO directly after APAP (250mg/kg
IV) and another 3h later. Mice were analyzed after 12h. (B) Assessment of liver
injury by serum ALT levels. (C) Quantification of liver MoMF and KC as percent
of liver leukocytes, determined by FACS analysis 12h after APAP. All data are
presented as mean ± SEM (n≥4).*p<0.05, **p<0.01, ***p<0.001 (unpaired
Student t test).
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Supplementary Figure 5. Cenicriviroc does not affect APAP hepatotoxicity,
metabolism or clearance. (A) LDH levels in the supernatant of primary murine
hepatocytes treated with APAP with or without cenicriviroc (CVC). (B)
Glutathione (GSH) concentrations in livers of mice that were fasted for 12h and
received either cenicriviroc (CVC) or control solution by oral gavage. Please note
that CVC does not increase hepatic GSH levels. (C) Induction of UGT1A reporter
gene constructs by CVC in a cell (HepG2) based reporter assay. Coffee extract
served as a positive control. Data summarize n=3 experiments per
assay.*p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
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... Whilst the role of inflammation in the initial liver injury of APAP ALI remains a subject of debate, it has been suggested in mice that the potency of the initial inflammatory immune response in APAP ALI is potentially maladaptive, aggravating liver injury. Monocytes are recruited by CCL2/CCR2 signalling, and use of CCR2-/-knockout mice, or CCL2 inhibition, demonstrates reduced liver injury after APAP, suggesting that the initial inflammatory response is not a required step in recovery after APAP toxicity (Mossanen et al. 2016). We can, therefore, infer that any impact of AAMs in reducing the initial inflammatory response if administered early in injury (rather than simply promoting resolution) is unlikely to be harmful. ...
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Acute liver injury (ALI) has a clear requirement for novel therapies. One emerging option is the use of alternatively activated macrophages (AAMs); a distinct subtype of macrophage with a role in liver injury control and repair. In this comprehensive review, we provide an overview of the current limited options for ALI, and the potential advantages offered by AAMs. We describe the evidence supporting their use from in vitro studies, pre-clinical animal studies, and human clinical trials. We suggest why the first evidence for the clinical use of AAMs is likely to be found in acetaminophen toxicity, and discuss the specific evidence for AAM use in this population, as well as potential applications for AAMs in other patient populations. The key domains by which the performance of AAMs for the treatment of ALI will be assessed are identified, and remaining challenges to the successful delivery of AAMs to clinic are explored.
... The BMDM were then cocultured with BMSCs or BMSCs-33 for 48 h. The protein CD68 represented total BMDM, Arg-1 and CD163 were M2-type BMDM markers and iNOS and IL-6 were M1-type BMDM markers [29][30][31]. ...
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Mesenchymal stem cells (MSCs) are highly effective in the treatment of acute liver failure (ALF). The efficacy of MSCs is closely related to the inflammatory environment. Therefore, we investigated the functional changes of MSCs in response to interleukin-33 (IL-33) stimulation. The results showed that bone marrow mesenchymal stem cells (BMSCs) pretreated with IL-33 had increased CCR2 expression, targeted CCL2 in the injured liver tissue, and improved the migration ability. Under LPS stimulation, the NF-κB pathway of BMDM was activated, and its phenotype polarized to the M1-type, while BMSCs pretreated with IL-33 inhibited the NF-κB pathway and enhanced M2 macrophage polarization. The M2-type macrophages could further inhibit hepatocytes inflammation, reduce hepatocytes apoptosis, and promote hepatocytes repair. These results suggest that IL-33 can enhance the efficacy of BMSCs in ALF and provide a new strategy for cell therapy of liver diseases.
... Hepatic macrophages (HMs), including monocyte-derived macrophages and resident Kupffer cells (KCs), play critical roles in regulating liver inflammation and injury [3,4]. Studies have shown that HMs contribute to the progression of liver injury by releasing proinflammatory cytokines and chemokines in several experimental murine models [5][6][7][8][9] or human clinical investigations [10][11][12]. Following injury, HMs can quickly transition to a proresolving phenotype to promote the restoration of the injured liver by producing anti-inflammatory mediators, such as VEGF-α and TGF-β [13]. ...
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Liver injury releases danger‐associated molecular patterns, which trigger the immune response. CD24 negatively regulates the immune response by binding with danger‐associated molecular patterns, but the specific role of CD24 in modulating macrophage‐related inflammation during liver injury remains largely unexplored. Here, we aimed to investigate the mechanisms of macrophage CD24 in the development of liver injury. Our results show that CD24 expression is upregulated primarily in hepatic macrophages (HMs) during acute liver injury. CD24‐deficient mice exhibited more severe liver injury and showed a significantly higher frequency and number of HMs, particularly Ly6Chi monocyte‐derived macrophages. Mechanistically, the CD24‐Siglec‐G interaction plays a vital role in mitigating acute liver injury. CD24‐mediated inhibitory signaling in HMs primarily limits downstream NF‐κB and p38 MAPK activation through the recruitment of SHP1. Our work unveils the critical role of macrophage CD24 in negatively regulating innate immune responses and protecting against acute liver injury, thus providing potential therapeutic targets for liver‐associated diseases.
... Newly infiltrated Ly-6C hi monocytederived macrophages have a pro-inflammatory phenotype, evidenced by increased expression of inflammatory cytokines such as tumor necrosis factor (TNF) or IL-1β (Zigmond et al. 2014). Therefore, inhibition of monocyte infiltration using CCL2 or CCR2 antagonists early after APAP administration can ameliorate liver injury (Mossanen et al. 2016). In the later stages of DILI, Ly-6C hi macrophages mature into Ly-6C lo reparative macrophages, which are characterized by the expression of anti-inflammatory cytokines (IL-10, IL-4, IL-13), regenerative growth factors, and matrix-degrading metalloproteinases (MMPs), thereby promoting injury resolution (Ju and Tacke 2016;Bourdi et al. 2002;Ju et al. 2002). ...
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Drug-induced liver injury (DILI) is an acute liver injury that poses a significant threat to human health. In severe cases, it can progress into chronic DILI or even lead to liver failure. DILI is typically caused by either intrinsic hepatotoxicity or idiosyncratic metabolic or immune responses. In addition to the direct damage drugs inflict on hepatocytes, the immune responses and liver inflammation triggered by hepatocyte death can further exacerbate DILI. Initially, we briefly discussed the differences in immune cell activation based on the type of liver cell death (hepatocytes, cholangiocytes, and LSECs). We then focused on the role of various immune cells (including macrophages, monocytes, neutrophils, dendritic cells, liver sinusoidal endothelial cells, eosinophils, natural killer cells, and natural killer T cells) in both the liver injury and liver regeneration stages of DILI. This article primarily reviews the role of innate immune regulation mediated by these immune cells in resolving inflammation and promoting liver regeneration during DILI, as well as therapeutic approaches targeting these immune cells for the treatment of DILI. Finally, we discussed the activation and function of liver progenitor cells (LPCs) during APAP-induced massive hepatic necrosis and the involvement of chronic inflammation in DILI.
... The initial NAPQI-induced direct hepatocyte damage results in the release of damage-associated molecular patterns that trigger a sterile inflammatory response [3]. This response includes the activation of cytokines and the formation of chemokines for the infiltration of immune cells in regions of hepatocyte damage, leading to further aggravation of liver injury in the early phase of APAP toxicity [4,5]. APAP-induced acute liver injury initiates a regenerative response; however, impairment of the resolution of liver inflammation and liver repair induces persistent liver injury, leading to acute liver failure. ...
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Background Acetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure. Macrophages are key players in liver restoration following APAP-induced liver injury. Thromboxane A 2 (TXA 2 ) and its receptor, thromboxane prostanoid (TP) receptor, have been shown to be involved in tissue repair. However, whether TP signaling plays a role in liver repair after APAP hepatotoxicity by affecting macrophage function remains unclear. Methods Male TP knockout ( TP −/− ) and C57BL/6 wild-type (WT) mice were treated with APAP (300 mg/kg). In addition, macrophage-specific TP-knockout ( TP △mac ) and control WT mice were treated with APAP. We explored changes in liver inflammation, liver repair, and macrophage accumulation in mice treated with APAP. Results Compared with WT mice, TP −/− mice showed aggravated liver injury as indicated by increased levels of alanine transaminase (ALT) and necrotic area as well as delayed liver repair as indicated by decreased expression of proliferating cell nuclear antigen (PCNA). Macrophage deletion exacerbated APAP-induced liver injury and impaired liver repair. Transplantation of TP -deficient bone marrow (BM) cells to WT or TP −/− mice aggravated APAP hepatotoxicity with suppressed accumulation of macrophages, while transplantation of WT-BM cells to WT or TP −/− mice attenuated APAP-induced liver injury with accumulation of macrophages in the injured regions. Macrophage-specific TP −/− mice exacerbated liver injury and delayed liver repair, which was associated with increased pro-inflammatory macrophages and decreased reparative macrophages and hepatocyte growth factor (HGF) expression. In vitro, TP signaling facilitated macrophage polarization to a reparative phenotype. Transfer of cultured BM-derived macrophages from control mice to macrophage-specific TP −/− mice attenuated APAP-induced liver injury and promoted liver repair. HGF treatment mitigated APAP-induced inflammation and promoted liver repair after APAP-induced liver injury. Conclusions Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury, which is associated with reduced accumulation of reparative macrophages and the hepatotrophic factor HGF. Specific activation of TP signaling in macrophages may be a potential therapeutic target for liver repair and regeneration after APAP hepatotoxicity.
... Alongside the in ammatory response, polymorphonuclear leukocytes (neutrophils) are rapidly recruited to the site of cell injury, providing a substantial source of reactive oxygen species (ROS) that promote liver repair 20 . Subsequently, there is a massive in ltration of in ammatory monocytes, leading to monocytopenia in the circulation, re ecting the degree of liver injury 21,22 . As these monocytes differentiate into macrophages, they assume an anti-in ammatory, wound-healing phenotype, initiating the healing process and facilitating the transition from the initial in ammatory phase of acute liver injury (ALI) to the resolution phase. ...
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Acute liver failure (ALF) is a rapidly progressing, life-threatening condition most commonly caused by an overdose of acetaminophen (paracetamol). The antidote, N-acetylcysteine (NAC), has limited efficacy when liver injury is established. If acute liver damage is severe, liver failure can rapidly develop with associated high mortality rates. We have previously demonstrated that alternatively activated macrophages are a potential therapeutic option to reverse acute liver injury in pre-clinical models. In this paper we present data using cryopreserved human alternatively activated macrophages (hAAMs) - which represent a potential, rapidly available, treatment suitable for use in the acute setting. In a mouse model of APAP-induced injury, peripherally injected cryopreserved hAAMs reduced liver necrosis, modulated inflammatory responses, and enhanced liver regeneration. hAAMs were effective even when administered after the therapeutic window for N-acetylcysteine. This cell therapy approach represents a potential treatment for APAP overdose when NAC is ineffective because liver injury is established.
... Inhibition of c-c motif chemokine receptor 2 (ccR2) reduced the accumulation, migration and infiltration of monocytes and macrophages to the liver, which decreased hepatic damage (215). ccR5 participates in activation of hepatic stellate cells following liver injury, further aggravating hepatic fibrosis (216). ...
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Monocytes are recruited from the blood to sites of inflammation, where they contribute to wound healing and tissue repair. There are at least two subsets of monocytes: classical or proinflammatory (CCR2(hi)CX3CR1(low)) and nonclassical, patrolling, or alternative (CCR2(low)CX3CR1(hi)) monocytes. Using spinning-disk confocal intravital microscopy and mice with fluorescent reporters for each of these subsets, we were able to track the dynamic spectrum of monocytes that enter a site of sterile hepatic injury in vivo. We observed that the CCR2(hi)CX3CR1(low) monocytes were recruited early and persisted for at least 48 h, forming a ringlike structure around the injured area. These monocytes transitioned, in situ, from CCR2(hi)Cx3CR1(low) to CX3CR1(hi)CCR2(low) within the ringlike structure and then entered the injury site. This phenotypic conversion was essential for optimal repair. These results demonstrate a local, cytokine driven reprogramming of classic, proinflammatory monocytes into nonclassical or alternative monocytes to facilitate proper wound-healing. © 2015 Dal-Secco et al.
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Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.
Article
The induction of acute hepatic damage by acetaminophen (N-acetyl-p-aminophenol [APAP]), also termed paracetamol, is one of the most commonly used experimental models of acute liver injury in mice. The specific values of this model are the highly reproducible, dose-dependent hepatotoxicity of APAP and its outstanding translational importance, because acetaminophen overdose is one of the most frequent reasons for acute liver failure (ALF) in humans. However, preparation of concentrated APAP working solutions, application routes, fasting period and variability due to sex, genetic background or barrier environment represent important considerations to be taken into account before implementing this model. This standard operating procedure (SOP) provides a detailed protocol for APAP preparation and application in mice, aimed at facilitating comparability between research groups as well as minimizing animal numbers and distress. The mouse model of acetaminophen poisoning therefore helps to unravel the pathogenesis of APAP-induced toxicity or subsequent immune responses in order to explore new therapeutic interventions for improving the prognosis of ALF in patients. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.
Article
Unlabelled: The liver is essential for inducing immunological tolerance toward harmless antigens to maintain immune system homeostasis. However, the precise cellular mechanisms of tolerance induction against particle-bound antigens, the role of the local hepatic microenvironment, and implications for therapeutic targets in immune-mediated diseases are currently unclear. In order to elucidate cellular mechanisms of tolerance induction in healthy and injured liver, we developed a novel in vivo system combining the systemic delivery of low-dose peptide antigens coupled to inert particles, immunological readouts, and sophisticated intravital multiphoton microscopy-based imaging of liver in mice. We show that liver resident macrophages, Kupffer cells (KCs), but not hepatic monocyte-derived macrophages or dendritic cells (DCs), are the central cellular scavenger for circulating particle-associated antigens in homeostasis. KC-associated antigen presentation induces CD4 T-cell arrest, expansion of naturally occurring Foxp3(+) CD25(+) interleukin-10-producing antigen-specific regulatory T cells (Tregs) and tolerogenic immunity. Particle-associated tolerance induction in the liver protected mice from kidney inflammation in T-cell-mediated glomerulonephritis, indicating therapeutic potential of targeting KC for immune-mediated extrahepatic disorders. Liver inflammation in two independent experimental models of chronic liver injury and fibrosis abrogated tolerance induction and led to an immunogenic reprogramming of antigen-specific CD4 T cells. In injured liver, infiltrating monocyte-derived macrophages largely augment the hepatic phagocyte compartment, resulting in antigen redistribution between myeloid cell populations and, simultaneously, KCs lose signature markers of their tolerogenic phenotype. Conclusions: Hepatic induction of tissue-protective immunological tolerance against particulate antigens is dependent on KCs as well as on a noninflamed liver microenvironment, thereby providing mechanistic explanations for the clinical observation of immune dysfunction and tolerance break in patients with advanced liver diseases.