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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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... However, exposure to pathogen-associated molecular patterns (PAMPs) breaks the tolerogenic environment, triggering an inflammatory cascade that recruits inflammatory immune cells to the liver, causing non-specific hepatocyte killing (10). Resident within the liver and sinusoids, and recruited early in the inflammatory process, is a significant monocyte population with the plasticity to develop into inflammatory macrophages (11,12). Given the central role of myeloid cells in the inflammatory process, we hypothesized the myeloid population within the liver may be an important source of the sexual dimorphism observed in the progression of chronic liver inflammation. ...
... With no differences in myeloid composition, we tested if the inflammatory potential of intrasinusoidal myeloid cells differed between males and females, as myeloid cells play key roles in liver inflammation and differentiate into inflammatory macrophages (11,12). To investigate this, we tested the response of monocytes to inflammatory stimuli. ...
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Background & AimMen have a higher prevalence of liver disease. Liver myeloid cells can regulate tissue inflammation, which drives progression of liver disease. We hypothesized that sex alters the responsiveness of liver myeloid cells, predisposing men to severe liver inflammation.Methods Luminex was done on plasma from Hepatitis B Virus infected patients undergoing nucleoside analogue cessation in 45 male and female patients. We collected immune cells from the sinusoids of uninfected livers of 53 male and female donors. Multiparametric flow cytometry was used to phenotype and characterize immune composition. Isolated monocytes were stimulated with TLR ligands to measure the inflammatory potential and the expression of regulators of TLR signaling.ResultsWe confirmed that men experienced more frequent and severe liver damage upon Hepatitis B Virus reactivation, which was associated with inflammatory markers of myeloid activation. No differences were observed in the frequency or phenotype of sinusoidal myeloid cells between male and female livers. However, monocytes from male livers produced more inflammatory cytokines and chemokines in response to TLR stimulation than female monocytes. We investigated negative regulators of TLR signaling and found that TOLLIP was elevated in female liver-derived monocytesConclusions Our data show that enhanced responsiveness of myeloid cells from the male liver predisposes men to inflammation, which was associated with altered expression of negative regulators of TLR signaling.
... TERN-201, a kind of potent VAP-1 inhibitor, is still undergoing clinical trials in China for the treatment of NASH (62,63,135). Circulating inflammatory monocytes are attracted to the hepatic microenvironment via their chemokine receptor C-C motif chemokine receptor 2 (CCR2), while the corresponding CCL2 is strongly expressed by various liver cells such LSECs and KCs (136). Cenicriviroc (CVC), a dual antagonist of CCR2 and CCR5, ameliorates hepatic inflammation in NASH mice models by reducing the recruitment of CCR2+ monocyte in the liver. ...
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Chronic liver injury can be caused by many factors, including virus infection, alcohol intake, cholestasis and abnormal fat accumulation. Nonalcoholic steatohepatitis (NASH) has become the main cause of liver fibrosis worldwide. Recently, more and more evidences show that hepatic microenvironment is involved in the pathophysiological process of liver fibrosis induced by NASH. Hepatic microenvironment consists of various types of cells and intercellular crosstalk among different cells in the liver sinusoids. Liver sinusoidal endothelial cells (LSECs), as the gatekeeper of liver microenvironment, play an irreplaceable role in the homeostasis and alterations of liver microenvironment. Many recent studies have reported that during the progression of NASH to liver fibrosis, LSECs are involved in various stages mediated by a series of mechanisms. Therefore, here we review the key role of crosstalk between LSECs and hepatic microenvironment in the progression of NASH to liver fibrosis (steatosis, inflammation, and fibrosis), as well as promising therapeutic strategies targeting LSECs.
... The Ly6C hi and Ly6C lo subsets exhibit functional heterogeneity, which is indicated by the high diversity in cellsurface marker, cytokine release and transcriptional profiles (14,(30)(31)(32). Indeed, Ly6C hi macrophages derived from circulating Ly6C hi monocytes are more enriched in the acute inflammatory response and show a proinflammatory ability (3). ...
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Macrophages originating from the yolk sac or bone marrow play essential roles in tissue homeostasis and disease. Bone marrow-derived monocytes differentiate into Ly6Chi and Ly6Clo macrophages according to the differential expression of the surface marker protein Ly6C. Ly6Chi and Ly6Clo cells possess diverse functions and transcriptional profiles and can accelerate the disease process or support tissue repair and reconstruction. In this review, we discuss the basic biology of Ly6Chi and Ly6Clo macrophages, including their origin, differentiation, and phenotypic switching, and the diverse functions of Ly6Chi and Ly6Clo macrophages in homeostasis and disease, including in injury, chronic inflammation, wound repair, autoimmune disease, and cancer. Furthermore, we clarify the differences between Ly6Chi and Ly6Clo macrophages and their connections with traditional M1 and M2 macrophages. We also summarize the limitations and perspectives for Ly6Chi and Ly6Clo macrophages. Overall, continued efforts to understand these cells may provide therapeutic approaches for disease treatment.
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Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has created a global pandemic infecting over 230 million people and costing millions of lives. Therapies to attenuate severe disease are desperately needed. Cenicriviroc (CVC), a C-C chemokine receptor type 5 (CCR5) and C-C chemokine receptor type 2 (CCR2) antagonist, an agent previously studied in advanced clinical trials for patients with HIV or nonalcoholic steatohepatitis (NASH), may have the potential to reduce respiratory and cardiovascular organ failures related to COVID-19. Inhibiting the CCR2 and CCR5 pathways could attenuate or prevent inflammation or fibrosis in both early and late stages of the disease and improve outcomes of COVID-19. Clinical trials using CVC either in addition to standard of care (SoC; e.g., dexamethasone) or in combination with other investigational agents in patients with COVID-19 are currently ongoing. These trials intend to leverage the anti-inflammatory actions of CVC for ameliorating the clinical course of COVID-19 and prevent complications. This article reviews the literature surrounding the CCR2 and CCR5 pathways, their proposed role in COVID-19, and the potential role of CVC to improve outcomes.
Thesis
Kupffer cells (KCs) are resident macrophages of the liver. Professional phagocytes of the innate immune system, they take part in the first line of defence against infections and injury. They also actively regulate liver homeostasis. Recent works have elucidated their origin. We now know that, like most other tissue resident macrophages, KCs develop during embryonic life from Erythro-Myeloid Progenitors (EMPs), seed the liver during development and persist there in adulthood. During inflammation, however, they can be joined by recently differentiated macrophages that arise from circulating monocytes belonging to the Haematopoietic Stem Cell (HSC) descendance. Here I studied the ability of mouse KCs to maintain themselves throughout life, and into old age. Using flow cytometry and fate mapping strategies, we showed that KC density decreased over time and was not compensated by recruitment of circulating cells. RNA sequencing, analysis of TicamLPS2 mutants and Poly (I:C)-induced repeated inflammation experiments highlighted the contribution of inflammation to the ageing phenotype. This phenotype correlated with lipid and senescent cell accumulation. We further studied KC maintenance in acetaminophen induced liver injury and after depletion induced by a CSF1R antagonist. In both contexts, KCs were able to maintain themselves through local proliferation without significant input from circulating cells. These experiments will provide a framework for the better characterisation of KC functions in injury and disease.
Chapter
Chronic liver inflammation leads to fibrosis. This chapter provides an in-depth description of the cellular and molecular mechanisms that link these events. Possible antifibrotic targets are highlighted.
Chapter
Obesity and sedentarism may cause fatty liver, leading to nonalcoholic steatohepatitis, fibrosis, cirrhosis, and even hepatocellular carcinoma. These frequent and increasing pathologies are reviewed in this chapter with a special focus on the cellular and molecular mechanisms involved. Possible therapeutic targets are highlighted.
Chapter
Macrophages are a heterogeneous population of innate immune cells and key cellular components of the liver. Hepatic macrophages consist of embryologically-derived resident Kupffer cells (KC), recruited monocyte-derived macrophages (MDM) and capsular macrophages. Both the diversity and plasticity of hepatic macrophage subsets explain their different functions in the maintenance of hepatic homeostasis and in injury processes in acute and chronic liver diseases. In this review, we assess the evidence for macrophage involvement in regulating both liver health and injury responses in liver diseases including acute liver injury (ALI), chronic liver disease (CLD) (including liver fibrosis) and hepatocellular carcinoma (HCC). In healthy livers, KC display critical functions such as phagocytosis, danger signal recognition, cytokine release, antigen processing and the ability to orchestrate immune responses and maintain immunological tolerance. However, in most liver diseases there is a striking hepatic MDM expansion, which orchestrate both disease progression and regression. Single-cell approaches have transformed our understanding of liver macrophage heterogeneity, dynamics, and functions in both human samples and preclinical models. We will further discuss the new insights provided by these approaches and how they are enabling high-fidelity work to specifically identify pathogenic macrophage subpopulations. Given the important role of macrophages in regulating injury responses in a broad range of settings, there is now a huge interest in developing new therapeutic strategies aimed at targeting macrophages. Therefore, we also review the current approaches being used to modulate macrophage function in liver diseases and discuss the therapeutic potential of targeting macrophage subpopulations as a novel treatment strategy for patients with liver disorders.
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Acetaminophen (APAP) -induced liver injury (AILI) is the most common cause of acute liver failure. Although the mechanisms which trigger AILI are well known, it is less understood how to halt AILI progression and facilitate liver recovery. Therefore, it is necessary to understand the pathophysiology of APAP hepatotoxicity in patients and to examine predictive/preventive markers. In a clinical study, we had a case in which AST and ALT levels increased in a patient with a low ratio of APAP glucuronide concentration (AP-G)/APAP plasma concentration. Then, a reverse translational study was conducted for clarifying this clinical question. The relationship between plasma AP-G/APAP concentration ratio and the levels of AST and ALT was examined by in vivo and in vitro experiments. In in vivo experiments, 10-week-old rats showed lower UGT activity, lower AP-G/APAP concentration ratios, and higher AST and ALT levels than 5-week-old rats. This suggests an inverse correlation between the AP-G/APAP concentration ratio and the AST, ALT levels in APAP-treated rats. Furthermore, as a result of in vitro experiment, it was confirmed that the cell viability decreased when the AP-G/APAP concentration ratio in the culture medium decreased. Since the decrease in the plasma AP-G/APAP concentration ratio appears earlier than the increase of AST and ALT levels, the ratio might be a presymptomatic marker of AILI. When APAP is used for a long time, it is recommended to perform therapeutic drug monitoring of AP-G/APAP concentration ratio, which is a predictive/preventive marker of AILI. This article is protected by copyright. All rights reserved.
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Acute liver injury (ALI) is characterized by massive hepatocyte necrosis and subsequent recruitment of myeloid cells to liver. Mesenchymal stem cells (MSCs) have therapeutic potential for ALI through their immunoregulation on macrophages, but the mechanism is not completely clear due to the heterogeneity and controversy of liver macrophages. Here, we detected the survival rate, biochemical indexes, histopathology, and inflammatory chemokine levels to assess the efficacy of MSC treatment on CCl4-induced ALI of C57BL/6 mice. Furthermore, flow cytometry and single-cell RNA sequencing (scRNA-Seq) were used to precisely distinguish macrophage populations and reveal the immunoregulation of MSCs. MSC treatment could effectively alleviate ALI and mitigate the recruitment of mononuclear phagocytes. Flow cytometry and scRNA-Seq analyses collectively indicated that there were monocytes with high Ly6C expression and heterogeneous monocyte-derived macrophages (MoMF) with low Ly6C expression in liver. Ly6Chi pro-inflammatory monocytes and Ly6Clo MoMF with powerful phagocytosis dominated during the acute injury period. MSC treatment promoted the transition from Ly6Chi to Ly6Clo population, inhibit the proinflammatory function of monocytes and promote the lysosomal function of MoMF. Furthermore, MSCs attenuated the recruitment of neutrophils by reducing the expression of CXCL2 of MoMF. MoMF with high expression of arginase 1 appeared during the recovery period, and MSCs could increase their expression of arginase 1, which may promote liver repair. To sum up, we demonstrated the characteristics of distinct MoMF during different periods of ALI and revealed their functional changes after MSC treatment, providing immunotherapeutic targets for MSC treatment of ALI.
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Background: Non-alcoholic steatohepatitis (NASH) is often accompanied by liver fibrosis, which can progress to cirrhosis; C-C chemokine receptors type 2 and 5 (CCR2/CCR5), which mediate interactions driving inflammation and fibrosis, are promising treatment targets. Cenicriviroc (CVC), a dual CCR2/CCR5 antagonist, has potent anti-inflammatory and antifibrotic activity in animal models; in HIV-positive subjects it reduced soluble CD14 levels, aspartate aminotransferase-to-platelet count ratio index, and non-invasive hepatic fibrosis risk scores; favorable tolerability was demonstrated in ~600 subjects. Efficacy and safety of CVC 150mg for treating NASH with liver fibrosis are being evaluated over 2years (primary endpoint at Year 1 [Y1]). Design: Phase 2b, randomized, double-blind, placebo-controlled, multinational study (CENTAUR; NCT02217475). Adults with histological evidence of NASH, non-alcoholic fatty liver disease activity score (NAS) ≥4, and liver fibrosis (Stages 1-3 NASH clinical research network system) enrolled. Subjects have increased risk of progression to cirrhosis due to ≥1 characteristic: type 2 diabetes; body mass index >25kg/m(2) with ≥1 feature of metabolic syndrome; bridging fibrosis and/or NAS ≥5. Liver biopsy evaluation at Screening, Y1, and Year 2 (Y2). Objectives: Assess histologic improvement (≥2-point in NAS with ≥1-point improvement in >1 category) without worsening of fibrosis at Y1 (primary); evaluate complete NASH resolution without worsening of fibrosis at Y2 (key secondary). Discussion: CENTAUR is the first prospective study evaluating an oral agent exclusively enrolling subjects with NASH and liver fibrosis, with increased risk of developing cirrhosis. It will compare shorter versus longer CVC treatment and assess correlations between decreased inflammation and fibrosis.
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Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them.
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The liver is a central immunological organ with a high exposure to circulating antigens and endotoxins from the gut microbiota, particularly enriched for innate immune cells (macrophages, innate lymphoid cells, mucosal-associated invariant T (MAIT) cells). In homeostasis, many mechanisms ensure suppression of immune responses, resulting in tolerance. Tolerance is also relevant for chronic persistence of hepatotropic viruses or allograft acceptance after liver transplantation. The liver can rapidly activate immunity in response to infections or tissue damage. Depending on the underlying liver disease, such as viral hepatitis, cholestasis or NASH, different triggers mediate immune-cell activation. Conserved mechanisms such as molecular danger patterns (alarmins), Toll-like receptor signalling or inflammasome activation initiate inflammatory responses in the liver. The inflammatory activation of hepatic stellate and Kupffer cells results in the chemokine-mediated infiltration of neutrophils, monocytes, natural killer (NK) and natural killer T (NKT) cells. The ultimate outcome of the intrahepatic immune response (for example, fibrosis or resolution) depends on the functional diversity of macrophages and dendritic cells, but also on the balance between pro-inflammatory and anti-inflammatory T-cell populations. As reviewed here, tremendous progress has helped to understand the fine-tuning of immune responses in the liver from homeostasis to disease, indicating promising targets for future therapies in acute and chronic liver diseases.
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In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine/'s activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide[middot]L-protein complex was solved and refined to 2.05[thinsp]A. It unveils the L-aptamer/'s intramolecular contacts and permits a detailed analysis of its structure-function relationship. Furthermore, the analysis of the intermolecular drug-target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.
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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.
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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.
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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.