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Intestinal Microbiota Protects against MCD Diet-Induced Steatohepatitis

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Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western countries, with a continuously rising incidence. Gut-liver communication and microbiota composition have been identified as critical drivers of the NAFLD progression. Hence, it has been shown that microbiota depletion can ameliorate high-fat diet or western-diet induced experimental Non-alcoholic steatohepatitis (NASH). However, its functional implications in the methionine-choline dietary model, remain incompletely understood. Here, we investigated the physiological relevance of gut microbiota in methionine-choline deficient (MCD) diet induced NASH. Experimental liver disease was induced by 8 weeks of MCD feeding in wild-type (WT) mice, either with or without commensal microbiota depletion, by continuous broad-spectrum antibiotic (AB) treatment. MCD diet induced steatohepatitis was accompanied by a reduced gut microbiota diversity, indicating intestinal dysbiosis. MCD treatment prompted macroscopic shortening of the intestine, as well as intestinal villi in histology. However, gut microbiota composition of MCD-treated mice, neither resembled human NASH, nor did it augment the intestinal barrier integrity or intestinal inflammation. In the MCD model, AB treatment resulted in increased steatohepatitis activity, compared to microbiota proficient control mice. This phenotype was driven by pronounced neutrophil infiltration, while AB treatment only slightly increased monocyte-derived macrophages (MoMF) abundance. Our data demonstrated the differential role of gut microbiota, during steatohepatitis development. In the context of MCD induced steatohepatitis, commensal microbiota was found to be hepatoprotective.
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International Journal of
Molecular Sciences
Article
Intestinal Microbiota Protects against MCD
Diet-Induced Steatohepatitis
Kai Markus Schneider 1, Antje Mohs 1, Konrad Kilic 1, Lena Susanna Candels 1, Carsten Elfers 1,
Eveline Bennek 1, Lukas Ben Schneider 1, Felix Heymann 1, Nikolaus Gassler 2, John Penders 3
and Christian Trautwein 1, *
1Department of Internal Medicine III, University Hospital RWTH Aachen, 52074 Aachen, Germany;
Kai.Markus.Schneider@gmail.com (K.M.S.); amohs@ukaachen.de (A.M.); konrad.kilic@gmail.com (K.K.);
lcandels@ukaachen.de (L.S.C.); celfers@ukaachen.de (C.E.); ebennek@ukaachen.de (E.B.);
Lukas.Ben.Schneider@gmx.de (L.B.S.); fheymann@ukaachen.de (F.H.)
2Department of Pathology, Klinikum Braunschweig, 38118 Braunschweig, Germany;
n.gassler@klinikum-braunschweig.de
3Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism,
Maastricht University Medical Center, 6200 MD Maastricht, The Netherlands;
j.penders@maastrichtuniversity.nl
*Correspondence: ctrautwein@ukaachen.de; Tel.: +241-80-80866; Fax: +241-80-82455
Received: 14 November 2018; Accepted: 8 January 2019; Published: 14 January 2019
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Abstract:
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western
countries, with a continuously rising incidence. Gut-liver communication and microbiota composition
have been identified as critical drivers of the NAFLD progression. Hence, it has been shown that
microbiota depletion can ameliorate high-fat diet or western-diet induced experimental Non-alcoholic
steatohepatitis (NASH). However, its functional implications in the methionine-choline dietary model,
remain incompletely understood. Here, we investigated the physiological relevance of gut microbiota
in methionine-choline deficient (MCD) diet induced NASH. Experimental liver disease was induced
by 8 weeks of MCD feeding in wild-type (WT) mice, either with or without commensal microbiota
depletion, by continuous broad-spectrum antibiotic (AB) treatment. MCD diet induced steatohepatitis
was accompanied by a reduced gut microbiota diversity, indicating intestinal dysbiosis. MCD
treatment prompted macroscopic shortening of the intestine, as well as intestinal villi in histology.
However, gut microbiota composition of MCD-treated mice, neither resembled human NASH, nor
did it augment the intestinal barrier integrity or intestinal inflammation. In the MCD model, AB
treatment resulted in increased steatohepatitis activity, compared to microbiota proficient control
mice. This phenotype was driven by pronounced neutrophil infiltration, while AB treatment only
slightly increased monocyte-derived macrophages (MoMF) abundance. Our data demonstrated
the differential role of gut microbiota, during steatohepatitis development. In the context of MCD
induced steatohepatitis, commensal microbiota was found to be hepatoprotective.
Keywords: NASH; Gut-liver-Axis; microbiota; MCD
1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western societies
and due to the obesity epidemic the incidence keeps rising [
1
3
]. The term NAFLD covers a spectrum
of disease manifestations ranging from liver steatosis over non-alcoholic steatohepatitis (NASH), liver
fibrosis, to advanced disease states, such as cirrhosis and hepatocellular carcinoma (HCC) [
2
]. Western
sedentary lifestyle and high caloric diets are the strongest and most significant risk factors for NAFLD
development [
4
]. Accordingly, 90% of obese patients are affected by hepatic steatosis, which usually
Int. J. Mol. Sci. 2019,20, 308; doi:10.3390/ijms20020308 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2019,20, 308 2 of 14
remains clinically asymptomatic. Thirty percent of patients diagnosed with NAFLD demonstrate
histological signs of inflammation, which causes liver cell damage and fuels disease progression
towards liver fibrosis and more advanced states, such as cirrhosis and HCC [
5
]. Given the pivotal role
of hepatic inflammation as a mediator of disease phase transition towards irreversible cirrhosis and
HCC, understanding the underlying mechanisms that perpetuate the inflammatory response in the
liver seems key, in order to design novel disease-modifying therapies.
Recent data identify infiltrating innate immune cells, such as monocyte-derived macrophages
(MoMFs) and neutrophil granulocytes, as mediators of the hepatic inflammation, during NASH [
6
9
].
Pharmacological inhibition of the MoMF infiltration ameliorates NASH development, in man and
mice [
9
,
10
]. These cells express high levels of intracellular and extracellular pathogen recognition
receptors (PRRs) and recognize damage-associated molecular patterns (DAMPs) released upon tissue
damage, as well as pathogen- or microbiota-associated molecular patterns (PAMPSs/MAMPs) that
reach the liver, via the portal circulation [
11
,
12
]. In NLRP3 and NLRP6 inflammasome deficient
mice, unfavorable intestinal microbiota has been linked to a loss of intestinal barrier integrity and
increased translocation of MAMPs into the liver, where they activate TLR4- and TLR9-mediated hepatic
inflammation [13].
These data indicate that translocation of bacterial products from the gut into the liver, contribute
to liver inflammation during NASH. In humans, unfavorable gut microbiota composition has been
identified, both as a regulator of body weight and body-fat composition, as well as a decisive factor
in the intestinal barrier impairment [
14
]. Obese individuals have significantly increased levels of
small intestinal bacterial overgrowth (SIBO), compared to healthy lean subjects, and may suffer from
increased gut permeability, prompting a translocation of lipopolysaccharides (LPS) [1517].
Similarly, mice fed with a high-fat diet become obese, develop insulin resistance, and demonstrate
intestinal barrier impairment and increased translocation of LPS into the portal vein—a phenotype
that closely reflects the disease mechanisms of human NASH [18].
Methionine-choline-deficient (MCD) diet represents another well-established rodent model of
non-alcoholic steatohepatitis, which results in hepatic steatosis, oxidative stress, inflammation, and
fibrosis [
19
]. On the one hand, a lack of choline in this diet, hampers the export of triglycerides
(TG) via a very low-density lipoprotein (VLDL) packaging from hepatocytes, resulting in hepatic
steatosis [
20
]. On the other hand, the essential amino acid methionine is required for the synthesis of
S-adenosylmethionine (SAM) and glutathione, which are both antioxidants [
21
]. Although the data
suggest an important role of sterile inflammation and the innate immune response, mediated by PRR
signaling in MCD-induced NASH [
13
,
22
,
23
], it is a matter of debate as to what extent gut microbiota
and gut-liver crosstalk contribute to steatohepatitis development in this model.
Here, we investigated the relevance of gut microbiota in MCD-induced experimental NASH.
2. Results
2.1. Microbiota Depletion Augments Steatohepatitis Development in the Murine MCD Model
To investigate the relevance of gut microbiota for the development and progression of MCD
induced steatohepatitis, 8 weeks old male mice were fed a methionine choline-deficient (MCD) diet for
8 weeks. One group of mice received a well-established cocktail of four non-absorbable broad-spectrum
antibiotics in their drinking water, for the whole feeding experiment, while the other group of age
and gender-matched mice received normal drinking water. After 8 weeks of dietary intervention,
the caecum of antibiotics-treated mice was strongly enlarged. As expected, liver hematoxylin and
eosin-stained liver sections of the MCD-fed mice displayed all hallmarks of NASH, including steatosis,
inflammation, and fibrosis (Figure 1A). Interestingly and other than expected, antibiotic treatment
(ABx) resulted in aggravated steatosis and significantly increased the inflammatory cell infiltration
(Figure 1A,B). In addition, the ABx-treated mice had a higher histopathological NAFLD activity score
(NAS) and a significantly higher liver-to-body weight ratio (Figure 1C,D).
Int. J. Mol. Sci. 2019,20, 308 3 of 14
Altogether, these data indicated that microbiota depletion results in a more severe liver injury.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 14
Figure 1. Antibiotic treatment (ABx) aggravates non-alcoholic steatohepatitis (NASH) in the murine
methionine-choline deficient (MCD) model. (A) Representative liver histology (hematotoxylin and
eosin staining) showing livers of the wild-type (WT) mice with (+ABx) and without (–ABx) treatment
on the normal chow diet (NCD) and after MCD treatment. (B) Increased “Inflammation” score in the
ABx treated mice. (C) ABx treated mice had a higher histopathological non-alcoholic fatty liver disease
(NAFLD) activity score (NAS). (D) ABx resulted in significantly increased Liver-to-Body-Weight
ratios. Data are expressed as the mean ± SD from 2–5 mice per group and were considered significant
if * p < 0.05, ** p < 0.01
2.2. Antibiotic Treatment Increases Hepatic Fat Accumulation in the MCD-Fed Mice, But Is Not Associated
with a Metabolic Phenotype Characteristic of the Human NASH
As previously reported, the MCD treatment resulted in a significant loss in the total body weight
and MCD feeding did not trigger increased fasting glucose levels. Interestingly, the ABx-treated mice
demonstrated an increased hepatic lipid accumulation shown by HE and Oil Red O stainings (Figures
1A and 2A), which was also reflected in the histopathological steatosis score (Figure 2B). A
colorimetric hepatic triglyceride assay confirmed the significantly increased triglyceride levels in the
ABx-treated mice, compared to the MCD-treated control mice (Figure 2C). Hence, antibiotic
treatment in the MCD-fed mice prompted an increased hepatic lipid storage, but was not associated
with the metabolic phenotype characteristic of the human NASH.
Figure 1.
Antibiotic treatment (ABx) aggravates non-alcoholic steatohepatitis (NASH) in the murine
methionine-choline deficient (MCD) model. (
A
) Representative liver histology (hematotoxylin and
eosin staining) showing livers of the wild-type (WT) mice with (+ABx) and without (–ABx) treatment
on the normal chow diet (NCD) and after MCD treatment. (
B
) Increased “Inflammation” score in the
ABx treated mice. (
C
)ABx treated mice had a higher histopathological non-alcoholic fatty liver disease
(NAFLD) activity score (NAS). (
D
) ABx resulted in significantly increased Liver-to-Body-Weight ratios.
Data are expressed as the mean
±
SD from 2–5 mice per group and were considered significant if
*p< 0.05, ** p< 0.01.
2.2. Antibiotic Treatment Increases Hepatic Fat Accumulation in the MCD-Fed Mice, But Is Not Associated
with a Metabolic Phenotype Characteristic of the Human NASH
As previously reported, the MCD treatment resulted in a significant loss in the total body weight
and MCD feeding did not trigger increased fasting glucose levels. Interestingly, the ABx-treated
mice demonstrated an increased hepatic lipid accumulation shown by HE and Oil Red O stainings
(Figures 1A and 2A), which was also reflected in the histopathological steatosis score (Figure 2B).
A colorimetric hepatic triglyceride assay confirmed the significantly increased triglyceride levels in the
ABx-treated mice, compared to the MCD-treated control mice (Figure 2C). Hence, antibiotic treatment
in the MCD-fed mice prompted an increased hepatic lipid storage, but was not associated with the
metabolic phenotype characteristic of the human NASH.
Int. J. Mol. Sci. 2019,20, 308 4 of 14
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 14
Figure 2. Antibiotic treatment increased the hepatic fat accumulation in the MCD-fed mice. (A)
Representative Oil Red O stainings demonstrated increased the hepatic lipid accumulation upon an
antibiotic treatment. (B) Steatosis score was higher in the ABx-treated mice, compared to the MCD-
fed control mice. (C) Colorimetric hepatic triglyceride assay confirmed significantly increased hepatic
triglycerides (TG) levels in +ABx group. Data are expressed as the mean ± SD from 2–5 mice per group
and were considered significant if ** p < 0.01
2.3. Microbiota Depletion Augments the Inflammatory Response During the MCD-Induced Steatohepatitis
Next, we investigated the relevance of intestinal microbiota for hepatic inflammation, during the
MCD-induced steatohepatitis. The Abx-treated mice demonstrated significantly higher inflammation
in the histopathological analyses of the HE sections (Figure 1B). To further analyze which cell type
mediated the pronounced inflammatory response, we first performed immunofluorescence staining
against the CD11b. Here, CD11b+ immune cells were increased in the livers of the Abx-treated mice,
compared to the MCD-fed controls (Figure 3A). To further dissect which cell type accounted for the
inflammatory response, we performed a flow cytometry (FACS) analysis of the liver homogenates.
MCD feeding induced a strong infiltration of the MoMFs (defined as Ly6G-, CD11b
hi
, F4/80
low
). In
contrast, both, the absolute and relative numbers of neutrophil granulocytes (defined as CD11b+,
Ly6G+) were only slightly increased in the MCD versus the NCD-fed mice (Figure 3B,C). While
antibiotic treatment only slightly augmented the MoMF infiltration in the MCD-fed mice (Figure 3C),
neutrophil granulocytes showed a significant almost two-fold increase in the MCD + ABx group,
compared to the MCDAbx group, which was reflected, both, in the absolute, as well as relative cell
numbers (Figure 3B). Together, these data demonstrated that antibiotic treatment in the MCD-
induced steatohepatitis triggered neutrophil infiltration.
The inflammatory response was orchestrated by a significantly increased mRNA expression of
pro-inflammatory genes, such as monocyte chemotactic protein 1 (Mcp1), tumor necrosis factor alpha
(Tnf), as well as interleukin 1 beta (Ilβ) in the Abx-treated mice, compared to the control mice (Figure
3D). Interestingly, this phenotype was also associated with a pronounced expression of PRRs,
including toll like receptor 2 (Tlr2), toll like receptor 4 (Tlr4), toll like receptor 9 (Tlr9), NLR family,
Figure 2.
Antibiotic treatment increased the hepatic fat accumulation in the MCD-fed mice.
(
A
) Representative Oil Red O stainings demonstrated increased the hepatic lipid accumulation upon an
antibiotic treatment. (
B
) Steatosis score was higher in the ABx-treated mice, compared to the MCD-fed
control mice. (
C
) Colorimetric hepatic triglyceride assay confirmed significantly increased hepatic
triglycerides (TG) levels in +ABx group. Data are expressed as the mean
±
SD from 2–5 mice per group
and were considered significant if ** p< 0.01.
2.3. Microbiota Depletion Augments the Inflammatory Response During the MCD-Induced Steatohepatitis
Next, we investigated the relevance of intestinal microbiota for hepatic inflammation, during the
MCD-induced steatohepatitis. The Abx-treated mice demonstrated significantly higher inflammation
in the histopathological analyses of the HE sections (Figure 1B). To further analyze which cell type
mediated the pronounced inflammatory response, we first performed immunofluorescence staining
against the CD11b. Here, CD11b+ immune cells were increased in the livers of the Abx-treated mice,
compared to the MCD-fed controls (Figure 3A). To further dissect which cell type accounted for the
inflammatory response, we performed a flow cytometry (FACS) analysis of the liver homogenates.
MCD feeding induced a strong infiltration of the MoMFs (defined as Ly6G-, CD11b
hi
, F4/80
low
).
In contrast, both, the absolute and relative numbers of neutrophil granulocytes (defined as CD11b+,
Ly6G+) were only slightly increased in the MCD versus the NCD-fed mice (Figure 3B,C). While
antibiotic treatment only slightly augmented the MoMF infiltration in the MCD-fed mice (Figure 3C),
neutrophil granulocytes showed a significant almost two-fold increase in the MCD + ABx group,
compared to the MCD
Abx group, which was reflected, both, in the absolute, as well as relative cell
numbers (Figure 3B). Together, these data demonstrated that antibiotic treatment in the MCD-induced
steatohepatitis triggered neutrophil infiltration.
The inflammatory response was orchestrated by a significantly increased mRNA expression
of pro-inflammatory genes, such as monocyte chemotactic protein 1 (Mcp1), tumor necrosis factor
alpha (Tnf ), as well as interleukin 1 beta (Il
β
) in the Abx-treated mice, compared to the control mice
(Figure 3D). Interestingly, this phenotype was also associated with a pronounced expression of PRRs,
including toll like receptor 2 (Tlr2), toll like receptor 4 (Tlr4), toll like receptor 9 (Tlr9), NLR family, pyrin
Int. J. Mol. Sci. 2019,20, 308 5 of 14
domain containing 3 (Nlrp3) and Caspase-1, which have all been implicated in the NASH pathogenesis
(Figure 3E).
Together, these data demonstrated that microbiota depletion in the MCD-fed mice
unleashes a strong hepatic inflammatory innate immune response, which is mediated by the
neutrophil granulocytes.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 14
pyrin domain containing 3 (Nlrp3) and Caspase-1, which have all been implicated in the NASH
pathogenesis (Figure 3E).
Together, these data demonstrated that microbiota depletion in the MCD-fed mice unleashes a
strong hepatic inflammatory innate immune response, which is mediated by the neutrophil
granulocytes.
Figure 3. Microbiota depletion augments the inflammatory response during the MCD-induced
steatohepatitis. (A) Representative immunofluorescence staining against CD11b, showing an
increased infiltration of the CD11b+ cells in the Abx group. (B) Flow cytometry (FACS) shows
increased infiltration of the neutrophils (CD11b+ Ly6G+ living leukocytes) after antibiotic treatment.
(C) Monocyte-derived macrophages (MoMFs) (defined as CD11b
hi
F4/80+ living leukocytes)
abundance is lower in the ABx group, compared to the MCD-fed control mice. (D) Pro-inflammatory
mRNA expression of the Mcp, Tnf, and Il1beta. GAPDH was used as a housekeeping gene. (E) ABx
treatment prompted pronounced mRNA expression of pathogen recognition receptors (PRRs),
including Tlr2, Tlr4, Tlr9, Nlrp3, and Caspase-1. GAPDH was used as a housekeeping gene. Data are
expressed as the mean ± SD from 2–5 mice per group and were considered significant if * p < 0.05, **
p < 0.01, *** p < 0.001. **** p < 0.0001
2.4. Intestinal Microbiota Protects Against Excessive Liver Fibrosis
Hepatic inflammation might lead to the activation of hepatic stellate cells, which
transdifferentiate into myofibroblasts-facilitating collagen deposition and, thus, contribute to tissue
remodeling and disease progression towards liver fibrosis. Next, we sought to investigate whether
Figure 3.
Microbiota depletion augments the inflammatory response during the MCD-induced
steatohepatitis. (
A
) Representative immunofluorescence staining against CD11b, showing an
increased infiltration of the CD11b+ cells in the Abx group. (
B
) Flow cytometry (FACS) shows
increased infiltration of the neutrophils (CD11b+ Ly6G+ living leukocytes) after antibiotic treatment.
(
C
) Monocyte-derived macrophages (MoMFs) (defined as CD11b
hi
F4/80+ living leukocytes)
abundance is lower in the ABx group, compared to the MCD-fed control mice. (
D
) Pro-inflammatory
mRNA expression of the Mcp, Tnf, and Il1beta. GAPDH was used as a housekeeping gene.
(
E
) ABx treatment prompted pronounced mRNA expression of pathogen recognition receptors (PRRs),
including Tlr2, Tlr4, Tlr9, Nlrp3, and Caspase-1. GAPDH was used as a housekeeping gene. Data are
expressed as the mean
±
SD from 2–5 mice per group and were considered significant if * p< 0.05,
** p< 0.01, *** p< 0.001. **** p< 0.0001.
2.4. Intestinal Microbiota Protects against Excessive Liver Fibrosis
Hepatic inflammation might lead to the activation of hepatic stellate cells, which transdifferentiate
into myofibroblasts-facilitating collagen deposition and, thus, contribute to tissue remodeling and
disease progression towards liver fibrosis. Next, we sought to investigate whether the increased liver
Int. J. Mol. Sci. 2019,20, 308 6 of 14
inflammation upon antibiotic treatment also translated into aggravated liver fibrogenesis. Indeed,
the depletion of the intestinal microbiota was associated with a strong increase in liver fibrosis,
as evidenced by the Sirius red stainings (Figure 4A). Histopathological quantification of collagen fibers
revealed an about 2,5-fold increase in the Sirius Red positive area (Figure 4B). Serum liver transaminases
Alanin-aminotransferase (ALT) and aspartate-aminotransferase (AST) were both significantly increased
after 8 weeks of the MCD treatment. While AST and ALT did not increase upon antibiotic treatment,
alkaline-phosphatase (AP) levels were higher in the +Abx group, compared to the MCD-fed control
animals (Figure 4C). In sum, these data showed that antibiotic treatment prompted excessive liver
fibrosis in the experimental MCD-induced NASH.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 14
the increased liver inflammation upon antibiotic treatment also translated into aggravated liver
fibrogenesis. Indeed, the depletion of the intestinal microbiota was associated with a strong increase
in liver fibrosis, as evidenced by the Sirius red stainings (Figure 4A). Histopathological quantification
of collagen fibers revealed an about 2,5-fold increase in the Sirius Red positive area (Figure 4B). Serum
liver transaminases Alanin-aminotransferase (ALT) and aspartate-aminotransferase (AST) were both
significantly increased after 8 weeks of the MCD treatment. While AST and ALT did not increase
upon antibiotic treatment, alkaline-phosphatase (AP) levels were higher in the +Abx group,
compared to the MCD-fed control animals (Figure 4C). In sum, these data showed that antibiotic
treatment prompted excessive liver fibrosis in the experimental MCD-induced NASH.
Figure 4. Antibiotic treatment fuels excessive liver fibrosis in experimentally-induced MCD-NASH.
(A) Representative Sirius red staining of the liver sections showing the collagen fibers in red. (B)
Quantification of the Sirius Red positive area, using the ImageJ software (at least 5 areas in 100×
magnification per mouse). (C) Serum liver function tests. Data are expressed as the mean ± SD from
2–5 mice per group and were considered significant if * p < 0.05, ** p < 0.01,
2.5. MCD Diet Impacts the Intestinal Homeostasis and Microbiota Composition
Steatohepatitis has been linked to intestinal dysbiosis. After eight weeks of MCD feeding, the small
intestines, as well as the colons, were atrophic and significantly shorter than those of the NCD-fed
control mice (Figure 5A). This phenotype was also reflected in the HE histology, which demonstrated
a shortening of the intestinal villi in the duodenum of the MCD-fed mice (Figure 5B). To investigate the
microbiota composition, we collected cecal microbiota samples to isolate the metagenomic DNA and
performed a 16s ribosomal gene (rDNA) amplicon sequencing of the V1–V3 hypervariable region, using
the 454 platform. Eight weeks of MCD treatment resulted in marked alterations in the microbiota
composition (Figure 5C). Among the genera that were differentially regulated between the NCD- and
MCD-fed mice, we identified a decrease in the potentially probiotic Lactobacillus, as well as Akkermansia,
and an increase in the Ruminococus, which has been linked to liver fibrosis (Figure 5C) [24]. Along with
changes in the individual bacterial communities, MCD feeding resulted in a strong overall decrease of
the microbiota alpha diversity metrics, such as observed species, as well as Chao1 (Figure 5D). Although
MCD feeding induced changes in the microbiota composition and a loss of species richness, we did not
observe a major decrease in the Occludin tight junction expression in the ileum of the MCD-fed mice
(Figure 5E). While the mRNA expression of Tnf in the ileum was even significantly decreased upon
A B
C
0
200
400
600
U/L
ALT
NCD MCD
** *
0
200
400
600
U/L
AST
NCD MCD
*
*
0
50
100
150
U/L
AP
- ABx
NCD MCD
+ ABx
P =0.11
**
0
2
4
6
8
10
% area fraction
Sirius Red
*
*
*
NCD MCD
- ABX
+ ABX
Figure 4.
Antibiotic treatment fuels excessive liver fibrosis in experimentally-induced MCD-NASH.
(
A
) Representative Sirius red staining of the liver sections showing the collagen fibers in red.
(
B
) Quantification of the Sirius Red positive area, using the ImageJ software (at least 5 areas in
100
×
magnification per mouse). (
C
) Serum liver function tests. Data are expressed as the mean
±
SD
from 2–5 mice per group and were considered significant if * p< 0.05, ** p< 0.01.
2.5. MCD Diet Impacts the Intestinal Homeostasis and Microbiota Composition
Steatohepatitis has been linked to intestinal dysbiosis. After eight weeks of MCD feeding, the small
intestines, as well as the colons, were atrophic and significantly shorter than those of the NCD-fed
control mice (Figure 5A). This phenotype was also reflected in the HE histology, which demonstrated
a shortening of the intestinal villi in the duodenum of the MCD-fed mice (Figure 5B). To investigate
the microbiota composition, we collected cecal microbiota samples to isolate the metagenomic DNA
and performed a 16s ribosomal gene (rDNA) amplicon sequencing of the V1–V3 hypervariable region,
using the 454 platform. Eight weeks of MCD treatment resulted in marked alterations in the microbiota
composition (Figure 5C). Among the genera that were differentially regulated between the NCD- and
MCD-fed mice, we identified a decrease in the potentially probiotic Lactobacillus, as well as Akkermansia,
and an increase in the Ruminococus, which has been linked to liver fibrosis (Figure 5C) [
24
]. Along with
changes in the individual bacterial communities, MCD feeding resulted in a strong overall decrease
of the microbiota alpha diversity metrics, such as observed species, as well as Chao1 (Figure 5D).
Although MCD feeding induced changes in the microbiota composition and a loss of species richness,
we did not observe a major decrease in the Occludin tight junction expression in the ileum of the
MCD-fed mice (Figure 5E). While the mRNA expression of Tnf in the ileum was even significantly
Int. J. Mol. Sci. 2019,20, 308 7 of 14
decreased upon MCD feeding, other inflammatory genes, including the Il1b and Mcp1 were unaffected,
both in the ileum and the colon (Figure 5F).
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 14
MCD feeding, other inflammatory genes, including the Il1b and Mcp1 were unaffected, both in the
ileum and the colon (Figure 5F).
Figure 5. MCD diet impacts intestinal homeostasis and microbiota composition. (A) MCD diets leads
to shortening of small and large intestines. (B) Representative histology of the paraffin-fixed
duodenum sections. (C) Clustered heatmap analysis of the microbiota composition of the normal
chow or the MCD-fed mice. (D) “Observed species” and “Chao1” alpha diversity metrics were
reduced, upon MCD feeding. (E) Occludin protein levels in the ileum tissue lysates. (F) Tnf, Il1beta,
Figure 5.
MCD diet impacts intestinal homeostasis and microbiota composition. (
A
) MCD diets leads
to shortening of small and large intestines. (
B
) Representative histology of the paraffin-fixed duodenum
sections. (
C
) Clustered heatmap analysis of the microbiota composition of the normal chow or the
MCD-fed mice. (
D
) “Observed species” and “Chao1” alpha diversity metrics were reduced, upon
MCD feeding. (
E
) Occludin protein levels in the ileum tissue lysates. (
F
) Tnf, Il1beta, and Mcp1 mRNA
expression, determined by the qRT-PCR in the ileum and the colon samples. * p< 0.05, ** p< 0.01,
*** p< 0.001.
Int. J. Mol. Sci. 2019,20, 308 8 of 14
Together, these data demonstrate that the MCD diet impacts the intestinal microbiota composition
and prompts both macroscopic and microscopic changes in intestinal architecture. This phenotype was
not associated with a strong suppression of tight junctions or increased inflammatory gene expression.
3. Discussion
Non-alcoholic Steatohepatitis (NASH) is a disease characterized by hepatic steatosis and
inflammation, which can further progress to fibrosis and HCC [
1
,
25
]. NASH is strongly associated
with obesity and the metabolic syndrome, and due to the obesity epidemic in western societies,
the incidence of NASH is rising [
26
]. Over- and malnutrition is widely accepted as the main cause of
NASH—however, not all obese Patients develop NASH and at the same time there are lean patients
suffering from active NASH [
27
]. This observation indicates that there must be additional mechanisms,
e.g., genetic or other environmental factors, which drive the transition from simple steatosis to
NASH [2830].
Recent data demonstrated that gut-liver communication and gut microbiota represent important
modulators of the liver disease [
12
,
30
32
]. Intestinal microbiota composition of NASH patients is
significantly different from healthy individuals and an emerging body of preclinical data support a
causal role of the gut microbiota in NASH development [
24
,
33
]. Patients suffering from NASH may
develop an intestinal barrier impairment, facilitating increased translocation of PAMPs and MAMPs,
through the portal vein into the liver [
16
]. Maintenance of the intestinal homeostasis and barrier
integrity relies on a complex interaction of the host immune system and commensal microbiota, which
may be hampered by environmental factors and affected by host genetics [
34
]. There is a huge body of
experimental evidence showing that depletion of the intestinal microbiota by antibiotic treatment or
in Germ-free (GF) mice, protects from a high-fat diet or western-style-diet induced NASH [
18
,
35
37
].
These dietary regimens nicely reflect human NAFLD, caused by the western sedentary lifestyle—the
mice become overweight, and develop insulin resistance and fatty liver disease [
38
,
39
]. In contrast to
the high-fat diet (HFD) feeding, mice fed with an MCD diet, actually loose body weight, and do not
develop insulin resistance [
39
]. Mechanistically, choline deficiency impairs the VLDL synthesis and
hepatic lipid export. Body and liver weight loss in the MCD model is induced by an increased energy
expenditure, without increased food consumption [
40
]. In our study, antibiotic treatment resulted in
pronounced liver remodelling and collagen deposition in the MCD-fed mice, which was also reflected
in an increased liver-to-bodyweight ratio, in these mice. The role of the intestinal microbiota in
MCD-induced steatohepatitis, is still a matter of debate. We, and other researchers have shown that
microbiota depletion using broad-spectrum antibiotics, protects mice from HFD or western-style,
diet-induced NASH [
18
]. Contrasting these data obtained in the HFD or the western-style diet (WSD)
models, we showed here that microbiota depletion in the MCD-fed mice, augments steatohepatitis,
by unleashing a strong, innate-immune response, orchestrated by neutrophil infiltration. In our study,
MCD feeding prompted intestinal dysbiosis, encompassing a reduced microbiota alpha diversity, likely
reducing probiotic bacteria, and inducing intestinal macroscopic, as well as microscopic, structural
changes. Still, a complete depletion of microbiota did not reverse the intestinal shortening and even
exacerbated steatohepatitis.
While our data clearly showed that a complete depletion of microbiota is detrimental in the MCD
model, Hanao-Mejia et al. demonstrated that a dysbiotic microbiota of the inflammasome-deficient
mice conferred susceptibility to the MCD-induced NASH, which was communicable via co-housing.
In contrast, the probiotic microbiota modulation has beneficial effects both in the HFD model, as well
as in the MCD-induced NASH. The VSL#3 probiotic treatment attenuated liver fibrosis in the MCD-fed
mice, without affecting the steatohepatitis and hepatic steatosis, by upregulation of the anti-fibrotic
transforming growth factor β(TGF-β) pseudoreceptor, Bambi [41].
Collectively, these data suggest a pathogenic role of the gut microbiota not only in the HFD models,
but also in the MCD-induced steatohepatitis. While our data showed that a complete depletion of
Int. J. Mol. Sci. 2019,20, 308 9 of 14
microbiota, exacerbates liver disease, an unfavorable microbiota composition might also drive the
disease progression and probiotic microbiota modulation might have a therapeutic potential.
There is a good body of evidence demonstrating that antibiotic treatment with Glycopeptid,
Aminopenicillin, Aminoglycosid, and Nitroimidazole, results in an almost complete depletion of the
intestinal microbiota [
42
44
]. As previously shown, mice receiving antibiotics showed a massively
enlarged caecum, which has also been described in the GF mice. Yet, we cannot exclude that the
antibiotic treatment caused an overgrowth with certain antibiotic resistant bacteria or fungi, which
might account for the observed phenotype.
Based on our current data, we can only speculate why an antibiotic treatment has such opposing
effects in the MCD model, compared to the HFD or the WSD treatment. Similar to our findings, a
beneficial role of the commensal microbiota, in preventing murine CCL4-induced liver fibrosis has
been shown [
45
]. Increased liver fibrosis was observed in the germ free (GF) mice, compared to the
conventional mice. Various pathogen recognition receptors (PRRs) signal via the Myd88/Trif and
a genetic deficiency of this important signaling node prompted a similar phenotype to what was
observed in the GF animals. Toll like receptor 2 (TLR2) and Toll like receptor 5 (TLR5) signal upstream
of the Myd88. While it has been shown that genetic deletion of the TLR2 and the TLR5 is associated
with enhanced steatohepatitis, upon MCD feeding [
46
,
47
], in the choline-deficient, I-amino-acid
defined (CDAA) dietary model and HFD models, TLR2 and TLR9 deletion were protective [
48
50
].
Interestingly, the TLR4 deficiency conferred a partial protection against NASH, in both the HFD and
MCD models [23,51].
Gut microbiota is strongly shaped by diet and represents an important source of the TLR ligands.
Compositional changes of microbiota and TLR ligands might explain the differential impact of the
various TLR pathways, depending on the dietary model [52].
In future studies, it might be interesting to investigate the role of the gut microbiota at
different time points—during early inflammatory initiation versus a later progression towards fibrosis.
Additionally, data on the MCD-induced steatohepatitis development in germ free animals or using
different regimens of antibiotic treatment, would complement our study.
A better understanding of how microbiota-mediated signals shape the hepatic inflammatory
response during steatohepatitis development and progression, might guide future, targeted, microbiota
modulation therapies.
4. Materials and Methods
4.1. Mice Experiments
All animal experiments were approved by the appropriate German authorities (LANUV, North
Rhine-Westphalia, Az 84-02.04.2012.A260, approved 03/26/2013). All animals received humane
care, according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”,
prepared by the National Academy of Sciences, and published by the National Institutes of Health
(NIH publication 86-23 revised 1985). C57BL/6 wild-type (WT) mice (C57BL/6 background) were
housed in filter-top cages. Mice that were 6–8 week old, were included in the experiments.
For 8 weeks, the mice were fed with the methionine-choline deficient diet (MCD (960439), MP
Biomedicals, Heidelberg, Germany). A normal chow diet (NCD) (provided by the Animal Facility at
the University Hospital RWTH Aachen, Germany) was used as a control diet.
Tissue and blood collection, RNA isolation, triglyceride measurement in liver tissue, cDNA
synthesis, real-time quantitative polymerase chain reaction, and measurement of serum parameters
(aminotransferases, glutamate dehydrogenase and alkaline phosphatase) were performed as described
previously [53,54].
Int. J. Mol. Sci. 2019,20, 308 10 of 14
4.2. Administration of the Broad-Spectrum Antibiotics
Eradication of intestinal microbiota in rodents, was performed using a well-established cocktail
of four broad-spectrum antibiotics (0.5 g/L Neomycin, 1 g/L Metronidazol, 1 g/L Vancomycin, 1 g/L
Ampicillin). Antibiotic treatment was initiated 2 weeks prior to the start of the experimental diet,
in 6 weeks old male mice. Antibiotics were administered in the drinking water for the whole dietary
feeding period and were changed every second day.
4.3. Immunofluorescence Analysis
Fixation of slides was performed in 4% PFA at room temperature. 5
µ
m liver cryosections were
stained with rat anti-mouse CD11b (BD Biosciences, Heidelberg, Germany).
Fluorescence signal was obtained using a secondary antibody conjugated with Cy3 (Jackson
Immunoresearch, West Grove, PA, USA). Mounting solution containing DAPI (Vector Laboratories,
Burlingame, CA, USA) was used to counterstain the nuclei of hepatocytes.
4.4. Microbiota—16S rRNA V1-V3 Amplicon Library Preparation
In order to extract the metagenomic DNA, 200mg of the cecal content were mechanically
homogenized. After a collum-based purification, the PSP SPIN Stool DNA plus kit (Stratec Molecular
GmbH, Berlin, Germany) was used to isolate the microbial DNA. The frozen cecal content was added to
sterile vials filled with Lysis buffer (Stratec Molecular, Berlin, Germany), 0.5 g of 0.1 mm zirconia/silica
beads (BioSpec, Bartlesville, OK, USA), and four 3.0–3.5 mm glass beads (BioSpec). Alternately,
by keeping the samples for one minute, on ice, in between the cycles, the samples were homogenized
in a Magna Lyser device (Roche, Basel, Switzerland), for one minute, at a speed of 5500 rpm three times.
The samples were isolated, afterwards, using the PSP SPIN Stool DNA plus kit and, according to the
manufacturer’s instructions, eluted in a final volume of the 200
µ
L. A barcoded sense primer, consisting
of the 454 Titanium platform A linker sequence (5
0
-CCATCCCTGCGTGTCTCCGACTCAG-3
0
),
a key (barcode) which was unique for each sample, the 16S rRNA 534R primer sequence
5
0
-ATTACCGCGGCTGCTGG-3
0
, and a reverse primer consisting of a 9:1 mixture of two oligonucleotides,
5
0
-B-AGAGTTTGATCMTGGCTCAG-3
0
, and 5
0
-B-AGGGTTCGATTCTGGCTCAG-3
0
, where B
represents the B linker (5
0
-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3
0
), followed by the 16S
rRNA 8F and 8F-Bif primers, was used to generate the amplicon libraries for pyrosequencing of the
16s rDNA V1-3 regions.
For the PCR amplification, we used 1x FastStart High Fidelity Reaction Buffer, 1.8 mM MgCl2,
1nM dNTP solution, 5U FastStart High Fidelity Blend Polymerase (from the High Fidelity PCR
System (Roche, Indiapolis, IN, USA)), 0.2 mM reverse primer, 0.2 mM of the barcoded forward primer
(unique for each sample), and 1 µL of template DNA. The following thermal cycling conditions were
used—an initial denaturation (94
C, 3 min), followed by 25 cycles of denaturation (94
C, 30 s),
annealing (51
C, 45 s), extension (72
C, 5 min), and a final elongation step (72
C, 10 min). Using
the AMPure XP purification (Beckman Coulter, Brea, CA, USA), subsequently, the amplicons were
purified, as instructed by the manufacturer, before elution in 1x low TE (10 mM Tris-HCl, 0.1 mM
EDTA, pH 8.0). To determine the concentration we applied the Quant-iT PicoGreen dsDNA reagent
kit (Invitrogen, New York, NY, USA), using a Victor3 Multilabel Counter (Perkin Elmer, Waltham, MA,
USA). To ensure proportional representation of each sample, the amplicons were mixed in equimolar
concentrations. The 454 sequencing run was performed on a GS Junior pyrosequencing system, using
Titanium chemistry (Roche, Branford, CT, USA).
4.5. Microbiota—Data Analysis
To minimize the error rate, raw pyrosequencing reads were passed through quality filters, using
Mothur version 1.32. For the further analysis, we retained only sequences matching the following
criteria—perfect proximal primer fidelity, a minimum average score of 25, over a window size of
Int. J. Mol. Sci. 2019,20, 308 11 of 14
50 nucleotides, a read length between the 200 and 590 nucleotides, a maximum of one ambiguous base
call, and a maximum homopolymer length of 6. The data were further analyzed using Quantitative
Insights Into the Microbial Ecology (QIIME) version 1.8 [
55
]. After de-multiplexing, sequences were
clustered by the UCLUST [
56
] algorithm into operational taxonomic units (OTUs), based on a 97%
sequence similarity against the Greengenes reference set version May 2013 [
57
]. The default parameters
for the UCLUST were applied, with the exception of the following parameters—maxrejects = 100 and
stepwords = 16. The influence of the pyrosequencing errors was minimized by disabling the creation
of the de novo OTUs for sequences that did not cluster to the reference sequences.
Observed OTUs (observed richness) and Chao1 index (estimated richness) have been calculated
as the metrics of species richness and diversity, within the communities (alpha-diversity).
MicrobiomeAnalyst was used for the hierarchical clustering and heatmap visualization, Ward’s
Clustering was performed on Genus level using the Euclidean distance [58].
4.6. Histology
For the tissue sections, 4% paraformaldehyde (PFA) was used as a fixative; these section were
then embedded in paraffin. For the hematoxylin and eosin (HE) staining, we cut the liver tissue into
2
µ
m thick sections. The staining then got reviewed by a board certified pathologist, whose scoring
was performed following a modified algorithm established for NASH, referred as the NAS-score [
59
].
Hepatocellular lipid deposits were scored in relation to the liver cells, with droplets (score 0: <5%;
1: 5–33%; 2: 33–66%; 3: >66%), and histologically, the inflammatory tissue activity was evaluated in a
three-level score (no inflammatory focus: 0; 1: 1; 2–4: 2; >4: 3) while a two-level score (0; 1) categorized
the degree of hepatocellular ballooning. Additionally, the paraffin-embedded tissue sections were
stained with Sirius Red, in order to evaluate the fibrosis development, as described previously [18].
4.7. Flow Cytometry Analysis of the Intrahepatic and Intestinal Leukocytes
Leukocytes were isolated from the fresh liver tissue, as previously described [
18
]. Liver leukocytes
were stained with 7-AAD, CD45, CD11b, CD11c, F4/80, Ly6G, and Ly6C. All samples were acquired
by flow cytometry (FACS Fortessa; BD Biosciences) and analyzed using the Flowjo software (Tree Star
Inc., Ashland, OR, USA).
4.8. Statistical Analysis
Data are expressed as the mean
±
standard error of the mean. Statistical significance was
determined via one-way analysis of variance, followed by a student’s t-test.
Author Contributions:
K.M.S. performed all experiments, analyzed the data and drafted the manuscript, A.M.
helped with experiments, K.K. contributed to tissue stainings, L.S.C. and C.E. helped with experiments, E.B. helped
with data analyses, L.B.S. helped with the performance of experiments, data analysis, and critically reviewed
the manuscript, F.H. supervised the flow cytometry experiments, N.G. analyzed liver histology and performed
the NAS scoring, J.P. detected microbiota composition, C.T. supervised the study, drafted the manuscript and
provided funds.
Funding:
This study was supported by the German Research Foundation TR 285/10-1 and SFB/TRR 57 to
C.T., the Federal Ministry of Education and Research (ObiHep grant #01KU1214A to C.T.), the Liver-LiSyM
Grant (BMBF) to C.T., The HDHL-INTIMIC Di-Mi-Liv to C.T. and K.M.S., the SFB 985 project C3 to C.T., the
Interdisciplinary Centre for Clinical Research (START Grant #691438) within the Faculty of Medicine at the RWTH
Aachen University.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... Methionine-choline-deficient (MCD) diet feeding results in steatosis, inflammation, fibrosis in the liver and shortening intestinal villi with disruption of small intestinal tight junctions (Kawauchi et al., 2019). Moreover, the intestinal microbiota can protect against MCD diet-caused liver injury (Schneider et al., 2019). Therefore, we aimed to investigate the possible protective effect of SB on the gut-liver axis in the MCD dietfed mice. ...
... It has been reported that MCD diet feeding could result in shortening of the small intestine and atrophy of the small intestinal villi (Schneider et al., 2019). The MCD diet-fed mice had consistently shorter small intestines than the NCD-vehicle group (p < 0.001). ...
... In this study, the MCD diet-fed mice showed shortening of the small intestines and the intestinal villi and narrowing of the intestinal crypts, which is consistent with a previous report by Schneider et al. (2019). The report by Schneider et al. showed only a shortening of the intestinal length and villi in the MCD diet-fed mice, whereas the gene expression of proinflammatory cytokines in both ileum and colon remained unchanged. ...
Article
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Non-alcoholic steatohepatitis (NASH) is affecting people worldwide. Changes in the intestinal microbiome are crucial to NASH. A previous study showed that eradicating intestinal fungi ameliorates NASH; however, the role of intestinal fungi in the development of NASH remains unclear. Saccharomyces boulardii (SB) , a dietary supplement yeast, has been reported to restore the integrity of the intestine. Here, we tested the effect of SB in the treatment of NASH. For this study, we fed eight-week-old C57/BL6 male mice either a methionine-choline deficient (MCD) diet or a normal chow diet (NCD) for eight weeks. Half of the MCD diet-fed mice were gavaged with SB (5 mg/day) once daily. The remainder of the NCD–fed mice were gavaged with normal saline as a control. The MCD diet-fed mice on SB supplement showed better liver function, less hepatic steatosis, and decreased inflammation. Both hepatic inflammatory gene expression and fibrogenic gene expression were suppressed in mice with SB gavage. Intestinal damage caused by the MCD diet was tampered with, intestine inflammation decreased, and gut permeability improved in mice that had been given the SB supplement. Deep sequencing of the fecal microbiome showed a potentially increased beneficial gut microbiota and increased microbiota diversity in the SB -supplemented mice. The SB supplement maintains gut integrity, increases microbial diversity, and increases the number of potentially beneficial gut microbiota. Thus, the SB supplement attenuates gut leakage and exerts a protective effect against NASH. Our results provide new insight into the prevention of NASH.
... There are many factors leading to fibrosis, including occupation, hereditary disease, lifestyle, aging [7,8], as well as physical, chemical, and biological factors, etc. [6,9,10]. For example, silicosis is a serious occupational disease in China. ...
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Tissue fibrosis is a key factor leading to disability and death worldwide; however, thus far, there are no approved treatments for fibrosis. Transforming growth factor (TGF)-β is a major pro-fibrotic cytokine, which is expected to become a target in the treatment of fibrosis; however, since TGF-β has a wide range of biological functions involving a variety of biological processes in the body, a slight change in TGF-β may have a systematic effect. Indiscriminate inhibition of TGF-β can lead to adverse reactions, which can affect the efficacy of treatment. Therefore, it has become very important to explore how both the TGF-β signaling pathway is inhibited and the safe and efficient TGF-β small molecule inhibitors or neutralizing antibodies are designed in the treatment of fibrotic diseases. In this review, we mainly discuss the key role of the TGF-β signaling pathway in fibrotic diseases, as well as the development of fibrotic drugs in recent years, and explore potential targets in the treatment of fibrotic diseases in order to guide subsequent drug development.
... NASH condition induced by MCD diet was markedly improved using the depletion of gut microbiota and consequent repopulation. Commensal microbiota appears to be hepatoprotective in that regard (147). Gastrointestinal dysbiosis associates with increased production of PAMPs and DAMPs, which then enter portal circulation and promote NLRP3 inflammasome activation and specifically inflammation in the liver most importantly by interacting with TLR4 (Figure 8) (148). ...
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The Transregional Collaborative Research Center “Organ Fibrosis: From Mechanisms of Injury to Modulation of Disease” (referred to as SFB/TRR57) was funded for 13 years (2009–2021) by the German Research Council (DFG). This consortium was hosted by the Medical Schools of the RWTH Aachen University and Bonn University in Germany. The SFB/TRR57 implemented combined basic and clinical research to achieve detailed knowledge in three selected key questions: (i) What are the relevant mechanisms and signal pathways required for initiating organ fibrosis? (ii) Which immunological mechanisms and molecules contribute to organ fibrosis? and (iii) How can organ fibrosis be modulated, e.g., by interventional strategies including imaging and pharmacological approaches? In this review we will summarize the liver-related key findings of this consortium gained within the last 12 years on these three aspects of liver fibrogenesis. We will highlight the role of cell death and cell cycle pathways as well as nutritional and iron-related mechanisms for liver fibrosis initiation. Moreover, we will define and characterize the major immune cell compartments relevant for liver fibrogenesis, and finally point to potential signaling pathways and pharmacological targets that turned out to be suitable to develop novel approaches for improved therapy and diagnosis of liver fibrosis. In summary, this review will provide a comprehensive overview about the knowledge on liver fibrogenesis and its potential therapy gained by the SFB/TRR57 consortium within the last decade. The kidney-related research results obtained by the same consortium are highlighted in an article published back-to-back in Frontiers in Medicine.
... These differences may be attributed to the different animal models used, i.e., differences in the knock-out mice and deleted genes. However, most studies have documented a state of gut dysbiosis in NAFLD [44][45][46][47][48][49][50][51][52][53][54]. Overall, human studies have found differential abundances among patients with NAFLD and especially among patients with severe NAFLD associated with fibrosis-and particularly among those staged ≥F2. ...
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Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. NAFLD begins as a relatively benign hepatic steatosis which can evolve to non-alcoholic steatohepatitis (NASH); the risk of cirrhosis and hepatocellular carcinoma (HCC) increases when fibrosis is present. NAFLD represents a complex process implicating numerous factors—genetic, metabolic, and dietary—intertwined in a multi-hit etiopathogenetic model. Recent data have highlighted the role of gut dysbiosis, which may render the bowel more permeable, leading to increased free fatty acid absorption, bacterial migration, and a parallel release of toxic bacterial products, lipopolysaccharide (LPS), and proinflammatory cytokines that initiate and sustain inflammation. Although gut dysbiosis is present in each disease stage, there is currently no single microbial signature to distinguish or predict which patients will evolve from NAFLD to NASH and HCC. Using 16S rRNA sequencing, the majority of patients with NAFLD/NASH exhibit increased numbers of Bacteroidetes and differences in the presence of Firmicutes, resulting in a decreased F/B ratio in most studies. They also present an increased proportion of species belonging to Clostridium, Anaerobacter, Streptococcus, Escherichia, and Lactobacillus, whereas Oscillibacter, Flavonifaractor, Odoribacter, and Alistipes spp. are less prominent. In comparison to healthy controls, patients with NASH show a higher abundance of Proteobacteria, Enterobacteriaceae, and Escherichia spp., while Faecalibacterium prausnitzii and Akkermansia muciniphila are diminished. Children with NAFLD/NASH have a decreased proportion of Oscillospira spp. accompanied by an elevated proportion of Dorea, Blautia, Prevotella copri, and Ruminococcus spp. Gut microbiota composition may vary between population groups and different stages of NAFLD, making any conclusive or causative claims about gut microbiota profiles in NAFLD patients challenging. Moreover, various metabolites may be involved in the pathogenesis of NAFLD, such as short-chain fatty acids, lipopolysaccharide, bile acids, choline and trimethylamine-N-oxide, and ammonia. In this review, we summarize the role of the gut microbiome and metabolites in NAFLD pathogenesis, and we discuss potential preventive and therapeutic interventions related to the gut microbiome, such as the administration of probiotics, prebiotics, synbiotics, antibiotics, and bacteriophages, as well as the contribution of bariatric surgery and fecal microbiota transplantation in the therapeutic armamentarium against NAFLD. Larger and longer-term prospective studies, including well-defined cohorts as well as a multi-omics approach, are required to better identify the associations between the gut microbiome, microbial metabolites, and NAFLD occurrence and progression.
... Analysis of the intestinal microbiota diversity was conducted according to the method described by Schneider et al. [28]. The analysis of microbial diversity in fecal samples was entrusted to GENEWIZ (Suzhou, China). ...
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Non-alcoholic fatty liver disease (NAFLD) has become a highly concerned health issue in modern society. Due to the attentions of probiotics in the prevention of NAFLD, it is necessary to further clarify their roles. In this study, the methionine and choline-deficient (MCD) diet induced NAFLD rats model were constructed and treated with strain L. plantarum MA2 by intragastric administration once a day at a dose of 1 × 108 cfu/g.bw. After 56 days of the therapeutic intervention, the lipid metabolism and the liver pathological damage of the NAFLD rats were significantly improved. The content of total cholesterol (TC) and total triglyceride (TG) in serum were significantly lower than that in the NAFLD group (p < 0.05). Meanwhile, the intestinal mucosal barrier and the structure of intestinal microbiota were also improved. The villi length and the expression of claudin-1 was significantly higher than that in the NAFLD group (p < 0.05). Then, by detecting the content of LPS in the serum and the LPS-TLR4 pathway in the liver, we can conclude that Lactobacillus plantarum MA2 could reduce the LPS by regulating the gut microecology, thereby inhibit the activation of LPS-TLR4 and it downstream inflammatory signaling pathways. Therefore, our studies on rats showed that L. plantarum MA2 has the potential application in the alleviation of NAFLD. Moreover, based on the application of the strain in food industry, this study is of great significance to the development of new therapeutic strategy for NAFLD.
... Several studies have shown the effect of probiotics via the repair of tight junctions and the integrity of the barrier in the gut [60][61][62]. Improvement of barrier integrity by probiotic bacteria in animal studies model NAFLD can reduce the level of liver steatosis by improving choline levels from the gut microbiota which correlates with decreased levels of fat accumulation in the liver and decreased steatosis [61][62][63]. Intestinal barrier repair can also improve the immune response in intestine to inhibit the growth and translocation of harmful bacteria to the liver and reduce the level of inflammation in the liver [36,[64][65][66]. ...
... 34 This includes metabolic disorders such as T2DM, dyslipidemia, and/or NAFLD. In animal models, whereas a healthy gut microbiota has been shown to be involved in protecting against NASH, 35 gut dysbiosis promotes the development of steatosis, inflammation, and fibrosis in the liver. 36,37 However, data on the possible role of gut microbiota in human NAFLD remain scarce. ...
Article
Full-text available
Nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the rising prevalence of obesity, leading to major health and socioeconomic consequences. To date, the most effective therapeutic approach for NAFLD is weight loss. Accordingly, bariatric surgery (BS), which produces marked reductions in body weight, is associated with significant histopathological improvements in advanced stages of NAFLD, such as nonalcoholic steatohepatitis (NASH) and liver fibrosis. BS is also associated with substantial taxonomical and functional alterations in gut microbiota, which are believed to play a significant role in metabolic improvement after BS. Interestingly, gut microbiota and related metabolites may be implicated in the pathogenesis of NAFLD through diverse mechanisms, including specific microbiome signatures, short chain fatty acid production or the modulation of one‐carbon metabolism. Moreover, emerging evidence highlights the potential association between gut microbiota changes after BS and NASH resolution. In this review, we summarize the current knowledge on the relationship between NAFLD severity and gut microbiota, as well as the role of the gut microbiome and related metabolites in NAFLD improvement after BS.
... For instance, Ye et al. found that MCD diet increased Firmicutes population but decreased Proteobacteria and Bacteroidetes populations [49]. Kai et al. found a decrease in the alpha diversity index (Chao1) in the NASH group [50]. However, our study was more comprehensive and systematic because we considered the host CYP450 enzymes and the gut microbiota. ...
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Full-text available
Introduction and Objectives Pharmacokinetic variability in disease state is common in clinical practice, but its underlying mechanism remains unclear. In this study, we aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH). Methods The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administrations of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol, after which the results were compared with those obtained from the control group. Thereafter, the pharmacokinetic variabilities of all drugs and their relations to the changes in gut microbiota and host Cyp450s were compared and analyzed. Results The exposures of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity in the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in the host Cyp2c65. Notably, gut microbiota and host Cyp450s exerted minimal effects on the pharmacokinetic variability of metoprolol. Conclusion Gut microbiota and host Cyp450s co-contribute to the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug.
... Choline is involved in biological processes in the liver, including lipid metabolism, signaling through lipid second messengers and enterohepatic circulation of bile and cholesterol [24]. Studies have shown that choline-deficient diets may result in obesity and hyperglycemia and are associated with NASH caused by prevention of the synthesis and secretion of very-lowdensity lipoprotein (VLDL), thus leading to the accumulation of hepatic triglycerides and liver steatosis [25][26][27][28]. ...
Article
Full-text available
Nonalcoholic fatty liver disease (NAFLD) is one of the most common and increasing liver diseases worldwide. NAFLD is a term that involves a variety of conditions such as fatty liver, steatohepatitis, or fibrosis. Gut microbiota and its products have been extensively studied because of a close relation between NAFLD and microbiota in pathogenesis. In the progression of NAFLD, various microbiota-related molecular and cellular mechanisms, including dysbiosis, leaky bowel, endotoxin, bile acids enterohepatic circulation, metabolites, or alcohol-producing microbiota, are involved. Currently, diagnosis and treatment techniques using these mechanisms are being developed. In this review, we will introduce the microbiota-related mechanisms in the progression of NAFLD and future directions will be discussed.
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Soy glycinin derived octapeptide (SGP8) is a peptide obtained from degradation of the soy glycinin, whose amino acid sequence is IAVPGEVA. To determine the effect of SGP8 on non-alcoholic fatty liver disease (NAFLD), steatosis HepG2 cells were induced by 1 mmol/L free fatty acid (FFA) and C57BL/6J mice were fed with methionine-choline deficient (MCD) diet for 3 weeks to establish NAFLD model. The results of oil red O staining and total cholesterol (TC)/triglyceride (TG) contents showed that SGP8 could significantly reduce the lipid content of steatosis HepG2 cells. In vivo, SGP8 lowered plasma alanine aminotransferase (ALT) and low density lipoprotein (LDL) content, normalized hepatic superoxide dismutase (SOD) and malondialdehyde (MDA) production, and reduced the severity of liver inflammation. The results of Western blotting showed that SGP8 increased expression of Sirtuin-1 (SIRT1) and phosphorylation level of AMP activated protein kinase (AMPK) in hepatocytes. Through activation of SIRT1/AMPK pathway, SGP8 downregulated the expression of sterol regulatory element binding protein 1c (SREBP-1c) and its target genes ACC and FAS expression levels, and increased the phosphorylation level of acetyl CoA carboxylase (ACC). Furthermore, SGP8 also upregulated the expression of transcription factor peroxisome proliferator activated receptor α (PPARα), which was regulated by SIRT1/AMPK pathway, and its target gene CPT1 level. In conclusion, SGP8 might improve NAFLD by activating the SIRT1/AMPK pathway. Our data suggest that SGP8 may act as a novel and potent therapeutic agent against NAFLD.
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Antibiotic-induced microbiome depletion (AIMD) has been used frequently to study the role of the gut microbiome in pathological conditions. However, unlike germ-free mice, the effects of AIMD on host metabolism remain incompletely understood. Here we show the effects of AIMD to elucidate its effects on gut homeostasis, luminal signaling, and metabolism. We demonstrate that AIMD, which decreases luminal Firmicutes and Bacteroidetes species, decreases baseline serum glucose levels, reduces glucose surge in a tolerance test, and improves insulin sensitivity without altering adiposity. These changes occur in the setting of decreased luminal short-chain fatty acids (SCFAs), especially butyrate, and the secondary bile acid pool, which affects whole-body bile acid metabolism. In mice, AIMD alters cecal gene expression and gut glucagon-like peptide 1 signaling. Extensive tissue remodeling and decreased availability of SCFAs shift colonocyte metabolism toward glucose utilization. We suggest that AIMD alters glucose homeostasis by potentially shifting colonocyte energy utilization from SCFAs to glucose.
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Backgrounds and Aims Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. Methods A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD)-plus-binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. Results HFD-plus-one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD-plus-multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses revealed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species (ROS) producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B-induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies revealed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interlukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. Conclusions The current data from a mouse model of HFD-plus-binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs.
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Intestinal microbiota and barrier functions seem to play an important role in the development of non-alcoholic fatty liver disease (NAFLD). However, whether these changes are an early event in the development of NAFLD or are primarily associated with later stages of the disease, has not yet been clarified. Using a pair-feeding model, we determined the effects of a short-term intake of a fat-, fructose- and cholesterol-rich diet (FFC) on the development of early hepatic steatosis and markers of intestinal barrier function in mice treated with and without non-resorbable antibiotics (AB). For four days, C57BL/6J mice were either pair-fed a control diet or a FFC diet ± AB (92 mg/kg body weight (BW) polymyxin B and 216 mg/kg BW neomycin). Hepatic steatosis and markers of inflammation, lipidperoxidation and intestinal barrier function were assessed. Lipid accumulation and early signs of inflammation found in the livers of FFC-fed mice were markedly attenuated in FFC + AB-fed animals. In FFC-fed mice the development of NAFLD was associated with a significant loss of tight junction proteins and an induction of matrix metalloproteinase-13 in the upper parts of the small intestine as well as significantly higher portal endotoxin levels and an induction of dependent signaling cascades in the liver. As expected, portal endotoxin levels and the expression of dependent signaling cascades in liver tissue were almost at the level of controls in FFC + AB-fed mice. However, FFC + AB-fed mice were also protected from the loss of zonula occludens-1 and partially of occludin protein in small intestine. Our data suggest that the development of early diet-induced hepatic steatosis in mice at least in part results from alterations of intestinal barrier function.
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The aim of this study was to evaluate cenicriviroc (CVC), a dual antagonist of CC chemokine receptor types 2 and 5, for treatment of nonalcoholic steatohepatitis (NASH) with liver fibrosis (LF). A randomized, double-blind, multinational phase 2b study enrolled subjects with NASH, a nonalcoholic fatty liver disease activity score (NAS) ≥4, and LF (stages 1-3, NASH Clinical Research Network) at 81 clinical sites. Subjects (N = 289) were randomly assigned CVC 150 mg or placebo. Primary outcome was ≥2-point improvement in NAS and no worsening of fibrosis at year 1. Key secondary outcomes were: resolution of steatohepatitis (SH) and no worsening of fibrosis; improvement in fibrosis by ≥1 stage and no worsening of SH. Biomarkers of inflammation and adverse events were assessed. Full study recruitment was achieved. The primary endpoint of NAS improvement in the intent-to-treat population and resolution of SH was achieved in a similar proportion of subjects on CVC (N = 145) and placebo (N = 144; 16% vs. 19%, P = 0.52 and 8% vs. 6%, P = 0.49, respectively). However, the fibrosis endpoint was met in significantly more subjects on CVC than placebo (20% vs. 10%; P = 0.02). Treatment benefits were greater in those with higher disease activity and fibrosis stage at baseline. Biomarkers of systemic inflammation were reduced with CVC. Safety and tolerability of CVC were comparable to placebo. Conclusion: After 1 year of CVC treatment, twice as many subjects achieved improvement in fibrosis and no worsening of SH compared with placebo. Given the urgent need to develop antifibrotic therapies in NASH, these findings warrant phase 3 evaluation. (Hepatology 2018;67:1754-1767).
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Non-alcoholic fatty liver disease (NAFLD) is commonly diagnosed in obese subjects; however, it is not rare among lean individuals. Given the absence of traditional risk factors, it tends to remain under-recognised. The metabolic profiles of lean NAFLD patients are frequently comparable to those of obese NAFLD patients. Though results from several studies have been mixed, it has been generally revealed that lean subjects with NAFLD have minor insulin resistance compared to that in obese NAFLD. Several genetic variants are associated with NAFLD without insulin resistance. Some data suggest that the prevalence of steatohepatitis and advanced fibrosis do not differ significantly between lean and obese NAFLD; however, the former tend to have less severe disease at presentation. The underlying pathophysiology of lean NAFLD may be quite different. Genetic predispositions, fructose- and cholesterol-rich diet, visceral adiposity and dyslipidaemia have potential roles in the pathogenic underpinnings. Lean NAFLD may pose a risk for metabolic disturbances, cardiovascular morbidity or overall mortality. Secondary causes of hepatic steatosis are also needed to be ruled out in lean subjects with NAFLD. The effectiveness of various treatment modalities, such as exercise and pharmacotherapy, on lean NAFLD is not known. Weight loss is expected to help lean NAFLD patients who have visceral obesity. Further investigation is needed for many aspects of lean NAFLD, including mechanistic pathogenesis, risk assessment, natural history and therapeutic approach.
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The widespread application of next-generation sequencing technologies has revolutionized microbiome research by enabling high-throughput profiling of the genetic contents of microbial communities. How to analyze the resulting large complex datasets remains a key challenge in current microbiome studies. Over the past decade, powerful computational pipelines and robust protocols have been established to enable efficient raw data processing and annotation. The focus has shifted toward downstream statistical analysis and functional interpretation. Here, we introduce MicrobiomeAnalyst, a user-friendly tool that integrates recent progress in statistics and visualization techniques, coupled with novel knowledge bases, to enable comprehensive analysis of common data outputs produced from microbiome studies. MicrobiomeAnalyst contains four modules - the Marker Data Profiling module offers various options for community profiling, comparative analysis and functional prediction based on 16S rRNA marker gene data; the Shotgun Data Profiling module supports exploratory data analysis, functional profiling and metabolic network visualization of shotgun metagenomics or metatranscriptomics data; the Taxon Set Enrichment Analysis module helps interpret taxonomic signatures via enrichment analysis against >300 taxon sets manually curated from literature and public databases; finally, the Projection with Public Data module allows users to visually explore their data with a public reference data for pattern discovery and biological insights. MicrobiomeAnalyst is freely available at http://www.microbiomeanalyst.ca.
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Conclusion: Pharmacological inhibition of CCR2+monocyte recruitment efficiently ameliorates insulin resistance, hepatic inflammation, and fibrosis, corroborating the therapeutic potential of CVC in patients with NASH. (Hepatology 2017).
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Macrophages represent a key cellular component of the liver, and are essential for maintaining tissue homeostasis and ensuring rapid responses to hepatic injury. Our understanding of liver macrophages has been revolutionized by the delineation of heterogeneous subsets of these cells. Kupffer cells are a self-sustaining, liver-resident population of macrophages and can be distinguished from the monocyte-derived macrophages that rapidly accumulate in the injured liver. Specific environmental signals further determine the polarization and function of hepatic macrophages. These cells promote the restoration of tissue integrity following liver injury or infection, but they can also contribute to the progression of liver diseases, including hepatitis, fibrosis and cancer. In this Review, we highlight novel findings regarding the origin, classification and function of hepatic macrophages, and we discuss their divergent roles in the healthy and diseased liver.
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Throughout the past century, we have seen the emergence of a large number of multifactorial diseases, including inflammatory, autoimmune, metabolic, neoplastic and neurodegenerative diseases, many of which have been recently associated with intestinal dysbiosis — that is, compositional and functional alterations of the gut microbiome. In linking the pathogenesis of common diseases to dysbiosis, the microbiome field is challenged to decipher the mechanisms involved in the de novo generation and the persistence of dysbiotic microbiome configurations, and to differentiate causal host–microbiome associations from secondary microbial changes that accompany disease course. In this Review, we categorize dysbiosis in conceptual terms and provide an overview of immunological associations; the causes and consequences of bacterial dysbiosis, and their involvement in the molecular aetiology of common diseases; and implications for the rational design of new therapeutic approaches. A molecular- level understanding of the origins of dysbiosis, its endogenous and environmental regulatory processes, and its downstream effects may enable us to develop microbiome-targeting therapies for a multitude of common immune-mediated diseases.