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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
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) [15–17].
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 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 (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 [28–30].
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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