hep27698-sheida

Original manuscript
Attenuated portal hypertension in germ-free mice:
function of bacterial flora on the development of
mesenteric lymphatic and blood vessels.
Sheida Moghadamrad1
, Kathy D. McCoy2
, Markus B. Geuking2
, Hans Sägesser1
,
Jorum Kirundi2
, Andrew J. Macpherson2,3
, Andrea De Gottardi1,3
Author’s affiliations:
1
Hepatology, Department of Clinical Research, University of Berne, Switzerland.
2
Gastroenterology, Department of Clinical Research, University of Berne, Switzerland.
3
Clinic of visceral surgery and medicine, Inselspital, Berne, Switzerland
Key words:
Portal hypertension, intestinal microbiota, angiogenesis, partial portal vein ligation, Paneth
cells.
Correspondence:
Dr. Andrea De Gottardi
Hepatology, Department of Clinical Research and Clinic of Visceral Surgery and Medicine
University of Berne
Murtenstrasse 35
3010 Berne, Switzerland
Phone: +41 31 632 35 70
FAX: +41 31 632 49 97
E-mail: andrea.degottardi@insel.ch
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/hep.27698
This article is protected by copyright. All rights reserved.

2
Abbreviations:
Ang-4 (Angiogenin-4), ASF (altered Schaedler flora), SPF (specific pathogen free), BT
(bacterial translocation), CFU (colony forming unit), GF (Germ-free), HCS (hyperdynamic
circulatory syndrome), MLN (mesenteric lymph nodes), Pla2g2a (phospholipase A2, group
IIA), PHT (portal hypertension), PP (portal pressure), PSS (portosystemic shunts), PPVL
(partial portal vein ligation), PAR2 (tissue factor protease receptor 2), VEGF (vascular
endothelial growth factor), FITC-Dextran (fluorescein isothiocyanate-dextran).
Financial support:
This work was supported by a grant from the Swiss National Science Foundation to ADG
(number 31003A_129842).
Author’s contributions:
SM: acquisition of data; analysis and interpretation of data; statistical analysis; drafting of the
manuscript
KMC: critical revision of the manuscript for important intellectual content; study supervision
MBG: Microbial composition analysis by high-throughput 16S amplicon sequencing
HS: technical support; critical revision of the manuscript for important intellectual content
JK: technical support; critical revision of the manuscript for important intellectual content
AMP: study concept and design; critical revision of the manuscript for important intellectual
content; study supervision
ADG: study concept and design; acquisition of data; analysis and interpretation of data;
drafting of the manuscript; statistical analysis; obtained funding; study supervision
Conflict of interests: The authors declare that there are no conflicts of interests.
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ABSTRACT
Intestinal bacterial flora may induce splanchnic hemodynamic and histologic
alterations that are associated with portal hypertension (PHT). We hypothesized that
experimental PHT would be attenuated in the complete absence of intestinal bacteria.
We induced prehepatic PHT by partial portal vein ligation (PPVL) in germ-free (GF) or
mice colonized with the altered Schaedler’s flora (ASF). After two or seven days we
performed hemodynamic measurements including portal pressure (PP) and porto-
systemic shunts (PSS) and we collected tissues for histomorphology, microbiology
and gene expression studies. Mice colonized with intestinal microbiota presented
significantly higher PP levels after PPVL compared to GF mice. The presence of
bacterial flora was also associated with significantly increased PSS and spleen
weight. However, there were no hemodynamic differences between sham-operated
mice in the presence or absence of intestinal flora. Bacterial translocation to the
spleen was demonstrated 2 days, but not 7 days, after PPVL. Intestinal lymphatic and
blood vessels were more abundant in colonized and in portal hypertensive mice as
compared to GF and sham-operated mice. The expression of the intestinal
antimicrobial peptide angiogenin-4 was suppressed in GF mice, but increased
significantly after PPVL, while other angiogenic factors remained unchanged.
Moreover, colonization of GF mice with the ASF 2 days after PPVL led to a significant
increase in intestinal blood vessels compared to controls. The relative increase in PP
after PPVL in ASF and SPF mice was not significantly different. In conclusion, we
demonstrate that in the complete absence of gut microbial flora PP is normal, but
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experimental PHT is significantly attenuated. Intestinal mucosal lymphatic and blood
vessels induced by bacterial colonization may contribute to the development of PHT.
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INTRODUCTION
The mammalian intestine is home to a highly abundant diversity of
microorganisms that provide nutritional, metabolic and immunologic benefits for
their host (1, 2). The relationship between intestinal bacterial flora and the host is
not merely commensal, but rather mutualistic or interdependent and appears
important for health. However, despite this symbiotic relationship, intestinal flora
can also contribute to the development of a number of diseases. These can
occur when intestinal permeability is impaired and bacteria or bacterial-derived
products translocate from the luminal space into other body compartments and
contribute to the pathogenesis of inflammatory or metabolic diseases (3, 4).
Bacterial overgrowth, increased permeability of the intestinal mucosal barrier due
to PHT, and host immune function impairment are common features of advanced
chronic liver disease. In this context bacterial translocation (BT) refers to the
passage of intestinal bacteria or bacterial-derived products from the gut lumen to
the mesenteric lymph nodes or to other extra intestinal organs and
compartments, leading to persistent infection and endotoxemia (5, 6).
One of the mechanisms that have been postulated to explain why PHT can be
maintained by bacterial translocation is splanchnic vasodilation. In agreement
with the forward flow theory, high portal pressure can be maintained by an
increased liver blood inflow resulting from splanchnic arterial vasodilatation (7).
This phenomenon leads to a hyperdynamic circulatory syndrome, which is
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characterized by PHT, peripheral and splanchnic vasodilation and increased
cardiac output.
The translocation of gut-derived bacterial products to the mesenteric lymph
nodes and systemic circulation triggers the activation of immune effectors in the
mucosal and mesenteric lymphoid tissues. The subsequent inflammatory
response includes an enhanced release of cytokines and vasodilatory mediators
such as tumor necrosis factor alpha, interleukin-6, nitric oxide and others (5, 8,
9). The eventual effect of this process on the splanchnic vasculature is arterial
vasodilation, which in turn leads to an increased portal blood inflow. Together
with hepatic vascular resistance, this parameter determines the degree of PHT
and its complications (10).
In parallel to increased hepatic resistance and portal inflow, mesenteric
angiogenesis has also been reported as an additional pathophysiological feature
associated with PHT. Several lines of evidence suggest not only that the density
of mesenteric blood vessels is proportional to the degree of portal pressure, but
also that inhibition of angiogenesis can ameliorate PHT (11). Whether and how
intestinal bacterial flora can contribute to the regulation of mesenteric perfusion
and blood vessel development in the context of PHT remains not fully elucidated.
Paneth cells are a particular epithelial cell lineage located in the base of intestinal
crypts that contribute to intestinal innate immunity by production of bactericidal
peptides. Ang-4 is produced by Paneth cells and can present both pro-
angiogenic features and microbicidal activity against intestinal flora (12, 13).
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Whether Ang-4 is modulated by the presence of PHT and intestinal flora remains
however unknown.
Current experimental evidence suggests that PHT can be modulated by altering
the composition of intestinal flora with antibiotics (14, 15). However, recent
findings also indicate that cirrhosis and PHT per se induce significant changes in
the composition of the gut microbiome (16). Based on the hypothesis that
experimental PHT would be attenuated in the complete absence of intestinal
microbial flora, we decided to investigate the development of PHT in germ-free
mice and we set out to explore the possible underlying mechanisms. We studied
altered Schaedler flora (ASF) and specific pathogen free flora (SPF) mice. The
ASF microbiota (17) contains eight species including Lactobacillus acidophilus,
Lactobacillus murinus, Bacteroides distasonis, Mucispirillum schaedleri,
Eubacterium plexicaudatum, a Fusiform-shaped bacterium and two Clostridium
species and has the advantage to limit the possible experimental variability that
can be expected with SPF.
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MATERIALS AND METHODS
Animals
GF male C57BL/6 mice, ASF (altered Schaedler flora) mice and specific
pathogen free (SPF) mice aged 10-12 weeks were used. All animals were kept
on a 12 hours dark-light cycle. Mice were born and maintained in flexible film
isolators under HEPA air and fed with autoclaved chow and water ad libitum. All
experimental protocols obtained the approval of the Research Animal Ethics
Committee of Canton Bern (authorization 53/10) and were performed according
to international guidelines concerning the conduct of animal experimentation.
Induction of portal hypertension by partial portal vein ligation (PPVL)
Midline laparotomy was performed under isoflurane anesthesia and
administration of buprenorphine 60 g/kgBW (Reckitt Benckiser, 0.3mg/ml), the
portal vein was isolated from the surrounding tissues and a 0.5 mm (26-gauge)
blunt-tipped needle was placed alongside the portal vein. Then a single 7-0 silk
ligature was tied around both needle and portal vein. Afterwards the blunt-tipped
needle was removed, leaving a calibrated stenosis on the portal vein. In sham-
operated animals the portal vein was isolated, but not ligated.
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Hemodynamic measurements
Hemodynamic measurements were performed 48 hours or 7 days after PPVL in
groups of 10 animals each. Under general anesthesia with isoflurane, a
laparotomy was performed and the portal vein was cannulated through an
ileocolic vein using a 26GA BD VasculonTM Plus cannula. The catheter was
connected to a highly sensitive pressure transducer and the signal was recorded
using a multichannel Power-lab instrumentation and Chart-7 Software (AD
Instruments).
Determination of the extent of portal systemic collateral formation
PSS were assessed by injecting 3x104 51
Cr-labeled micro-spheres into the ileo-
colic vein and then measured using a Canberra Packard Cobra II Auto-Gamma
Counter. Animals were then sacrificed and the ratio of radioactivity in the liver
and lungs was quantified using the equation PSS(%) = [pulmonary
radioactivity/(pulmonary radioactivity + liver radioactivity)] x 100 (18).
Assessment of bacterial translocation and mucosal immune compartment
integrity
MLN and spleen were dissected aseptically and weighed. The cecum was then
opened and an aliquot of cecal content was collected and weighed. Organs were
homogenised in 0.5% Tergitol/PBS using a Tissuelyser (Qiagen) and sterile
stainless-steel ball bearings. Cecal contents and organ (MLN and spleen)
suspensions were then plated on Luria Bertani agar (LB, Sigma) and blood agar
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(anaerobe agar and sheep blood defibrinated, Wilkins-Chalgren, Oxoid) plates
for aerobic and anaerobic culture respectively and incubated 48 hours at 37˚C for
CFU quantification. Bacterial colonies were counted as normalized for milligrams
of tissue. In order to evaluate intestinal permeability, we quantified fecal albumin
loss using a mouse albumin ELISA kit (Bethyl laboratories) and we measured
serum concentration of fluorescein isothiocyanate-dextran (FITC-Dextran) as
described in supplementary materials and methods.
Matrix Assisted Laser Desorption/Ionization (MALDI-TOF)
MLN and spleen were homogenized and then plated on LB and blood agar
plates. Single bacterial colonies were isolated for MALDI-TOF analyses as
previously described (19).
Intestinal histology
Segments of distal small intestine were either snap frozen or fixed in 4% buffered
formalin, embedded in paraffin blocks and tissue slides were prepared for
standard histology (H&E staining) or immunohistochemical analyses. The 5- m
paraffin sections were deparaffinized in xylol and rehydrated in graded alcohol
series. Antigen retrieval was performed by boiling the sections in citrate buffer
(10mM Citric acid, PH 6.0) for 15 minutes. After endogenous peroxidase
blockage with 0.6% H2O2 in methanol, slides were incubated with normal goat
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serum blocking solution (2.5%) for CD31 and in normal bovine serum albumin
(10%) for Lyve-1 for one hour at room temperature. Immunohistochemistry was
performed using rabbit anti-CD31, a marker of endothelial cells (Acris antibodies,
CD31 /PECAM1) or biotinylated goat-anti mouse Lyve-1, a marker of lymphatic
vessels (R&D Systems). Slides were incubated overnight with anti CD31 (1:100)
or Lyve-1 (1:30) at 4 °C. The CD31 slides were then incubated with biotinylated
goat anti-rabbit IgG (1:200) and detection was performed with horseradish
peroxidase streptavidin (Vector laboratories) and 3’-3-diaminobenzidene (DAB;
Vector lab.) following hematoxylin counterstaining.
For Lyve-1, after overnight incubation, the slides were then treated with the
Avidin/biotinylated enzyme complex reagents (VECTASTAIN Elite ABC kit,
Vector lab.).The Lyve-1 detection was performed using the AEC peroxidase (3-
amino-9 ethylcarbazole) substrate kit (Vector lab) and the sections were then
counterstained with hematoxylin.
Quantitative Real-Time PCR
Total RNA was extracted from 30 to 50 mg of intestine using the RNeasy Plus
Mini Kit (Qiagen). Reverse transcription was performed with M-MLV Reverse
transcriptase (Invitrogen) and a random hexamer mix. Probes and primers for the
detection of mouse PAR2, Angiogenin-4 and Pla2g2a were purchased from
Applied Biosystems. Quantitative PCR was performed using an ABI TaqMan
7500 Sequence Detection System and TaqMan universal PCR Master Mix
(Applied Biosystems) according to standard protocols. Each reaction was carried
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out in triplicate. All transcripts were normalized using glyceraldehyde 3-
phosphate dehydrogenase as a housekeeping gene.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software. Data are
expressed as mean ± SD. p values were considered statistically significant at
<.05. Comparisons between 2 groups were performed using the Mann-Whitney U
test. Multiple comparisons were performed by ANOVA followed by Kruskal-Wallis
test.
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RESULTS
Portal pressure after partial portal vein ligation (PPVL)
To verify whether the absence of intestinal flora had splanchnic hemodynamic
effects we performed partial ligation of the portal vein. This surgical intervention
induced a significant increase of portal pressure in ASF-PPVL (p<0.0001) as well
as in GF-PPVL mice (p=0.0081), as measured 2 and 7 days after surgery.
However the increase in portal pressure in GF-PPVL mice was significantly lower
than ASF-PPVL mice. In sham-operated mice there was no significant difference
in portal pressure between ASF-sham and GF-sham mice (ASF-Sham 6.6±1.2
mmHg vs GF-sham 6.3±1.4 mmHg, p=0.632). However, following PPVL, portal
hypertension was significantly higher in ASF-PPVL mice compared to GF-PPVL
mice both 2 and 7 days after surgery (ASF-PPVL2d 11.1±1.7mmHg vs GF-
PPVL2d 7.4±1.3mmHg, p=0.0003) and ASF-PPVL7d 10.8±2.7mmHg vs GF-
PPVL7d 8.2±1.3mmHg, p=0.0384) (Fig. 1A). The relative increase in portal
pressure was not significantly different when PPVL was performed in SPF
compared to ASF mice (Supplementary Fig.1).
Moreover, we gavaged mice with ASF 2 days after PPVL and observed only a
slight, but not statistically significant increase in PHT after 7 days. Control group
7.1±1.9mmHg vs gavaged animals 8.8±2mmHg (Supplementary Fig.2).
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Development of porto-systemic collaterals
Due to the fact that PHT is associated with an enhanced angiogenesis, we
evaluated the extent of porto-systemic shunts in GF and ASF mice after PPVL.
Our data showed a significant increase in PSS in ASF-PPVL (p=0.0010), but not
in GF-PPVL mice (p=0.152). ASF-sham 0.3±0.1% vs GF-sham 0.3±0.1%,
p=0.315) and 2 days after PPVL the collateralization began slightly in ASF-PPVL
mice but not GF-PPVL mice (ASF-PPVL2d 2.6±3.7% vs GF-PPVL2d 0.4±0.2%,
p=0.278) and increased significantly in ASF-PPVL mice 7 days after PPVL (ASF-
PPVL7d 14.7±10% vs GF-PPVL7d 0.9±1.4%, p=0.0013) (Fig. 1B).
Spleen size
Spleen size can be increased as a consequence of PHT. Consequently, we
measured the ratio of spleen to the body weight in order to evaluate this
surrogate marker of PHT. The results showed a significant increase in the spleen
size of ASF-PPVL mice (p=0.0012), but not in GF-PPVL mice (p=0.397). In
sham-operated mice there was no significant difference in spleen size, ASF-
sham 0.28±0.1% vs GF-sham 0.30±0.04%, p=0.367 but after 2 days PPVL, ASF-
PPVL2d 0.33±0.11% vs GF-PPVL2d 0.27±0.05%, p=0.0007 and after 7 days
PPVL, ASF-PPVL7d 0.41±0.08% vs GF-PPVL7d 0.26±0.12%, p=0.0006
significant differences were observed (Fig. 1C). These results were consistent
with the direct measurements of portal pressure and porto-systemic shunts.
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Bacterial translocation after partial portal vein ligation
To detect bacterial translocation after PPVL we harvested and weighed
mesenteric lymph nodes and spleen tissues under sterile conditions. Then we
plated the homogenized organs on LB agar (Luria Bertani agar) for aerobic
bacteria grown or blood-agar plates for anaerobic bacterial grown at 37ْ C for 48
hours. Bacterial colonies were counted and normalized for milligrams of tissue.
We observed a significant bacterial translocation in the spleen of ASF-PPVL
mice (p=0.0088) only as a consequence of acute PHT (2 days after PPVL) on
LB-agar plates (Fig. 2A). A trend towards increased bacterial translocation after
PPVL was observed in mesenteric lymph nodes of ASF-PPVL mice (Fig. 2B). To
evaluate whether PHT was affecting the total number of bacteria in the intestine,
we plated stool samples from the cecum and observed no significant difference
in the total number of bacteria after PPVL (Fig. 2C). We also assessed the
composition of microflora by 16s rRNA gene sequencing and observed no
significant differences due to PHT (Supplementary Fig.3).
The assessment of intestinal permeability by measuring fecal albumin and serum
FITC-Dextran did not show any significant differences after PPVL
(Supplementary Fig.4 A,B).
Next, bacterial species isolated from the spleen were analysed using MALDI-
TOF. This procedure allowed the identification of Lactobacillus species L.
murinus as the only one of the 8 bacterial components of the altered Schaedler
flora that translocated to the spleen in this model of PHT.
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Immunohistochemistry
Since it has been reported that both the presence of intestinal bacteria and
PHT can induce mesenteric and intestinal vascular proliferation (20-22) we
evaluated by immunohistochemistry semi-quantitatively the intestinal positivity
for anti-CD31 (Fig. 3A). Single blood vessels were counted per 100 crypts.
Intestinal vascular density resulted significantly increased after PPVL in ASF-
PPVL (p=0.0185) compared to GF-PPVL (p=0.340) mice. Under baseline
conditions there was a significant difference in the intestinal vascular density
between ASF and GF sham mice. ASF-sham 64±18 blood vessels/100 crypts
vs GF-sham 45±11, p<0.0001 and ASF-PPVL2d 80±22 vs GF-PPVL2d 52±17,
p<0.0001 and after 7days PPVL, ASF-PPVL7d 77±25 vs GF-PPVL7d 54±19,
p=0.0041 (Fig. 3B).
In germ-mice gavaged with ASF 2 days after PPVL we observed that the
abundance of intestinal blood vessels was significantly higher in gavaged
animals (63±10 blood vessels/100 crypts vs 45±9, p=0.0028) suggesting that
intestinal flora positively stimulated vascular proliferation (Supplementary Fig.
5).
Further we investigated by staining intestinal tissue using the specific marker
Lyve-1 whether also the number of lymphatic vessels was affected by the
presence of intestinal flora (Fig. 4A).
Lymphatic vessel count for 100 crypts revealed that, in contrast with GF-PPVL
mice (p=0.084), lymphatic vessels in ASF-PPVL mice (p=0.025) increased
significantly after PPVL. ASF-sham 75±17 lymphatic vessels/100 crypts vs GF-
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sham 63±9, p=0.0045 and ASF-PPVL2d 85±13 vs GF-PPVL2d 72±16, p=0.0008
then after 7 days PPVL, ASF-PPVL7d 87±20 vs GF-PPVL7d 72±19, p=0.0172
(Fig.4B). Similarly to blood vessels, there was a remarkable difference in the
number of lymphatic vessels between ASF and GF mice even in baseline
conditions.
To confirm the selectivity of the two markers CD31 and Lyve-1 for blood and
lymphatic vessels, respectively, we stained two adjacent intestinal sections,
which clearly showed that only vessels containing red blood cells resulted
positive for CD31 (Fig. 5).
Expression of angiogenic factors in the small intestine
To gain further insight into the processes regulating the relative abundance of
blood and lymphatic vessels in the small intestine of hypertensive mice in the
presence or absence of gut flora, we performed mRNA expression analyses of
factors regulating angiogenesis including VEGFs and VEGF receptors,
angiopoeitin, neuropilin-2, tissue factor and its receptors PAR1 and PAR2. In
addition, we measured the expression of the antimicrobial intestinal peptide
angiogenin-4 and the Paneth cell marker Pla2g2A.
A significant up-regulation of PAR2 mRNA was observed in ASF-PPVL mice
(p=0.024), but not in GF-PPVL mice (p=0.72) (Fig.6A), whereas PAR1 mRNA
expression remained unchanged. The levels of expression of the remaining
angiogenic factors were not significantly altered (Supplementary Fig. 6 A,B,C).
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We measured furthermore a significant up-regulation of Ang-4 (p=0.0035) and
Pla2g2a (p=0.0074) mRNA expression in GF-PPVL mice, but not in ASF-PPVL
mice (Fig. 6B,C). Under baseline conditions the expression of Ang-4 (p=0.0007)
and Pla2g2a (p=0.0002) was significantly lower in GF than in ASF mice. Finally,
we counted the Paneth cells and observed a significantly higher number of
positive intestinal crypts in colonized than in GF mice and this difference
persisted after PPVL (Fig.7).
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DISCUSSION
We observed in this study that the increase in portal pressure following partial
portal vein ligation was significantly attenuated in the absence of intestinal
bacterial flora. Secondary indicators of PHT such as the amount of porto-
systemic collaterals and the spleen size were in line with the results obtained by
direct measurement of portal pressure in the ileo-colic vein. In confirmatory
experiments including SPF mice and the gavage of germ-free mice with ASF
after PPVL, we did not observe any significant difference in PHT. To evaluate the
forward component of PHT we measured mesenteric artery blood flow and we
found a significant increase after PPVL, but no difference between control and
ASF-gavaged animals (Supplementary Fig.7). We hypothesize that this was due
to the short colonization time and that a longer observation time or repeated
gavaging may lead to an increased mesenteric blood flow and PHT. Since
bacterial translocation is considered an important mechanism in the
pathogenesis of PHT (23), because bacterial-derived products such as
lipopolysaccharide can activate immune (24) and hepatic stellate cells (16), we
checked abdominal organs for the presence of microbes and identified
Lactobacillus murinus in the spleen of ASF-PPVL mice. The presence of this
gram-positive bacterium that belongs to the altered Schaedler’s flora and does
not produce lipopolysaccharide was only detected in acute PHT (2 days after
PPVL) and only in the spleen. Therefore, although other gram-positive-derived
molecular patterns, such as peptidoglycan and lipoteichoic acid (25-28) may
have contributed to exacerbate PHT in the mouse model used here, evidence for
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significant bacterial translocation was lacking. This was also supported by the
observation that intestinal permeability was not significantly increased after PPVL
(Supplementary Fig. 4).
To investigate whether other mechanisms known to be important in the
regulation of portal pressure were affected by the presence of microbes in the
intestinal lumen, we studied vascular spread in the intestine. Several lines of
evidence indicate that differences in portal pressure can be related to blood
vessel proliferation (11, 21) and that bacterial microbiota can induce intestinal
angiogenesis (29).
Therefore we quantified the abundance of intestinal capillaries in germ-free and
colonized mice and found that the number of blood and lymphatic vessels was
significantly higher in the presence of bacterial flora.
Only under the stress of PPVL, portal pressure increased more in colonized
animals that presented a higher density of intestinal vessels. Based on these
results on blood capillaries, we extended our observations to intestinal lymphatic
vessels and found similar results, indicating that both blood and lymphatic
capillaries are more abundant in both the presence of intestinal microbiota and in
PHT. Thus, we reasoned that the attenuation of PHT could also be related to a
less developed lymphatic and blood intestinal capillary system as a consequence
of sterility of the intestine.
To support this hypothesis, we measured whether the expression of several
angiogenic factors in the intestine was altered by the presence of bacterial flora
and after PPVL. In these experiments we did not observe any significant
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alterations in the expression of angiogenic factors apart from protease activated
receptor-2 and angiogenin-4. PAR2 was significantly up-regulated in ASF-
colonized mice after PPVL. Tissue factor can activate PAR2 signaling and
consequently stimulate angiogenesis particularly under inflammatory conditions
(30, 31). In the present work PAR2 up-regulation after PPVL was consistent with
the increased intestinal vasculature in colonized mice.
Angiogenin-4 was significantly downregulated in the absence of intestinal
bacteria. This peptide exerts a double function as an antimicrobial ribonuclease
stored in Paneth cell granules (32) and as a factor stimulating angiogenesis (11,
33). Here we confirmed previous observations, which demonstrated that
intestinal angiogenesis was in part dependent on the presence of bacterial flora
(29). In our experiments the expression of Ang-4 increased after PPVL,
suggesting that PHT per se could induce Ang-4. In a subsequent step we
hypothesized that the upregulation of Ang-4 was related to a higher number of
Paneth cells or of their intracellular granules. To demonstrate this assumption we
quantified the expression of Pla2g2a and found a parallel increase to Ang-4,
suggesting that PHT could be a driver of Paneth cell granule development and
Ang-4 expression in germ-free mice. These findings are compatible with the
hypothesis that the relative deficiency in Paneth cell-derived Ang-4 in germ-free
mice contributed to the attenuated portal pressure through a decreased vascular
density. Therefore, Paneth cells may contribute to the regulation of vascular
development and PHT. The possible contribution of Paneth cells to the
development of intestinal lymphatic vessels remains to be investigated (34, 35).
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In conclusion, the results of this study showed an attenuation of PHT in germ-
free mice, suggesting that, besides the known mechanism of translocation of
bacterial-derived products, also the mere presence of bacteria in the intestinal
lumen, could significantly contribute to the regulation of PHT through its potential
effects on intestinal vasculature.
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FIGURE LEGENDS
Figure 1. Portal pressure measurements after PPVL.
Portal pressure increased significantly in ASF-PPVL and GF-PPVL mice 2 and 7
days after partial portal vein ligation (PPVL). GF-PPVL mice demonstrated an
attenuated portal hypertension in comparison to ASF-PPVL mice. n=10 per group
(1A). Porto-systemic shunts measurement. The formation of collaterals after
PPVL in ASF-PPVL mice increased significantly compared GF-PPVL. n=10 mice
per group (1B). Splenomegaly. Spleen size increased in ASF-PPVL mice 2 and
7 days after PPVL but there was not any significant increase in GF-PPVL mice.
n=15 mice per group (1C).The data presented here were compared with ANOVA
test following Kruskal-Wallis and are expressed as mean ± SD. * p<0.05,
** p<0.005, *** p<0.0005. ns: not significant. GF: germ-free mice, ASF mice:
altered Schaedler flora.
Figure 2. CFU in ASF mice. A significant bacterial translocation was observed
2 days after PPVL in the spleen of ASF-PPVL mice (2A). In MLN the number of
bacterial colonies increased after PPVL, but this was not statistically significant
(2B). PPVL did not affect significantly the total number of intestinal bacteria in the
feces (2C). In each group (n= 6-8) mice were used. Data are presented on a
logarithmic scale (log10). The geometric mean was calculated. Data were
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compared by the Mann-Whitney U test. ** p<0.005. ns: not significant. The
dashed lines indicate the detection limit (D.L).
Figure 3. CD31 immunostaining in mouse intestine. Paraffin sections of
mouse intestine were stained using CD31 antibodies in GF (left panel) and
ASF mice (right panel). The endothelial layer of intestinal blood vessels stained
in brown (arrows) (3A). Intestinal blood vessel density in ASF and GF mice.
Vessels were counted per 100 crypts (3B). Data are expressed as mean ± SD.
* p<0.05, ** p<0.005, *** p<0.0005. n=5 mice per group.
Figure 4. Lyve-1 immunostaining in mouse intestine. Paraffin sections of GF
mouse intestine were stained using Lyve-1 antibodies. The endothelial layer of
intestinal lymphatic vessels stained in red, while blood vessels did not show any
staining (green arrow) (4A). Intestinal lymphatic vessel density in ASF and
GF mice. Lymphatic vessels were counted per 100 crypts (4B). Data are
expressed as mean ± SD. Data are expressed as mean ± SD. * p<0.05, ** p<
0.005, *** p<0.0005. n=5 mice per group.
Figure 5. Staining of two adjacent sections by CD31 and Lyve-1 in mouse
intestine. CD31 (left panel) and Lyve1 (right panel) selectively stained blood
vessels and lymphatic vessels, respectively.
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Figure 6. Overexpression of PAR2 in ASF- PPVL mice intestine. A significant
increase in PAR2 expression was observed 7 days after PPVL in ASF-PPVL
mice, but not in GF-PPVL mice. PAR2 expression was not different between ASF
and GF sham (6A). Overexpression of Ang-4 and Pla2g2a in GF-PPVL mice
intestine. The expression of Ang-4 (6B) and Pla2g2a (6C) increased significantly
in GF-PPVL but not ASF-PPVL mice. In sham animals Ang-4 and Pla2g2a
expression was significantly higher in ASF than in GF sham mice. * p<0.05, ** p<
0.005, *** p<0.0005, n= 8 mice per group.
Figure 7. Paneth cell positive crypts in GF and ASF mice intestine. In sham
animals the number of Paneth cell positive crypts was higher in ASF than in GF
sham mice. This increased number of Paneth cells in ASF mice persisted 2 days
and 7 days after PPVL. * p<0.05 , n= 5 mice per group.
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26
AKNOWLEDGEMENTS
The authors would like to thank Dr. R. Gäumann from the Institute of Infectious
Diseases of the University of Berne for help in the MALDI-TOF facility and Dr. S.
Karaman from the Institute of Pharmaceutical Sciences of the Swiss Federal
Institute of Technology of Zurich, for help in immuno-staining of lymphatic
vessels, Dr. F. Ronchi and M. Wyss for help in high-throughput 16S amplicon
sequencing on the Iontorrent PGM platform and Dr. M. Gomez de Agüero for
help in gavaging of the mice and FITC-Dextran assay.
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Figure 1. Portal pressure measurements after PPVL.
Portal pressure increased significantly in ASF-PPVL and GF-PPVL mice 2 and 7 days after partial portal vein
ligation (PPVL). GF-PPVL mice demonstrated an attenuated portal hypertension in comparison to ASF-PPVL
mice. n=10 per group (1A). Porto-systemic shunts measurement. The formation of collaterals after PPVL in
ASF-PPVL mice increased significantly compared GF-PPVL. n=10 mice per group (1B). Splenomegaly. Spleen
size increased in ASF-PPVL mice 2 and 7 days after PPVL but there was not any significant increase in GF-
PPVL mice. n=15 mice per group (1C).The data presented here were compared with ANOVA test following
Kruskal-Wallis and are expressed as mean ± SD. * p<0.05, ** p<0.005, *** p<0.0005. ns: not
significant. GF: germ-free mice, ASF mice: altered Schaedler flora,
220x184mm (300 x 300 DPI)
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Figure 2. CFU in ASF mice. A significant bacterial translocation was observed 2 days after PPVL in the spleen
of ASF-PPVL mice (2A). In MLN the number of bacterial colonies increased after PPVL, but this was not
statistically significant (2B). PPVL did not affect significantly the total number of intestinal bacteria in the
feces (2C). In each group (n= 6-8) mice were used. Data are presented on a logarithmic scale (log10). The
geometric mean was calculated. Data were compared by the Mann-Whitney U test. ** p<0.005. ns: not
significant. The dashed lines indicate the detection limit (D.L).
128x178mm (300 x 300 DPI)
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Figure 3. CD31 immunostaining in mouse intestine. Paraffin sections of mouse intestine were stained using
CD31 antibodies in GF (left panel) and ASF mice (right panel). The endothelial layer of intestinal blood
vessels stained in brown (arrows) (3A).
249x135mm (300 x 300 DPI)
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Figure 3. Intestinal blood vessel density in ASF and GF mice. Vessels were counted per 100 crypts (3B).
Data are expressed as mean ± SD. * p<0.05, ** p<0.005, *** p<0.0005. n=5 mice per group.
138x117mm (300 x 300 DPI)
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Figure 4. Lyve-1 immunostaining in mouse intestine. Paraffin sections of GF mouse intestine were stained
using Lyve-1 antibodies. The endothelial layer of intestinal lymphatic vessels stained in red, while blood
vessels did not show any staining (green arrow) (4A).
245x138mm (300 x 300 DPI)
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Figure 4. Intestinal lymphatic vessel density in ASF and GF mice. Lymphatic vessels were counted per 100
crypts (4B). Data are expressed as mean ± SD. Data are expressed as mean ± SD. * p<0.05, ** p< 0.005,
*** p<0.0005. n=5 mice per group.
145x120mm (300 x 300 DPI)
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Figure 5. Staining of two adjacent sections by CD31 and Lyve-1 in mouse intestine. CD31 (left panel) and
Lyve1 (right panel) selectively stained blood vessels and lymphatic vessels, respectively.
253x130mm (300 x 300 DPI)
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Figure 6. Overexpression of PAR2 in ASF- PPVL mice intestine. A significant increase in PAR2 expression was
observed 7 days after PPVL in ASF-PPVL mice, but not in GF-PPVL mice. PAR2 expression was not different
between ASF and GF sham (6A). Overexpression of Ang-4 and Pla2g2a in GF-PPVL mice intestine. The
expression of Ang-4 (6B) and Pla2g2a (6C) increased significantly in GF-PPVL but not ASF-PPVL mice. In
sham animals Ang-4 and Pla2g2a expression was significantly higher in ASF than in GF sham mice. *
p<0.05, ** p< 0.005, *** p<0.0005, n= 8 mice per group.
137x260mm (300 x 300 DPI)
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Figure 7. Paneth cell positive crypts in GF and ASF mice intestine. In sham animals the number of Paneth
cell positive crypts was higher in ASF than in GF sham mice. This increased number of Paneth cells in ASF
mice persisted 2 days and 7 days after PPVL. * p<0.05 , n= 5 mice per group.
142x116mm (300 x 300 DPI)
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Supplementary Information
Supplementary Figure 1. Comparison of portal hypertension between ASF
and SPF mice. Increase in portal pressure after PPVL in ASF colonized mice
(n=10) versus SPF mice (n=5). SPF: specific pathogen free, PPVL: partial portal
vein ligation, ASF: altered Schaedler flora.
Supplementary Figure 2. Portal pressure measurements after PPVL. Portal
pressure, porto-systemic shunts and spleen size measured 7 days after PPVL in
germ-free (GF) mice as a control group versus GF mice gavaged with ASF at the
day 2 post PPVL n=10 per group. A slight increase in portal pressure was
observed in GF mice gavaged with ASF compared to control animals but the
difference was not statistically significant. Crt: control group, Gav: gavaged with
ASF group. GF: germ-free mice, ASF: altered Schaedler flora, PPVL: partial
portal vein ligation.
Supplementary Figure 3. Microbial community analysis. No difference in
ASF microbiota composition. The microbial composition in fecal samples of sham
operated ASF mice (n=4) or 2 (n=5) and 7 days (n=5) post PPVL at the phyla
and genus level was determined by high throughput 16S amplicon analysis (A).
To assess b diversity between the different groups principal coordinates analysis
on weighted UniFrac distances was performed on all operational taxonomic units
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(OUT) (B & C). p values to determine the statistical significance of clustering
were calculated using Anosim. Analysis was performed using QIIME 1.8.0.
Supplementary Figure 4. Mouse Albumin Elisa in feces. There were no
significant changes in the albumin content of feces, suggesting that the
permeability of the intestinal mucosa was not altered in the presence of portal
hypertension (A). Assessment of intestinal permeability by FITC-Dextran.
Serum FITC-dextran concentration in ASF sham n=3, GF-PPVL2d n=3, ASF-
PPVL2d n=5, SPF n=3, SPF-PPVL2d n=5, 3 hours post-gavaged is shown. Mice
with a significant damaged of epithelial barrier (3% DSS in drinking water for 5
days) used as positive controls n=5 (B).The dashed lines indicate the detection
limit (D.L). GF: germ-free mice, ASF: altered Schaedler flora, PPVL: partial portal
vein ligation.
Supplementary Figure 5. Intestinal blood vessel density in GF mice versus
gavaged mice with ASF. Paraffin sections of mouse intestine were stained
using CD31 antibodies. Vessels were counted per 100 crypts. (Data are
expressed as mean ± SD, ** p < 0.005, n=10 mice per group. Crt: control group
Gav: gavaged group.
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Supplementary Figure 6. Quantification of small intestine mRNA
expression levels of TF, PAR1 and Angiopoietin 1,2 (A). VEGF receptors
(VEGF-R1, VEGF-R2, VEGF-R3) and Neuropilin-2 (B). VEGF A,B,C and D (C).
No significant changes were observed in mRNA expression of these angiogenic
factors after PPVL neither in ASF nor in GF mice. GF: germ-free mice, ASF:
altered Schaedler flora, PPVL: partial portal vein ligation.
Supplementary Figure 7. Blood flow measurement in the mesenteric artery.
Mesenteric blood flow in GF mice, sham group n= 5, GF-PPVL7d as a control
group n=10 and GF-PPVL mice gavaged with ASF n=10 (A) and SPF mice n=5
(B) measured using the ultrasound Doppler technique. Mesenteric blood flow
(MBF) increased significantly after PPVL. Data are expressed as mean ± SD. * p
< 0.05, ** p < 0.005, *** p < 0.0005. GF: germ-free mice, ASF: altered Schaedler
flora, PPVL: partial portal vein ligation. Crt: control group, Gav: gavaged group.
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Supplementary materials and methods:
Altered Schaedler flora gavage
The entire cecal content of an ASF mouse was isolated aseptically under
anaerobic chamber and diluted in 7ml of reduced (1x) sterile phosphate buffer
saline (PBS) and mixed well. The suspension was left to sediment for 5 mins
and then briefly centrifuged (3000rpm, 10sec) to remove particulate material.
The supernatant was passed through a 100μm mesh and 500μl was gavaged
per mouse.
Microbial community analysis
DNA was isolated from fecal pellets using the Qiagen Stool Kit (Qiagen). The
16S rRNA gene segments spanning the variable V5 and V6 regions were
amplified using the barcoded forward fusionprimer 5’-CCA TCT CAT CCC
TGC GTG TCT CCG ACT CAG BARCODE ATT AGA TAC CCY GGT AGT
CC-3’ in combination with the reverse fusionprimer 5’-CCT CTC TAT GGG
CAG TCG GTG AT ACG AGC TGA CGA CAR CCA TG-3’. The sequences in
italic are Iontorrent PGM-specific adaptor sequences. The 16S V5-V6
amplicons were purified and prepared for sequencing on an Iontorrent PGM
system 316v2 chip according to the manufacturers instructions (Life
Technologies). Analysis of microbial composition and beta diversity was
performed using the QIIME pipeline version 1.8.0 (1). 20’000 – 100’000 reads
per sample were obtained. Operational taxonomic units (OUT) were picked
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based on 97% sequence identity followed by taxonomy assignment by
blasting representative sequences against a custom database containing 16
sRNA gene sequences from the eight ASF members. Beta diversity between
groups was assessed by weighted UniFrac-based Principal Coordinate
Analysis (PCoA) comparing OTUs form all groups or by pairwise comparison
of two groups at a time. P values for clustering significance in pairwise
comparisons was calculated by the ANOSIM method using 9999
permutations (1).
Assessment of intestinal permeability by fluorescein isothiocyanate-
dextran (FITC-dextran)
The mice were fasted for 4 hours and then gavaged with FITC-dextran 4000
(0.6mg/g body weight; Sigma). FITC-dextran in the serum was assessed 3
hours post-gavage by determining the OD at excitation 492nm and emission
525nm using Tecan Reader infinite 200 fluorometer. FITC-Dextran diluted in
normal mouse serum was used for the standard.
Dextran sulfate sodium salt (DSS) colitis
The mice were treated with 3% DSS (MP biomedicals, LLC) in drinking water
for 5 days and intestinal permeability was assessed by FITC-Dextran in the
serum following oral gavage.
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Measurement of mesenteric artery blood flow
Mesenteric artery blood flow was measured by placing a transonic blood flow
probe (400-series flow-meters MA-0.5 PSB) around the mesenteric artery
then connected with a Power-lab instrumentation (AD Instruments).
1.Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer
N, et al. QIIME allows analysis of high-throughput community sequencing data. Nat
Methods 2010;7:335-336.
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