Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6Clo macrophages

Accepted Manuscript Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6Clo macrophages Akito Yada, Yuji Iimuro, Naoki Uyama, Yugo Uda, Toshihiro Okada, Jiro Fujimoto PII: DOI: Reference:

S0168-8278(15)00337-2 http://dx.doi.org/10.1016/j.jhep.2015.05.010 JHEPAT 5682

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

19 January 2015 4 May 2015 5 May 2015

Please cite this article as: Yada, A., Iimuro, Y., Uyama, N., Uda, Y., Okada, T., Fujimoto, J., Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6Clo macrophages, Journal of Hepatology (2015), doi: http://dx.doi.org/10.1016/j.jhep.2015.05.010

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6Clo macrophages.

Akito Yada, Yuji Iimuro, Naoki Uyama, Yugo Uda, Toshihiro Okada, Jiro Fujimoto.

Department of Surgery, Hyogo College of Medicine, Nishinomiya, JAPAN

Corresponding author:Yuji Iimuro MD, PhD, Department of Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, 663-8501 Japan E-mail: [email protected] Phone: +81-798-45-6582 Fax:

+81-798-45-6581

Electronic word count: 5985 (Abstract: 250) Number of figures and tables: 8 figures, 0 table, and 3 supplementary figures Abbreviations: TAA, thioacetamide; MMP-9, matrix metalloproteinase -9; α-SMA, alpha smooth muscle actin; EpCAM, epithelial cell adhesion molecule; DAPI, 4',6-Diamidino-2-Phenylindole; HPLC, High-Performance Liquid Chromatography; CXCL9, chemokine (C-X-C motif) ligand 9; CXCR3, chemokine (C-X-C motif) receptor 3; Sfrp1, Secreted frizzled-related protein 1; Dkk2 Dickkopf 2; CTNNB1, catenin (cadherin-associated protein) beta 1; Dll4, delta like ligand 4; Jag2, jagged-2; Hes5, transcription factor HES-5; Hey1, hairy/enhancer-of-split related with YRPW motif protein 1.

Key words: splenectomy, liver fibrosis, monocyte/macrophage, progenitor cell Conflict of interest: The authors who have taken part in this study declared that they do not have anything to disclose regarding or conflict of interest with respect this manuscript.

Financial support: This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS), 25461966 and 22591510.

Author’s contributions: Study concept and design: AY, YI, TO, JF. Acquisition of data: AY, NU, YU. Drafting of the manuscript: AY, YI. Statistical analysis: AY. Study supervision: YI, JF. 1

Abstract Background & Aims: Splenectomy in cirrhotic patients has been reported to improve liver

function; however the underlying mechanism remains obscure. In the present study, we investigated

the mechanism using a murine model, which well represents the compensated liver cirrhosis. Methods: C57BL/6 male mice were allowed to drink water including thioacetamide (TAA: 300

mg/l) ad libitum for 32 weeks. After splenectomy at 32 weeks, mice were sacrificed on days 1, 7,

and 28, respectively, while TAA-administration was continued. Perioperative changes in peripheral

blood and liver tissues were analyzed. Results: TAA treatment of mice for 32 weeks reproducibly achieved advanced liver fibrosis with

splenomegaly, thrombocytopenia, and leukocytopenia. After splenectomy, liver fibrosis was

attenuated, and macrophages/monocytes were significantly increased in peripheral blood, as well as

in the liver. Progenitor-like cells expressing CK-19, EpCAM, or CD-133 appeared in the liver after

TAA treatment, and gradually disappeared after splenectomy. Macrophages/monocytes accumulated

in the liver, most of which were negative for Ly-6C, existed adjacent to the hepatic progenitor-like

cells, and quantitative RT-PCR indicated increased canonical Wnt and decreased Notch signals. As a result, a significant amount of β-catenin accumulated in the progenitor-like cells. Moreover, Ki67-positive relatively small hepatic cells were significantly increased. Protein expression of

2

MMP-9, to which Ly-6G-positive neutrophils contributed, was also increased in the liver after

splenectomy. Conclusions: The hepatic accumulation of macrophages/monocytes, most of which are Ly-6Clo,

the reduction of fibrosis, and the gradual disappearance of hepatic progenitor-like cells possibly play

significant roles in the tissue remodeling process in cirrhotic livers after splenectomy.

3

Introduction

Liver cirrhosis is the result of excessive accumulation of extracellular matrix in the liver, and is

one of the major causes of morbidity and mortality in many countries. Liver cirrhosis often leads to

portal hypertension and splenomegaly, and finally results in liver failure. Liver transplantation is the

only curative treatment for advanced cirrhosis. Meanwhile, splenectomy has been performed as a

palliative surgical treatment to attenuate hypersplenism [1, 2]. Recently, it has been reported that

liver function parameters improved after splenectomy, in addition to the amelioration of

thrombocytopenia in several clinical settings [3-6]. However, the mechanism underlying such a

phenomenon remains unclear, and the significant role of platelets in liver fibrosis has been

emphasized in some reports [7, 8]. One obstacle for the elucidation of the mechanism is that there

has been no suitable animal model of liver fibrosis that resembles a human situation, in which

thrombocytopenia appears in accordance with the advancement of liver fibrosis. Thus, we tried to

produce such a suitable animal model and found that thioacetamide (TAA) treatment of mice via

drinking water for a long period well reproduces thrombocytopenia in addition to advanced liver

fibrosis. In the present study, we analyzed the effect of splenectomy on liver fibrosis and its function,

and tried to investigate the underlying mechanism using this model. In this report, we indicate that

splenectomy induced the accumulation of macrophages/monocytes as well as platelets in the liver in

a TAA-induced murine liver fibrosis model, accelerating the resolution of fibrosis. The accumulated

4

macrophages/monocytes (F4/80+) were negative for Ly-6C; the macrophage subset which has been

reported to play important roles in resolution of liver fibrosis [9]. Meanwhile, the increased number

of hepatic progenitor-like cells after TAA-treatment gradually decreased after splenectomy with

up-regulated Wnt and down-regulated Notch signals in the liver.

5

Methods

Animal models

C57BL/6 male mice (4 weeks old) were obtained from Japan SLC Inc. (Shizuoka, Japan). Mice

were fed with a control chow diet and were allowed to drink water including thioacetamide (TAA;

300 mg/l, Sigma-Aldrich, St Louis, MO) ad libitum for 32 weeks. After the TAA treatment,

splenectomy was performed. The mice were sacrificed 1, 7 and 28 days after the operation, and

blood samples and liver tissues were collected. Control mice underwent sham operations without

splenectomy. TAA-treatment was continued even after splenectomy or sham operation until sacrifice.

All animals received human care, and all animal experiments were performed in accordance with the

guidelines of the Institutional Animal Care Committee, Hyogo College of Medicine.

Splenectomy

For splenectomy, the abdominal wall of mice was opened through a left subcostal minimal

incision under ether anesthesia. The splenic arteries and veins were ligated at the splenic hilum with

3-0 silk and divided. The resected spleen was removed, and the abdominal incision was closed. All

of the surgical procedures were performed under sterilized conditions. Under the sham operation, the

abdominal wall was similarly opened, and the wall was closed immediately after identifying the

spleen.

Microarray analysis

6

RNA was extracted from liver tissue using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA

degradation was analyzed by electrophoresis using a 2100 Bioanalyzer (Agilent Technologies, Santa

Clara, CA). One microgram of each total RNA sample was amplified using Ambion Amino Allyl

aRNA kit (Ambion, Carlsbad, CA). The amplified anti-sense RNAs were labeled with Cyanin5 (GE,

Amersham, Little Chalfont, Buckinghamshire, UK) and were subjected to hybridization. The

hybridization was performed using Mouse Oligo chips 24k (Toray industries Inc., Tokyo, Japan) The

signal for each gene was normalized using a global normalization method (median Cy3/Cy5 ratio =

1).

Quantitative RT-PCR (Real-time PCR)

Total RNA was extracted from liver tissues using the ISOGEN kit (NIPPON GENE, Tokyo,

Japan). Reverse transcription was carried out using the High-capacity cDNA archive kit (Applied

Biosystems, Foster City, CA). Quantitative RT-PCR was performed using the Applied Biosystems

7300 Real-Time PCR System (Applied Biosystems). Taqman gene expression assays including the

primers for Cxcl9 and Cxcr3 were purchased from Applied Biosystems. A predeveloped TaqMan

assay reagent for 18S ribosomal RNA (18S rRNA) (Applied Biosystems) was used as an internal

control. For analysis of the mRNA expression in the Wnt signaling pathway, PrimerArray® Wnt

signaling pathway (Takara Bio, Otsu, Japan) was employed. For analysis of the mRNA expression in

7

the Notch signaling pathway, RT 2 Profiler TM PCR Array Mouse Notch Signaling Pathway (Qiagen,

Hilden, Germany) was used.

Flow cytometry (FACS)

Cell surface expression analysis of immune cells was performed using the following antibodies: T

lymphocytes, B lymphocytes, monocytes and macrophages/monocytes were reacted with

anti-murine CD3e-APC, CD45R/B220-PerCp-Cy5.5, CD14-PE（BD Bioscience, San Diego, CA),

and F4/80-FITC (Biomedicals, Augst, Switzerland), respectively. Cells were incubated for 15

minutes on ice with anti-CD16/CD32 monoclonal antibody (Biolegend, San Diego, CA) to reduce

nonspecific binding. Cells were washed in buffer and incubated with each antibody for 30 min and

subjected to flow cytometry using the FACS Calibur (BD Biosciences, San Diego, CA). Liver

lymphomyeloid cells were obtained from mice liver as previously reported [10].

Histological analysis

Histological analysis was performed as previously reported [11]. Tissues were fixed in 10%

buffered formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin and

examined using a light microscopy. Sirius red-fast green staining was also performed to evaluate

liver fibrosis. For the detection of activated hepatic stellate cells, a monoclonal antibody against α-SMA (Diagnostic Biosystems, Pleasanton, CA) was employed. Macrophages/monocytes or platelets were detected using an anti-F4/80 monoclonal antibody (BMA Biomedicals, Augst,

8

Switzerland) or a polyclonal anti-thrombocyte antibody (Acris Antibodies Inc., San Diego, CA),

respectively. For the detection of progenitor-like cells in the liver, a monoclonal anti-CK-19 (Abcam,

Cambridge, MA), a monoclonal anti-EpCAM (BD Biosciences, San Jose, CA), and a polyclonal

anti-CD133 (eBioscience, San Diego, CA) antibodies were used. Similarly, anti-β-catenin (Santa Cruz Biotechnology Inc, Dallas, TX), anti-Wnt-2 (Atlas Antibodies, Stockholm, Sweden), anti-Ki67

(Cell Signaling Technology Inc, Beverly, MA), or anti-MMP-9 (R&D Systems, Minneapolis, MN)

was used for each immunostaining. Frozen sections were used for MMP-9 immunostaining. For the

assessment of the number of Ki-67-positive cells, cells with Ki-67-positive nuclei were counted in

10 fields (x 200) randomly selected in each sample, and the mean values were calculated.

Image analysis

Image analysis was employed for semi-quantitative assessment of each immunostaining. At a

magnification of x40, x100, or x200, the ratio of the area positive for each immunostaining including

Sirius-red, α-SMA, F4/80, thrombocyte, CK-19, EpCAM, CD133 or MMP-9 was defined in 10

fields randomly selected in each sample using free software available from the National Institutes of

Health (Image J; http://rsb.info.nih.gov/ij). The mean value of the 10 fields was used as a

representative data point of the sample.

Immunofluorescence staining

9

Fluorescence immunohistochemistry was performed as previously reported [11]. An anti-CK19

mouse monoclonal antibody (Abcam) and an anti-F4/80 rat monoclonal antibody (BMA

Biomedicals,

Augst,

Switzerland)

were

used

to

investigate

the

relationship

between

macrophages/monocytes and CK19-positive cells in the liver. Alexa Fluor 555 tyramide (Invitrogen,

Carlsbad, CA, USA) was used to visualize the CK-19-positive cells. Alexa Fluor 488-conjugated

streptavidin (Invitrogen) was employed to visualize the F4/80-positive cells. DAPI staining was used

for the counterstaining of nuclei. In the same manner, fluorescence immunostaining was also

performed using an anti-Wnt2 mouse monoclonal antibody (Atlas Antibodies, Stockholm, Sweden),

an anti-β-catenin (CTNNB-1) antibody (Santa Cruz Biotechnology Inc.), an anti-MMP9 (R&D Systems) antibody, an anti-Ly6C antibody (Abcam), anti-CXCL9 antibody (Bioss Inc., Woburn, MA,

USA), or an anti-Ly-6G antibody (BD Biosciences). Fluorescence immunostaining was observed

using a confocal laser scanning microscope, LSM510 (Carl Zeiss, Jena, Germany).

Measurement of hydroxyproline content

Liver tissues were hydrolyzed with 12 N HCL for 12 h. After centrifugation, the hydroxyproline

content of these tissues was quantified using a High-Performance Liquid Chromatography (HPLC)

system (Nihon Bunko, Tokyo, Japan).

Statistical analysis

10

Data were expressed as the mean + SD, and the statistical significance of differences among groups

were assessed by Student’s t-test or the Mann–Whitney U-test as appropriate. P-values < 0.05 were

regarded as statistically significant.

11

RESULTS

A murine liver fibrosis model suitable for analyzing the effect of splenectomy in hypersplenism

After the treatment of mice with TAA in drinking water for 32 weeks, remarkable liver fibrosis was detected by Sirius-red staining (Suppl. Fig. 1A). Immunostaining for α-SMA also revealed a significant increase in the number of activated myofibroblasts in the liver (Suppl. Fig. 1A). The

spleen/body weight ratio was significantly increased, reaching almost twice (0.58% + 0.08) that of

normal untreated controls (0.32 + 0.01) (Suppl. Fig. 1B). Most importantly, the counts of platelets

and leukocytes were significantly decreased after the treatment, concomitant with a significant

increase in the serum concentrations of total bilirubin, resembling the situation of hypersplenism

with liver fibrosis in humans (Fig. 1D and Suppl. Fig. 1C). Additionally, the serum concentration of

albumin did not change, and the increase in the concentrations of transaminases was moderate after

TAA treatment. These results indicate that this TAA-induced murine liver fibrosis model seems to

resemble well the compensated liver cirrhosis with hypersplenism in humans as reported previously

[12].

Splenectomy attenuates the TAA-induced liver fibrosis as well as thrombocytopenia

After splenectomy, liver fibrosis was markedly attenuated (Fig. 1A). This phenomenon was also confirmed by semi-quantitative analysis of the Sirius red- or α-SMA-positive area in the liver (Fig. 1B) and by the quantitative measurement of the hydroxyplorine content in the liver (Fig. 1C). The

12

decreased counts of platelets and leukocytes in peripheral blood after TAA treatment were

dramatically increased after splenectomy with a peak around day 7, similar to the clinical situation in

humans (Fig. 1D). Interestingly, the increase in leukocytes was much more drastic (almost three

times) than that in platelets. Moreover, the increased concentration of serum total bilirubin was

significantly decreased after splenectomy, while the concentration of serum albumin did not

significantly change (Fig. 1D). While thrombocytosis has been well documented after splenectomy

in previous reports, the drastic increase in the leukocyte counts in peripheral blood was more

intriguing.

The mRNA expression of CXCL-9 is increased in the liver after splenectomy

Comparison of the mRNA expression in the TAA-treated liver before and 1 week after

splenectomy using microarray analysis revealed that only the mRNA expression of CXCL-9, a

chemokine mainly produced by macrophages or monocytes [13], was increased over 1.5-fold after

splenectomy. Real-time PCR using the mRNA extracted from liver tissues revealed a mild increase

in the mRNA expression of CXCL-9 after splenectomy (Suppl. Fig. 3A), while the mRNA

expression of CXCR3, the receptor of CXCL-9, did not change (data not shown). These results

implicated the possible participation of macrophages or monocytes during the attenuation process of

liver fibrosis after splenectomy in this model. However, double immunofluorescence staining for

13

CXCL9 and F4/80 or CXCL9 and Ly-6G revealed that the source of increased CXCL9 protein was

not macrophages/monocytes but neutrophils (Suppl. Fig. 3B) in this model.

The percentage of macrophages/monocytes is increased in the liver and peripheral blood after

splenectomy

To analyze the compositional change in the increased counts of leukocytes, we employed FACS to

analyze the peripheral and liver leukocytes. The percentage of F4/80-positive cells was significantly

increased in the peripheral blood on day 7 (Fig. 2A). In addition, the percentage of F4/80-positive

liver macrophages/monocytes was also significantly increased 7 days after splenectomy (Fig. 2B).

We also analyzed the changes in the percentage of T or B cells in peripheral and liver leukocytes.

The percentages of T or B cell in peripheral blood or in the liver did not change significantly (Fig.

2C). The percentage of other leukocytes, which consist largely of neutrophils, did not change

significantly in the peripheral blood or liver. Therefore, we thereafter focused on the dynamic change

in macrophages/monocytes in the liver tissues, rather than on the mild increase in hepatic neutrophil

infiltration, which was the source of CXCL9.

Macrophages as well as platelets accumulate in the fibrotic liver after splenectomy

To investigate the possible participation of macrophages/monocytes during the attenuation process

of liver fibrosis after splenectomy, we focused on these cells. Immunohistochemical analysis of liver

tissues revealed that F4/80-positive macrophages/monocytes (Fig. 3A) as well as platelets (Fig. 3C)

14

remarkably accumulated in the liver after splenectomy. Semi-quantitative image analysis of the

stain-positive area confirmed that the macrophages/monocytes and platelets accumulated in the liver

with a peak approximately 7 days after splenectomy, while the macrophage accumulation

predominated that of platelets (Fig. 3B and D). Interestingly, TAA treatment itself decreased the

intrahepatic accumulation of macrophages/monocytes but not platelets in this model, suggesting a

possible relationship between fibrosis and macrophages/monocytes in the liver. Intriguingly, the

detailed observation of the pattern of macrophage/monocyte accumulation in the liver revealed that

these cells predominantly existed along with the injured hepatic area including a fibrotic scar on day

7 and that they gradually became less obvious coinciding with the attenuation of liver fibrosis on day

28 (Fig. 3A). This accumulation pattern was not obvious concerning platelets on day 7 (Fig. 3C).

Appearance of hepatic progenitor-like cells after the TAA treatment

The participation of hepatic progenitor cells in the recovery process of advanced liver fibrosis has

been well documented recently [14, 15]. Thus, we investigated the existence of such cells in our

model. Immunohistochemical analysis revealed that the number of cells expressing hepatic

progenitor markers, including CK-19, EpCAM, or CD-133, was dramatically increased along with

the chronically injured hepatic area after TAA treatment (Fig. 4A). Interestingly, the number of these

cells was gradually decreased after splenectomy accompanied by the accumulation of F4/80-positive

macrophages/monocytes in the area (Fig. 4A and B). Double fluorescence immunostaining for

15

CK-19 and F4/80 revealed a close interaction between macrophages/monocytes and hepatic

progenitor-like cells in the injured hepatic area after splenectomy (Fig. 5A right), while only a few

F4/80-positive cells infiltrated into the area without splenectomy (Fig. 5A left).

The macrophages/monocytes accumulated in the liver were negative for Ly-6C One subset of macrophages responsible for the hepatic tissue remodeling, which express F4/80int Ly-6Clo, have been recently reported [9]. Thus, we analyzed whether the macrophages/monocytes

accumulated in the liver after splenectomy expressed Ly-6C in our model. Double

immunofluorescence staining for Ly-6C and F4/80 revealed that only small part of the accumulated

macrophages/monocytes expressed Ly-6C at day 7 after splenectomy (Fig. 5B middle), suggesting that these cells (Ly-6Clo) were responsible for tissue remodeling in this model. Meanwhile, a large

part of the splenic F4/80-positive macrophages expressed Ly-6C (Fig. 5B right). Intriguingly,

macrophages/monocytes marginally detected in the sham-operated liver did not express Ly-6C,

possibly reflecting no or mild acute inflammation in our TAA-induced animal model.

Canonical Wnt signaling is up-regulated after splenectomy in the TAA-treated liver

The interaction between infiltrated liver macrophages and hepatic progenitor-like cells through Wnt

signaling in murine liver fibrosis has been previously reported [16]. Thus, we analyzed changes in

Wnt signaling after splenectomy in this model. Real-time RT-PCR using mRNA obtained from

sham- or splenectomy-treated TAA-induced cirrhotic mice revealed that the mRNA expression of the

16

canonical Wnt molecules, including Wnt9b, Wnt2,

and Wnt16, dramatically increased after

splenectomy (Fig. 6A left). By contrast, the mRNA expression of Wnt4, a non-canonical Wnt

molecule, and negative regulators of Wnt signaling, including Secreted frizzled-related protein (Sfrp)

1 and Dickkopf (Dkk) 2, decreased more than 3 times after splenectomy (Fig. 6A right). These

results indicate that splenectomy up-regulates canonical Wnt signaling in the TAA-induced fibrotic

liver. Down-regulation of Notch signaling pathway is also reported in such a situation [16], so we

also performed real-time RT-PCR for Notch signals. The results showed the down-regulated Notch

signals, including Dll4, Jag2, Notch3, Notch4, Hes5, and Hey1 (Fig. 6B). To investigate the cell

sources of the canonical Wnt molecules in the liver after splenectomy, double immunofluorescence

staining for F4/80 and Wnt2 was performed. The expression of Wnt2 was markedly increased 7 days

after splenectomy, while only slight expression of Wnt2 was detected in sham-operated animals (Fig.

6C). This double immunofluorescence staining revealed that most of F4/80-positive cells expressed

Wnt2 after splenectomy, while other cells, including sinusoidal endothelial cells, were also positive

for Wnt2. Additionally, double immunofluorescence staining for CK19 and CTNNB1 (β-catenin) clearly demonstrated increased accumulation of CTNNB1 in the cytoplasm of CK19-positive

progenitor-like cells, indicating the activated canonical Wnt signaling pathway in these cells (Fig.

6D).

Ki67-positive cells increases after splenectomy in the TAA-treated liver

17

Next, we investigated whether the up-regulated Wnt signaling resulted in hepatic cell proliferation

after splenectomy in this model. TAA treatment for 32 weeks induced the occasional appearance of

Ki67-positive cells along with the injured area in the liver (Fig. 7A). After splenectomy, the number

of Ki67-positive cells started to increase along with the injured area and peaked on day 7, declining

thereafter (Fig. 7A and B). Interestingly, the number of Ki67-positive cells with relatively small

nuclei and cytoplasm was increased on days 1 and 7 after splenectomy, implying the stimulated

proliferation of immature or small hepatocytes (Fig. 7A). The mean number of Ki67-positive cells at

each time point was summarized in Fig. 7B. Co-expression of Ki67 and CTNNB1 was further

immunohistochemically analyzed in the liver (Fig. 7C). Brown-colored, Ki67-positive, relatively

small, round nuclei were merely overlapped with orange-colored CTNNB1-positive cells even after

splenectomy (white triangles), suggesting that proliferating hepatic cells were already not

CTNNB1-positive progenitor-like cells. Meanwhile, double immunohistochemistry for Ki67 and

EpCAM revealed that some of the cells with relatively small and round Ki67-positive nuclei weakly

expressed EpCAM at day 7 after splenectomy (Fig. 7D, white triangle), and that these cells existed

close or adjacent to narrow-shaped cells strongly expressing EpCAM, namely progenitor-like cells.

Expression of MMP-9 in the liver increases after splenectomy

Intrahepatic expression of MMP-9 was immunohistochemically analyzed. Expression of MMP-9

was occasionally detected after TAA treatment even without splenectomy (Fig. 8A). Additionally,

18

the expression obviously increased after splenectomy along with the chronically injured hepatic area.

Semi-quantitative image analysis revealed that the MMP-9 expression significantly increased after

splenectomy, peaking around day 7 and lasting until day 28 (Fig. 8B). Double immunofluorescence

staining for MMP-9 and F4/80 or Ly-6G revealed that MMP-9 was expressed on Ly-6G-positive

infiltrated neutrophils but not on F4/80-positive macrophages/monocytes after splenectomy (Fig. 8C

and D), suggesting a significant participation of neutrophils in the resolution process of liver fibrosis

in this model.

19

Discussion

Liver function parameters have been often improved after splenectomy, in addition to the

amelioration of thrombocytopenia in several clinical reports [3-6]; however the underlying

mechanism remains unclear. To investigate the mechanism, an appropriate animal model resembling

the clinical situation is required. In a popular liver fibrosis model induced by chronic intra-peritoneal

injection of CCl4, acute inflammation accompanied by CCl4 administration cannot be avoided [12]. Indeed, in our preliminary experiments, repeated i.p. injection with CCl4 for 8 weeks often caused peritonitis and acute liver damage, while the counts of platelets and leukocytes in peripheral blood

was rather increased compared with normal controls (data not shown), consistent with a previous

report [7]. Therefore, we used a murine liver fibrosis model induced by chronic treatment with TAA

in drinking water (300mg/l) in the present study [12]. In this model, a mild increase in ALT and AST

has been reported irrespective of the duration of TAA treatment without obvious parenchymal

damage. Moreover, thrombocytopenia in addition to liver fibrosis was well reproducible after a

32-week treatment with TAA in the present study. The spleen/body weight ratio was also

significantly increased, and even leukocytopenia was observed. Thus, we assumed that this murine

model is suitable for analyzing liver fibrosis with hypersplenism.

A significant role of thrombocytosis during the improving process of liver function after

splenectomy was suspected [7], and several reports have emphasized the role of platelets

20

accumulated in the liver or platelet-derived growth factors including HGF in this process [8, 17].

Indeed, we also observed the accumulation of platelets in the liver after splenectomy in the present

study. More intriguingly, we detected the drastic accumulation of macrophages/monocytes in fibrotic

livers after splenectomy for the first time, and the accumulation of these cells was prominent along

with the injured hepatic area. Although the sensitivity of microarray analysis employed in the present

study seemed insufficient, only CXCL9 was detected as an up-regulated gene in the liver after

splenectomy, leading us to focus on the significant role of macrophages/monocytes. Even though the

source of CXCL9 was neutrophils in our model, drastic accumulation of macrophages/monocytes in

the liver was confirmed. The accumulation of macrophages/monocytes was followed by the

resolution of liver fibrosis and cell proliferation. A similar observation was reported in a

macrophage-infusion experiment in mice, in which bone marrow-derived macrophages were

therapeutically delivered into injured fibrotic livers, improving fibrosis and stimulating regeneration

[18].

Ly-6C, a cell surface glycoprotein used to identify murine circulating monocytes, is differentially

expressed in diseased tissues, implying existence of functionally distinct macrophage population [19, 20]. As for the liver, an Ly-6Chi intrahepatic macrophage population, derived from recruitment of circulating Ly-6Chi monocytes, is critical for fibrogenesis [21]. Meanwhile, it was recently reported that an F4/80int Ly-6Clo macrophage population plays significant roles in liver fibrosis resolution [9].

21

Therefore, we analyzed whether the macrophages/monocytes accumulated in the liver after splenectomy in our model were Ly-6Chi or Ly-6Clo. The results showed that only small part of the

accumulated macrophages/monocytes expressed Ly-6C, suggesting that most of the accumulated macrophages/monocytes (Ly-6Clo) were responsible for tissue remodeling in this model. On the

other hand, most of the F4/80-positive splenic macrophages/monocytes expressed Ly-6C. From these results, it is possible to speculated that Ly-6Chi macrophages, which were increased in the circulating blood pool after splenectomy, were recruited to the liver, and differentiated to a Ly-6Clo

population, similar to the phenomenon observed in previous report [9].

In the present study, accumulated macrophages/monocytes were detected close to the hepatic

progenitor-like cells expressing CK-19, EpCAM, or CD-133. The pivotal roles of macrophages in

specifying hepatic progenitor cell fate have been reported in chronic liver injury [16]. In that report,

canonical Wnt signaling was determined to be a macrophage-derived factor that promotes the

progenitor’s specification to hepatocytes, opposing Notch signaling. In our model, the mRNA

expression of some canonical Wnt signaling molecules, such as Wnt2, Wnt9b, and Wnt16, was

dramatically increased in the liver after splenectomy. Moreover, an obvious increase in the protein

expression

of

Wnt2

was

also

histologically

confirmed,

and

many

F4/80-positive

macrophages/monocytes participated in its expression. Additionally, Notch signaling, including Dll4,

Jag2, Notch3, Notch4, Hes5, and Hey1, was markedly suppressed after splenectomy. As a result, the

22

increased protein accumulation of CTNNB1 (β-catenin) was detected in the cytoplasm of CK19-positive progenitor-like cells after splenectomy, indicating up-regulated Wnt signals in these

cells. It should be noted that cells undergoing Wnt signaling may display an overall rise in CTNNB1

protein without a clear nuclear preference in many cases [22]. The number of Ki67-positive

proliferating cells was significantly increased after splenectomy; particularly, the appearance of

relatively small-sized nuclei positive for Ki67 was prominent 7 days after splenectomy. However,

those Ki67-positive small cells were already negative for CTNNB1, implying those cells represent

transit-amplifying cells rather than hepatic progenitor cells [23]. Double immunohistochemistry for

Ki67 and EpCAM revealed that some of the cells with relatively small and round Ki67-positive

nuclei weakly expressed EpCAM, and that these cells existed close or adjacent to the narrow-shaped

EpCAM-positive progenitor-like cells. These phenomena may suggest the proliferation of immature

hepatocytes that were possibly derived from progenitor-like cells as a result of canonical Wnt-signal

activation. Although we could not provide direct evidence for such differentiation in our experiment,

the gradual decrease in the number of CK19-positive cells and increase in the number of

Ki67-positive cells after splenectomy suggest that the progenitor-like cells may possibly play

significant role in the regeneration process in cirrhotic livers after splenectomy.

How the macrophages/monocytes were recruited into the liver after splenectomy was unclear in

this study. One possible explanation is that the hepatic accumulation of these cells may be a passive

23

phenomenon in response to the prominent increase in F4/80-positive cells in the peripheral blood

after splenectomy. A significant increase in the number and percentage of monocytes and B cells in

peripheral blood has been reported in humans in earlier investigations [24-26]. The reason for this

increase has been explained such that cells homing to and circulating through the spleen accumulate

in the relatively small peripheral blood pool after splenectomy. An increased number of monocytes

in peripheral blood possibly resulted in the accumulation of these cells in the liver. The participation

of chemokines such as CCL2, a macrophage chemoattractant, in this process is strongly speculated

[18]. An alternative possible explanation for the macrophage/monocyte recruitment is that platelets

accumulated in the liver after splenectomy stimulated leukocyte recruitment into the liver. However,

immunohistochemical analysis of the liver in the present study revealed that the response after

splenectomy was much quicker in macrophages/monocytes than in platelets, and that

macrophages/monocytes accumulated mostly in the injured area but not platelets. Thus, the latter

explanation seems less persuasive. A more detailed investigation will be required to solve this issue

in the future.

In addition to cell proliferation, another important observation in the present study was the

resolution of liver fibrosis after splenectomy. We analyzed the expression of MMP-9, one of the

MMPs responsible for the degradation of the hepatic extracellular matrix [27]. The protein

expression of MMP-9 quickly increased after splenectomy and lasted until day 28. Additionally,

24

MMP-9 protein was produced by Ly-6G-positive neutrophils infiltrated into the liver, but not by

accumulated macrophages/monocytes. This phenomenon was similar to that observed in a previous

report concerning the macrophage-infusion experiment mentioned above [18, 27], implying the

important role of macrophage-mediated neutrophil recruitment in fibrosis resolution. Scar-associated

macrophages producing CXCL9 has been recently reported to contribute to the fibrosis resolution

process, expressing MMP-13 [28]. Meanwhile, neutrophils, but not macrophages, were positive for

CXCL9 in our model, and produced significant amount of MMP-9 after splenectomy. This

discrepancy is possibly derived from the difference in the animal models.

In summary, we newly detected macrophage/monocyte accumulation in the murine fibrotic liver

with hypersplenism after splenectomy, and these cells participated in the activation of the canonical

Wnt signaling pathway, which is a key component of tissue homeostasis. As a result, CTNNB1

accumulated in the neighboring hepatic progenitor-like cells. Additionally, the number of

Ki67-positive proliferating cells increased, and some of the cells were positive for EpCAM,

suggesting the important roles of progenitor-like cells in the regeneration process after splenectomy.

This mechanism, in addition to the resolution of fibrosis, possibly contributes to the ameliorating

liver function after splenectomy in cirrhotic patients with hypersplenism. It remains unclear whether

advanced liver fibrosis and portal hypertension completely disappears after radical eradication of

hepatitis viruses B or C. In such a situation, splenectomy might become a clinical choice if the

25

surgical procedure is minimally invasive and safe.

26

Acknowledgement

The authors thank Miss Keiko Mitani for her technical assistance in immunohistochemistry.

27

References

[1.]

Sugawara Y, Yamamoto J, Shimada K, Yamasaki S, Kosuge T, Takayama T, et al.

Splenectomy in patients with hepatocellular carcinoma and hypersplenism. J Am Coll Surg

2000; 190:446-450.

[2.]

Tomikawa M, Hashizume M, Akahoshi T, Shimabukuro R, Gotoh N, Ohta M, et al. Effects

of splenectomy on liver volume and prognosis of cirrhosis in patients with esophageal

varices. J Gastroenterol Hepatol 2002; 17:77-80.

[3.]

Elsebae MM, Abu-Zekri NB. A study of the effect of splenectomy on hepatic functional

reserve and structural damage in patients with chronic hepatitis C virus infection by

non-invasive serum markers. A prospective study. Int J Surg 2008; 6:362-366.

[4.]

Imura S, Shimada M, Utsunomiya T, Morine Y, Ikemoto T, Mori H, et al. Impact of

splenectomy in patients with liver cirrhosis: Results from 18 patients in a single center

experience. Hepatol Res 2010; 40:894-900.

[5.]

Ushitora Y, Tashiro H, Takahashi S, Amano H, Oshita A, Kobayashi T, et al. Splenectomy in

chronic hepatic disorders: portal vein thrombosis and improvement of liver function. Dig

Surg 2011; 28:9-14.

[6.]

Nomura Y, Kage M, Ogata T, Kondou R, Kinoshita H, Ohshima K, et al. Influence of

splenectomy in patients with liver cirrhosis and hypersplenism. Hepatol Res 2013.

28

[7.]

Watanabe M, Murata S, Hashimoto I, Nakano Y, Ikeda O, Aoyagi Y, et al. Platelets

contribute to the reduction of liver fibrosis in mice. J Gastroenterol Hepatol 2009; 24:78-89.

[8.]

Kodama T, Takehara T, Hikita H, Shimizu S, Li W, Miyagi T, et al. Thrombocytopenia

exacerbates

cholestasis-induced

liver

fibrosis

in

mice.

Gastroenterology

2010;

138:2487-2498, 2498 e2481-2487.

[9.]

Ramachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, et al. Differential

Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the

regression of murine liver fibrosis. Proc Natl Acad Sci U S A 2012; 109:E3186-3195.

[10.]

Goossens PL, Jouin H, Marchal G, Milon G. Isolation and flow cytometric analysis of the

free lymphomyeloid cells present in murine liver. J Immunol Methods 1990; 132:137-144.

[11.]

Asano Y, Iimuro Y, Son G, Hirano T, Fujimoto J. Hepatocyte growth factor promotes

remodeling of murine liver fibrosis, accelerating recruitment of bone marrow-derived cells

into the liver. Hepatol Res 2007; 37:1080-1094.

[12.]

Salguero Palacios R, Roderfeld M, Hemmann S, Rath T, Atanasova S, Tschuschner A, et al.

Activation of hepatic stellate cells is associated with cytokine expression in

thioacetamide-induced hepatic fibrosis in mice. Lab Invest 2008; 88:1192-1203.

[13.]

Lalor PF, Shields P, Grant A, Adams DH. Recruitment of lymphocytes to the human liver.

Immunol Cell Biol 2002; 80:52-64.

29

[14.]

Greenbaum LE, Wells RG. The role of stem cells in liver repair and fibrosis. Int J Biochem

Cell Biol 2011; 43:222-229.

[15.]

Williams MJ, Clouston AD, Forbes SJ. Links between hepatic fibrosis, ductular reaction,

and progenitor cell expansion. Gastroenterology 2014; 146:349-356.

[16.]

Boulter L, Govaere O, Bird TG, Radulescu S, Ramachandran P, Pellicoro A, et al.

Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in

chronic liver disease. Nat Med 2012; 18:572-579.

[17.]

Takahashi K, Murata S, Fukunaga K, Ohkohchi N. Human platelets inhibit liver fibrosis in

severe combined immunodeficiency mice. World J Gastroenterol 2013; 19:5250-5260.

[18.]

Thomas JA, Pope C, Wojtacha D, Robson AJ, Gordon-Walker TT, Hartland S, et al.

Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis,

regeneration, and function. Hepatology 2011; 53:2003-2015.

[19.]

Lin SL, Castano AP, Nowlin BT, Lupher ML, Jr., Duffield JS. Bone marrow Ly6Chigh

monocytes are selectively recruited to injured kidney and differentiate into functionally

distinct populations. J Immunol 2009; 183:6733-6743.

[20.]

Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, et al.

The healing myocardium sequentially mobilizes two monocyte subsets with divergent and

complementary functions. J Exp Med 2007; 204:3037-3047.

30

[21.]

Karlmark KR, Weiskirchen R, Zimmermann HW, Gassler N, Ginhoux F, Weber C, et al.

Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes

hepatic fibrosis. Hepatology 2009; 50:261-274.

[22.]

Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006; 127:469-480.

[23.]

Okabe M, Tsukahara Y, Tanaka M, Suzuki K, Saito S, Kamiya Y, et al. Potential hepatic

stem cells reside in EpCAM+ cells of normal and injured mouse liver. Development 2009;

136:1951-1960.

[24.]

McBride JA, Dacie JV, Shapley R. The effect of splenectomy on the leucocyte count. Br J

Haematol 1968; 14:225-231.

[25.]

Durig M, Landmann RM, Harder F. Lymphocyte subsets in human peripheral blood after

splenectomy and autotransplantation of splenic tissue. J Lab Clin Med 1984; 104:110-115.

[26.]

Ferrante A, Drew PA, Kiroff GK, Zola H. Peripheral blood leucocyte subpopulations in

patients splenectomized for trauma. Clin Exp Immunol 1987; 70:158-163.

[27.]

Roderfeld M, Weiskirchen R, Wagner S, Berres ML, Henkel C, Grotzinger J, et al.

Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice. FASEB J

2006; 20:444-454.

[28.]

Yang L, Kwon J, Popov Y, Gajdos GB, Ordog T, Brekken RA, et al. Vascular endothelial

growth factor promotes fibrosis resolution and repair in mice. Gastroenterology 2014;

31

146:1339-1350 e1331.

32

Figure legends Figure 1. Splenectomy attenuated liver fibrosis, thrombocytopenia, and leukocytopenia. A:

Sirius-red staining of the liver tissues on days 1, 7 and 28 are shown. The original magnification was

x40.

B: Semi-quantitative analysis of the Sirius-red- or α-SMA-positive area in the liver is

summarized. C: The hydoxyproline contents in the liver (µg/g) on days 7 and 28 are shown. Perioperative changes in platelets (Plt), white blood cells (WBCs), serum total bilirubin (T.Bil), or serum albumin (s-Alb) are summarized in D. White or black bars represent the sham-operated or

splenectomy-performed mice, respectively. * P < 0.05, ** P< 0.01 compared with mice treated with

thiocetamide (TAA) for 32 weeks (TAA32W). Each experimental group contains 6-10 animals. Figure 2.

Flow cytometry analysis of peripheral or hepatic leukocytes revealed the increased

number of F4/80-positeive cells. A: Flow cytometry analysis of peripheral white blood cells. Red

circles indicate CD3-netative, CD220-negative, and F4/80-positive cells, and the percentages of these cells are summarized in the bar graphs in the right side. B: Flow cytometry analysis of hepatic

white blood cells. The percentages of CD3-negative, CD220-negative, and F4/80-positive cells are

similarly summarized. The percentages of T, B cells, or other leukocytes in peripheral and hepatic white blood cells are summarized in C. * P < 0.05 compared with sham-operated mice. Each

experimental group contains 4 animals. Figure 3. Immunohistochemical analysis of the liver indicated significant accumulation of

33

F4/80-positive macrophages/monocytes and platelets in the liver after splenectomy. A:

Immunostaining for F4/80 of the liver before and after splenectomy is shown. The original

magnifications are x 200 or x 400, as indicated. Results of the semi-quantitative image analysis of the F4/80-positive area (%) are summarized in B. Each experimental group contains 6-10 animals. White or black bars represent the sham-operated or splenectomy-performed mice, respectively. C:

Immunostaining for thrombocytes of the liver before and after splenectomy is shown. The original

magnifications are x 200 or x 400, as indicated. Results of the semi-quantitative image analysis of the thrombocyte area (%) are summarized in D. Each experimental group contains 6-10 animals.

White or black bars represent the sham-operated or splenectomy-performed mice, respectively. * P <

0.05, ** P< 0.01 compared with mice treated with thiocetamide (TAA) for 32 weeks (TAA32W). Figure 4. Immunohistochemical analysis of progenitor-like cells in the liver revealed the increased

number of progenitor-like cells after TAA treatment and their gradual disappearance after splenectomy. A: Immunostaining of the livers for CK-19, EpCAM, or CD-133 before and after splenectomy is shown. The original magnification was x 100. B: Semi-quantitative analysis of each

immunostainig (%) is summarized in B. Each experimental group contains 6-10 animals. White or

black bars represent the sham-operated or splenectomy-performed mice, respectively. * P < 0.05, **

P< 0.01 compared with mice treated with thiocetamide (TAA) for 32 weeks (TAA32W). ## P < 0.01

compared with sham-operated mice.

34

Figure 5. Double fluorescence immunostaining revealed the close interaction between

F4/80-positive macrophages/monocytes and CK-19-positive progenitor-like cells in the liver.

Representative figures in sham-operated control (Fig. 5A left) and splenectomy-performed mice (Fig.

5A right) on day 7 are shown. The original magnification was x 400. Double fluorescence

immunostaining for Ly-6C and F4/80 is shown in Fig. 5B. Only small part of the accumulated

macrophages/monocytes expressed Ly-6C at day 7 after splenectomy (Fig. 5B middle), while a large

part

of

the

splenic

F4/80-positive

macrophages

expressed

Ly-6C

(Fig.

5B

right).

Macrophages/monocytes detected in the sham-operated liver at day 7 did not express Ly-6C (Fig. 5B

left). The original magnification was x 400. Figure 6. Canonical Wnt signaling was up-regulated in the liver after splenectomy. A: Quantitative

RT-PCR for genes included in the Wnt signaling pathway revealed the up-regulated mRNA

expression of Wnt2, 9b, and 16 after splenectomy. Additionally, the mRNA expression of a

non-canonical Wnt molecule, Wnt4, or negative regulators of Wnt signaling, including Secreted

frizzled-related protein (Sfrp) 1 and Dickkopf (Dkk) 2, was decreased. White or black bars represent

the sham-operated or splenectomy-performed mice, respectively. Black bars represent mean + SD of three distinct experiments. B: Quantitative RT-PCR for genes included in the Notch signaling

pathway revealed the down-regulated mRNA expression of Dll4, Jag2, Notch3, Notch4, Hes5, and Hey1 after splenectomy. C: Double fluorescence immunostaining for Wnt2 and F4/80 of the liver in

35

sham-operated or splenectomy-performed mice is shown. Splenectomy increased the protein

expression of Wnt2, and most of the F4/80-positive cells participated in the expression. The original

magnification was x 400 or x 1200, as indicated. The area outlined with a square is enlarged on the right side. D: Double fluorescence immunostaining for CK-19 and β-catenin indicated that a significant amount of β-catenin accumulated in the CK-19-positive cells after splenectomy. The original magnification was x 800. Figure 7. Splenectomy stimulated the proliferation of relatively small hepatocytes. A:

Immunostaining for Ki67 of the liver is shown. The number of Ki67-positive nuclei was markedly

increased in the liver after splenectomy. The original magnification was x 200 or x 400, as indicated.

Perioperative changes in the mean number of Ki67-positive nuclei in high power fields (HPFs x200) are summarized in B. White or black bars represent the sham-operated or splenectomy-performed

mice, respectively. Each experimental group contains 6-10 animals. * P < 0.05 compared with

untreated normal mice. # P < 0.05, ## P < 0.01 compared with mice treated with thiocetamide (TAA) for 32 weeks (TAA32W). C: Double immunostaining for β-catenin and Ki67 indicated that cells with Ki67-positive nuclei merely overlapped with β-catenin-expressing cells. White triangles indicate relatively small hepatocytes with Ki67-positive nuclei. The original magnification was x 400. D: Double immunostaining for EpCAM and Ki67 indicated that some of the cells with

relatively small and round Ki67-positive nuclei expressed EpCAM at day 7 after splenectomy (Fig.

36

7D, white triangle). The area indicated by rectangle in x 400 (left) was enlarged in X 1000 (right).

The original magnification was x 400 or x100, as indicated. Figure 8.

Splenectomy up-regulated MMP-9 protein expression in the liver. A: Immunostainings

for MMP-9 of the liver are shown. The original magnification was x 200 or x 400, as indicated. Semi-quantitative image analysis of MMP-9 protein expression (%) is summarized in B. White or

black bars represent the sham-operated or splenectomy-performed mice, respectively. Each group

contains 6-10 animals. ** P< 0.01 compared with mice treated with thiocetamide (TAA) for 32

weeks (TAA32W). ## P < 0.01 compared with sham-operated mice. Double fluorescence

immunostaining for MMP-9 and F4/80 (C) or MMP-9 and Ly-6G (D) revealed that Ly-6G-positive

neutrophils participated in the MMP-9 expression after splenectomy. The original magnification was

x 400.

37

Percentage of liver fibrosis area αSMA % % Sirius-red 30

14

＊

＊

12

Sham op

25

10

20

8

X40

15

6

＊

＊

4

splenectomy

10 ＊

2

0

×103/㎜3

1200

250

1000 200 ＊＊

＊＊

30000 ＊＊

＊

＊＊

0.07

20000

0.06

800

50

200

0

0 Day7

day 28

day 7

day 1

3.5 3 ＊＊

2.5 ＊＊

0.05

＊

0.04

1

0.02 5000

0.5

0.01

0

0

0

2 1.5

0.03

10000

400

＊

0.09

25000

600

100

g/dl

4

0.08

15000

150

0.1 ＊＊

＊＊

1400

300

㎎/dl

/㎕

35000

1600

350

hydroxyplorin

TAA 32w

D

μg/g

＊

5

0

C

＊<0.05

day28

B

Day28

day7

Day7

day1

Day1

TAA32W

A

Day28

Pｌｔ

WBC

T.Bil

S-Alb

Yada et al. Figure 1

A

splenectomy

sham

%

B

blood

splenectomy

sham

%

＊

12

30

10

25

8

20

6

15

4

10

2

5

0

0

% T cell - blood

25 20 15 10

5 0

% T cell - liver

25 20

15 10 5 0

%

B cell - blood

＊

＊<0.05

＊<0.05

C

liver

% B cell - liver

% Other cells - blood

% Other cells - liver

50

50

50

45

45

45

40

40

40

35

35

35

30

30

30

25

25

25

20

20

20

15

15

15

15

10

10

10

10

5

5

5

0

0

0

35 30 25 20

5 0

Yada et al. Figure 2

A

B Day1

TAA32W

＊<0.05 ＊＊<0.01

%

4.5

TAA32W

＊＊

4 3.5

＊ ＊＊

3 X200 Day7

X400

X200

2

Day7

Day28

2.5

1.5 1 0.5 X200

X400

X200

C Day1

TAA32W

0 normal TAA32W Day1

Day7

D 3%

TAA32W

＊<0.05

＊

2.5

＊

2 X200 Day7

X200

X400

Day28

1.5

Day7

Day28

1 0.5

X200

X200

X400

0 normal TAA32W Day1

Day7

Day28

Yada et al. Figure 3

A

normal

TAA32W

Day1

Day7

Day28

CK19

x100

x100

x100

x100

x100

x100

x100

x100

x100

x100

x100

x100

x100

x100

x100

EpCAM

CD133

%

6 ＊

5 ##

##

＊＊

＊＊

4 ##

3

＊＊

2

## ＊＊

1

EpCAM

Day28

Day7

Day1

Day28

0 TAA32W

CK-19

＊＊

7

＊

Day7

Day7

Day1

＊＊

##

Day28

##

%

Day1

＊

＊

9 8 7 6 5 4 3 2 1 0

TAA32W

%

TAA32W

B

18 16 14 12 10 8 6 4 2 0

* <0.05

＊＊<0.01

## ＜0.01

CD-133

Yada et al. Figure 4

A

CK19

F4/80

CK19

F4/80

DAPI

MERGE

DAPI

MERGE

X400

TAA32w + sham (day 7)

B

Ly6C

DAPI

X400

TAA32w + splenectomy (day 7) F4/80

MERGE

X400

TAA32w + sham; liver (day 7)

Ly6C

F4/80

Ly6C

F4/80

DAPI

MERGE

DAPI

MERGE

X400

TAA32w + splenectomy; liver (day 7)

X400

TAA32W; spleen

Yada et al. Figure 5

A

C Wnt9b 30

Wnt4

TAA32w + sham (day 7)

TAA32w + splenectomy (day 7)

Wnt2

F4/80

Wnt2

F4/80

DAPI

MERGE

DAPI

MERGE

1.0

20 0.5 10 0.0

0

Wnt2 20 16 12 8 4 0

Sfrp1 1.0

0.5

X400

X400

X1200

0.0

Wnt16 15

MERGE

Dkk2 1.0

D

TAA32w + sham (day 7) CK19

TAA32w + splenectomy (day 7) CTNNB1

CK19

CTNNB1

10 0.5 5 0

0.0

B 1

DAPI

MERGE

DAPI

MERGE

0.5

0

X800

X800

Yada et al. Figure 6

A

B TAA32W

normal

35

Day1

* ##

30

X200

X200

Day7

Day28

X200 Day7

/ x200 HPFs

25

*#

20

*

*

* *

*

15 10 5 0

X200

C

X200

D

Sham Day7

X400

Sham Day7

CTNNB1 (orange), Ki67(brown)

Day28 # <0.05

## ＜0.01

Sham Day7

Ｘ1000

Ｘ400 Splenectomy Day7

X400

Day7

* <0.05

X400 Splenectomy Day7

normal TAA32W Day1

Splenectomy Day7

Ｘ400 EpCAM (orange), Ki67(brown)

Ｘ1000

Yada et al. Figure 7

A

B

MMP-9 TAA32W

normal

Day1

MMP-9

% 2

＊＊ ## ＊＊ ##

1.5

X200

X200

Day7

X200

Day2 8

＊＊ ##

1

Day7 0.5

0 X200

X200

C

X400

normal TAA32W Day1

Day28

＊＊<0.01, ## <0.01

MMP-9

F4/80

DAPI

MERGE

D

MMP-9

Ly-6G

DAPI

MERGE

X400

TAA32w + splenectomy (day 7)

Day7

X400

TAA32w + splenectomy (day 7)

Yada et al. Figure 8