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Interleukin-17A plays a pivotal role after partial hepatectomy

in mice

Shinji Furuya, MD, Hiroshi Kono, MD, PhD,

*

Michio Hara, MD, Kazuyoshi Hirayama, MD,

Masato Tsuchiya, MD, PhD, and Hideki Fujii, MD, PhD

First Department of Surgery, University of Yamanashi, Yamanashi, Japan

a r t i c l e i n f o

Article history:

Received 29 October 2012 Received in revised form 1 February 2013

Accepted 12 March 2013 Available online 3 April 2013 Keywords:

Interleukin-17A

Interleukin-17A knockout mouse Cytokine Kupffer cell Spleen Partial hepatectomy Splenectomy a b s t r a c t

Background: Liver regeneration after partial hepatectomy (PH) is regulated by tumor necrosis factor (TNF)-a derived from the Kupffer cell. Furthermore, it was reported from our laboratory that interleukin (IL)-17A enhances the production of TNF-a by the Kupffer cell, suggesting that IL-17A may play a role in liver regeneration.

Objective: The purpose was to determine the role of IL-17A and the spleen in liver regen-eration after PH.

Methods: Two mouse models including the wild-type (WT) mice or the IL-17A knockout (KO) mice underwent PH. Animals were killed at the designated time points; liver tissues were harvested for further investigation. Proliferation of hepatocytes was evaluated. Further-more, the messenger RNA and protein expression of TNF-a and IL-6 were measured in the liver. In another set of experiments, the two animal models underwent splenectomy before PH. In an in vitro study, CD4-positive lymphocytes in the spleen were isolated from mice, and the number of IL-17Aepositive cells was investigated.

Results: Liver regeneration was significantly impaired in the KO mice compared with the WT mice. This was associated with suppression of cell proliferation assessed by cell proliferation markers in the KO mice. In the WT mice that underwent splenectomy, liver regeneration was significantly delayed compared with animals without splenectomy. In contrast, splenectomy did not affect liver regeneration in the KO mice. IL-17Aepositive lymphocytes increased significantly in the spleen in the WT mice after PH.

Conclusions: These results indicate that IL-17A derived from CD4-positive lymphocytes in the spleen is a key regulator in liver regeneration after PH.

ª 2013 Elsevier Inc. All rights reserved.

1.

Introduction

Liver regeneration is a physiological phenomenon of recovery from loss of liver volume to compensate for impaired hepatic function after partial hepatectomy (PH), liver injury, viral hepatitis, or ischemic damage[1e5]. Because insufficient liver regeneration is potentially fatal for these patients, investiga-tion of the physiological mechanism of liver regenerainvestiga-tion

could lead to decreased morbidity and mortality, leading to increased opportunities for therapy against critical liver disease.

The hepatic macrophage, the Kupffer cell, is known to produce a variety of growth and immunomodulating medi-ators that have stimulatory and inhibitory effects on liver regeneration[6]. It was reported that hepatic macrophages are the major source of tumor necrosis factor (TNF)-a and

* Corresponding author. First Department of Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan. Tel.:þ81 55 273 7390; fax: þ81 55 273 6751.

E-mail address:[email protected](H. Kono).

Available online atwww.sciencedirect.com

journal homepage: www.J ournalofS urgicalR esearch.com

0022-4804/$e see front matter ª 2013 Elsevier Inc. All rights reserved.

interleukin (IL)-6 and depletion of hepatic macrophages impaired liver regeneration after PH[7]. Because TNF-a and IL-6 have been implicated as important factors for initiating the regenerative responses after PH, they have been postu-lated to play a key role in liver regeneration. The cytokine IL-17A may also affect hepatic regeneration. IL-17A was originally described and cloned by Rouvier et al. [8] and named cytotoxic T lymphocyteeassociated serine esterase-8 (CTLA8). It was reported from this laboratory that IL-17A increases TNF-a production by isolated Kupffer cells in vitro [1]. These results support the hypothesis that IL-17A may be involved in liver regeneration after PH; however, the role of IL-17A in liver regeneration has not been fully eluci-dated. Therefore, the specific purpose of the present study was to investigate the role of IL-17A in liver regeneration after PH.

2.

Materials and methods

2.1. Animals

Male wild-type (WT) mice (C57BL/6, 20e25 g, 8e9 wk of age, obtained from Jackson Laboratories, Bar Harbor, ME) and male IL-17A knockout (KO) mice (backcrossed onto C57BL/6) were housed in a clean, temperature-controlled environment with a 12-h lightedark cycle and were given free access to regular laboratory chow diet and water for several days. All animals received humane care, and the study protocols were approved by the Committee of Laboratory Animals at University of Yamanashi according to institutional guidelines.

2.2. Operative procedure

Mice underwent 70% liver resection under diethyl ether anesthesia (n¼ 8 at each time point) [9]. After PH, animals were killed at designated time points, and remnant liver tissues were collected to assess the regenerative response by the methods outlined below. The remnant liver was weighed, and the relative liver-weight was calculated as the actual liver-weight/body-weight.

To further clarify the role of the spleen as a source of IL-17A in liver regeneration, WT and KO mice underwent splenec-tomy or sham operation 7 d before PH.

2.3. Collection of samples

Blood samples were collected via the portal vein at designated times after PH, centrifuged, and stored at80_{C until assay.}

Tissue samples were also collected at designated times after PH and were stored at 80_{C for further analysis. Samples}

were fixed in formalin or acetone, embedded in paraffin, and serially sectioned.

2.4. Immunohistochemistry for Ki-67 and 5-bromo-2’-deoxyuridine

The liver was removed, sliced, and fixed in 10% neutral buff-ered formalin, processed routinely, embedded in paraffin, and sectioned at 5 mm. Liver sections were denatured for 45 min in

boiling 10 mmol citric acid (pH 9.0). Endogenous peroxidases were quenched by incubation at room temperature in 0.3% H2O2followed by rinsing in phosphate-buffered saline. Mouse

anti-Ki-67 primary antibody (DAKO, North America, CA) in 0.1% bovine serum albumin was added to the tissue sections at a 1:50 dilution. Sections were incubated overnight at 4C and then rinsed in phosphate-buffered saline. Avidin and biotin blocking steps were performed using a Vectastain avidinebiotin blocking kit (Vector Laboratories, Burlingame, CA) in accordance with the manufacturer’s instructions. The labeling index was expressed as the number of Ki-67-positive nuclei/1000 nuclei. Results were expressed as means standard errors of random five sections per mouse (eight mice per group).

Mice received an intraperitoneal injection of 100 mg of 5-bromo-2’-deoxyuridine (BrdU; Invitrogen, Carlsbad, CA) 2 h before killing. The liver was removed, sliced, and fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin, and sectioned at 5 mm. Staining steps were per-formed using an Invitrogen BrdU Staining Kit (Invitrogen) in accordance with the manufacturer’s instructions. The labeling index was expressed as the number of BrdU-positive nuclei/1000 nuclei.

2.5. Immunohistochemistry for IL-17A

Immunohistochemical staining for IL-17A in the liver, spleen, and small intestine was performed on frozen sections fixed in acetone for 10 min, followed by postfixation immersion for 15 min at room temperature in 0.3% H2O2methanol to block

endogenous peroxidase, and incubation with a 1:50 dilution of rat anti-mouse IL-17 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

2.6. Western blotting analysis

Whole liver protein extract (50 mg) was separated by 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The following antibodies were used as primary antibodies: b-actin (Santa Cruz Biotechnology) and Cyclin D1 (Santa Cruz Biotech-nology). Densitometric analysis of the Cyclin D1 protein expression was used as sample signals at 72 h (n¼ 4 at each group). Densitometric evaluation was done using Scion Image for Windows program, as distributed by the Scion Corporation (Frederick, MD).

2.7. RNA isolation and real-time reverse transcriptionepolymerase chain reaction

About 30 mg of frozen liver tissue was harvested from each animal and homogenized in 600 mL RLT buffer (Qiagen, Valencia, CA) containing 1% b-mercaptoethanol. Lysate was centrifuged for 3 min at 13,000 rpm. RNA was isolated using an RNeasy kit (Qiagen) as per the manufacturer’s protocol. Total RNA (10 mg) was reverse transcribed using random primers and the high capacity complementary DNA (cDNA) archive kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. The resulting cDNA was stored at20_C.

The following TaqMan (Applied Biosystems) gene ex-pression assays were used for quantitative real-time poly-merase chain reaction (PCR): TNF-a (Mm00443258_ml), IL-6 (Mm00446190_m1), and 18s rRNA (4310893E). Reactions were performed in a 96-well assay format. Each plate contained one experimental gene and a housekeeping gene. Each reac-tion contained 2 mL of cDNA template, 18.0 mL of RNase-free H2O, 2.5 mL of TaqMan gene expression assay primer, and

25 mL of TaqMan universal PCR master mix (Applied Bio-systems). Reactions were processed for 1 cycle of 2 min at 50C and 10 min at 95C, followed by 40 cycles of 15 s at 95C and 1 min at 60C on the AB 7500 Real-Time PCR System (Applied Biosystems). The cycle threshold (Ct) for each sample was determined from the linear region of the amplification plot. The DCt value for TNF-a and IL-6 relative to the control gene 18s rRNA was determined. The DDCt values were calcu-lated using treated group means relative to control group means. Fold change data were calculated from the DDCt values.

2.8. Measurement of serum cytokines

Blood samples were collected via the portal vein at designated time points after PH (n¼ 8 for each time point). The samples were centrifuged at 3000 rpm for 10 min at 4C, and serum was stored at80_{C until the assays. Serum IL-17A levels were}

measured using enzyme-linked immunosorbent assay kits (eBioscience, Inc., San Diego, CA).

2.9. Isolation of CD4þlymphocytes in the liver, spleen, and small intestine

Seventy-two hours after PH, liver and spleen tissues were pressed through a 200-gauge stainless steel wire mesh and collected in media[10]. After all macroscopic Peyer Patches were removed, small intestine tissues were opened and cut laterally into small pieces. Fragments were then transferred to flasks holding 15 mL HBSS-containing collagenase D (90 U/ mL; Sigma, St Louis, MO) and were incubated at 37C in an orbital incubator for 25 min. The resultant soup was pressed through a 200-gauge stainless steel wire mesh and collected in media [11]. Next, the dispersed tissue suspension was transferred to a 50 mL conical centrifuge tube and centrifuged at 300g for 10 min. The pellet was resuspended in media, carefully layered on Histopaque 1083 (Sigma), and centrifuged at 400g for 30 min at room temperature. Mononuclear cells at and above the opaque interface were collected and centri-fuged at 300g for 10 min at room temperature. Cell viability was >95% in all cases, as determined by trypan blue exclusion.

2.10. Measurement of intracellular cytokine staining for IL-17A in CD4þlymphocytes and gd-T cells

Detection of IL-17A in CD4þlymphocytes and gd-T cells was determined by intracellular cytokine staining [12]. Before fixation and permeabilization, cells were stained with allophycocyanin-conjugated anti-CD4 antibodies (eBio-science, Inc.) and allophycocyanin-conjugated anti-gd-TCR antibodies (eBioscience, Inc.) to detect cell surface antigens.

Afterward, cells were fixed and permeabilized with the Fixation/Permeabilization buffer (eBioscience, Inc.) for 30 min at 4C before intracellular staining. Cells were then stained with allophycocyanin-conjugated IL-17A anti-bodies (eBioscience, Inc.). Flow cytometric analysis was performed with a Cytomics FC500 (Beckman Coulter, Full-erton, CA), and the cells were analyzed using CellQuest Pro software (BD, Biosciences, San Jose, CA).

2.11. Isolation of Kupffer cells and assessment of TNF-a production

Kupffer cells were isolated as detailed elsewhere with minor modifications [13]. Briefly, mice were anesthetized with sodium pentobarbital (80 mg/kg body weight, intraperitone-ally), the abdomen was opened, and the portal vein was cannulated. The liver was perfused in situ for 5 min with Ca2þ_/Mg2þ_{-free liver perfusion medium (LPM-1:}

8000 mg/L NaCl, 400 mg/L KCl, 88.17 mg/L NaH2PO4-2H2O,

120.45 mg/L Na2HPO4, 2380 mg/L HEPES, 350 mg/L NaHCO3,

190 mg/L ethylenediaminetetraacetic acid, 900 mg/L glucose, 6 mg/L phenol red; pH 7.4, 37C) and was then perfused with complete liver perfusion medium (LPM-2: same as LPM-1 except without ethylenediaminetetraacetic acid and glucose, but with 560 mg/L CaCl2-2H2O; pH 7.4, 37C)

con-taining 0.06% collagenase type IV (Sigma) for an additional 15 min. After perfusion, the liver was removed, cut into small pieces, and homogenized. After passing through a gauze filter (mesh size w60 mm), cells were washed twice with warm Gey’s balanced salt solution (370 mg/L KCl, 210 mg/L MgCl2

-6H2O, 70 mg/L MgSO4-7H2O, 150 mg/L NaH2PO4-2H2O, 30 mg/

L KH2PO4, 1090 mg/L glucose, 227 mg/L NaHCO3, 225 mg/L

CaCl2-2H2O, 6 mg/L phenol red, 8000 mg/L NaCl, 100 U/L

streptomycin, and 1  105 _{U/L penicillin G; pH 7.4) and}

centrifuged over a 16% (wt/vol) Nycodenz (Axis-Shields, Oslo, Norway) gradient for 20 min at 1900g at 4C. Kupffer cells were collected from under the interface, washed with Gey’s balanced salt solution, and resuspended at a concentration of 1 106cells/mL in Dulbecco’s modified Eagle medium media (Invitrogen). Isolated Kupffer cells were seeded (1 106_cells

per well) in 24-well dishes and cultured in Dulbecco’s modi-fied Eagle medium media for 12 h. After changing the media to remove the nonadherent cells, cultures were maintained for an additional 24 h, and the media was collected and stored at80C. Viability of the cells was confirmed by trypan blue exclusion and was >95% in all experiments. Five to 20 million Kupffer cells were obtained from each mouse. TNF-a production from Kupffer cells was assayed using enzyme-linked immunosorbent assay kits according to the manufacturer’s protocol (Biosource, Camarillo, CA). In all experiments, Kupffer cells were isolated from four different mice.

2.12. Statistical analysis

Data were expressed as mean  standard error. Statistical differences between mean values were analyzed by analysis of variance with Bonferroni post hoc test. A P value<0.05 was considered significant.

3.

Results

3.1. Evaluation of liver regeneration after PH

To evaluate liver regeneration after PH, the liver-weight/ body-weight ratio was assessed (Fig. 1). The liver-weight/ body-weight ratio gradually recovered to the basal preopera-tive level in the WT mice. Although the ratio also gradually recovered to the basal level in the KO mice, it was significantly lower compared with the WT at 72 h and 5 d after PH, sug-gesting that hepatic regeneration was impaired in the KO mice.

In the WT animals that underwent splenectomy, the liver-weight/body-weight ratio was significantly lower compared with animals that underwent the sham operation at 72 h after PH. In contrast, in the KO mice that underwent splenectomy, the ratio was not different compared with animals that underwent the sham operation.

3.2. Evaluation of proliferation of the hepatocyte after PH

To evaluate the proliferation of the hepatocyte, immunohis-tochemistry for Ki-67 and BrdU was performed (Fig. 2A). The number of Ki-67epositive hepatocytes increased markedly at 72 h after PH in the WT mice (Fig. 2B). In contrast, the value was significantly lower in the KO mice compared with the WT mice at 72 h and 5 d after PH. In the WT animals that under-went splenectomy, the number of Ki-67epositive hepatocytes was significantly lower compared with animals that under-went the sham operation at 72 h after PH. In contrast, there were no differences in the number of KO mice with or without splenectomy.

The number of BrdU-positive hepatocytes increased markedly at 72 h after PH in the WT mice (Fig. 2C). However, the number was significantly lower in the KO mice compared with the WT mice at 72 h after PH. In the WT animals that underwent splenectomy, the number of BrdU-positive hepa-tocytes was significantly lower compared with the animals that underwent the sham operation at 72 h after PH. However, there were no differences in the number of KO mice with or without splenectomy.

Western blot analysis demonstrated that expression of Cyclin D1 signaling was detected at 72 h after PH in the WT mice (Fig. 3). In contrast, it was significantly reduced in the KO mice compared with the WT mice. In the WT animals that underwent splenectomy, Cyclin D1 signaling was reduced compared with the animals that underwent the sham operation at 72 h after PH. In contrast, there were no differ-ences in its expression in the KO mice with or without splenectomy.

3.3. Evaluation of messenger RNA expression of TNF-a and IL-6 in the liver

The messenger RNA (mRNA) expression of TNF-a increased markedly at 2 h after PH in the WT mice (Fig. 4A). In contrast, the expression was significantly lower in the KO mice compared with the WT mice at 1 and 2 h after PH. In the WT

animals that underwent splenectomy, the mRNA expression of TNF-a was significantly lower compared with the animals that underwent the sham operation at 2 h after PH. In contrast, there were no differences in its expression in the KO mice with or without splenectomy.

The mRNA expression of IL-6 increased markedly at 2 h after PH in the WT mice (Fig. 4B). Although the expression was higher in the KO mice, there were no significant differences between the WT mice and the KO mice. In the two animal models that underwent splenectomy, the mRNA expression of IL-6 was not significantly different compared with the animals that underwent the sham operation.

3.4. Serum IL-17A level after PH

Serum IL-17A was not detected as expected in the KO animals with sham operation or splenectomy (data not shown). Serum IL-17A levels peaked at 24 h after PH in the WT mice that underwent the sham operation (Fig. 5). In contrast, levels were significantly lower in the WT mice that underwent splenec-tomy compared with those of the WT mice with the sham operation at 24 and 72 h after PH.

3.5. Investigation of the population and distribution of IL-17Aeproducing cells in the liver, spleen, and small intestine

To evaluate the population and distribution of IL-17Ae producing cells, immunohistochemistry for IL-17A was per-formed in the liver, spleen, and small intestine (Fig. 6). In the Fig. 1e The liver-weight/body-weight ratio after PH. The liver-weight/body-weight ratio after PH was determined as described in detail in the Materials and methods (n [ 8 in each group). Open circle, WT mice; closed circle, KO mice; closed triangle, WT mice that underwent splenectomy; and open square, KO mice that underwent splenectomy. Data represent mean ± standard error. *P < 0.01 compared with KO mice; #P < 0.01 compared with WT mice that

underwent splenectomy; and **P < 0.01 compared with KO mice that underwent splenectomy by analysis of variance with Bonferronipost hoc test.

WT and the KO mice, IL-17Aeproducing cells were not detected in the liver and small intestine at 72 h after PH (data not shown). In the spleen, IL-17Aeproducing cells were

markedly detected at 72 h after PH in the WT mice (Fig. 6A and B). In contrast, they were not detected in the spleen from the KO mice (Fig. 6C and D).

A

C

B

Fig. 2e Immunohistochemical staining for Ki-67 and BrdU, the number of Ki-67epositive hepatocytes and BrdUepositive hepatocytes after PH. (A) Immunohistochemical staining for Ki-67 and BrdU of the liver after PH. Livers were harvested from animals at 72 h after PH (n [ 8 in each group). Representative photomicrographs are shown. Original magnification, 3400. WT (Spx), WT mice that underwent splenectomy and KO (Spx), KO mice that underwent splenectomy. Black arrows showed Ki-67epositive hepatocytes. White arrows showed BrdU-positive hepatocytes. The number of Ki-67epositive hepatocytes and BrdU-positive hepatocytes was evaluated as described in detail in the Materials and methods (n [ 8 in each group). (B) Number of Ki-67epositive hepatocytes after PH and (C) Number of BrdU-positive hepatocytes after PH. Black bars, WT mice; white bars, KO mice; gray bars, WT mice that underwent splenectomy; and plaid bars, KO mice that underwent

splenectomy. Data represent mean ± standard error. #P < 0.05 compared with the WT mice and *P < 0.01 compared with the WT mice by analysis of variance with Bonferronipost hoc test.

3.6. Analysis of IL-17Aeproducing cells by intracellular cytokine staining

Intracellular cytokine staining for IL-17A was performed by flow cytometrical analysis to detect IL-17Aeproducing cells in the liver, spleen, and small intestine. In the WT mice, IL-17Aeproducing cells were no different in the liver and small intestine at 72 h after PH (data not shown). The percentage of IL-17Aepositive cells to CD4þ_{lymphocytes in the spleen was}

2.0% 0.5% before PH and 6.2%  1.1% 72 h after PH (Fig. 7B). In the WT mice, the percentage of IL-17Aepositive cells to CD4þ

lymphocytes was significantly increased in the spleen 72 h after PH compared with the animals pre-PH. In contrast, the percentage of IL-17Aepositive cells to gd-T cells was no different 72 h after PH compared with pre-PH (data not shown).

4.

Discussion

4.1. Role of IL-17A in liver regeneration after PH

IL-17A is a cytokine produced by various cells, such as Th17, gd-T cells, and CD8-positive T cells,[14]which is elevated in inflammatory conditions such as inflammatory myopathies,

rheumatoid arthritis, asthma, ulcerative colitis, and multiple sclerosis[15e18]. However, the role of IL-17A in liver regen-eration is still unclear. TNF-a is known to play a key role in liver regeneration after PH[19]. Importantly, it was reported from this laboratory that IL-17A enhances the production of TNF-a by the Kupffer cell[1]. This result leads to the hypoth-esis that IL-17A could be involved in hepatic regeneration. Indeed, in the present study, the mRNA expression of TNF-a decreased significantly in the KO mice compared with the WT mice after PH (Fig. 4). Furthermore, liver regeneration was significantly impaired in the IL-17A KO mice compared with the WT mice after PH (Figs. 1e3). Thus, IL-17A plays an important role in liver regeneration after PH.

A

B

Fig. 3e Expression of Cyclin D1 in the liver after PH. (A) Expression of Cyclin D1 was evaluated by western blotting analysis as described in detail in the Materials and methods. Representative blots are shown. (B)

Densitometrical analysis of the Cyclin D1 expression. Black bar, WT mice; white bar, KO mice; gray bar, WT mice that underwent splenectomy; and plaid bar, KO mice that underwent splenectomy. Data represent mean ± standard error. *P < 0.05 compared with the WT mice and #P < 0.01 compared with the WT mice by analysis of variance with Bonferronipost hoc test.

Ra ti o T N F -αα m RNA / G AP DH m RNA pre-PH 1h 2h 0.1 1 10

*

*

*

* *

A

B

Fig. 4e mRNA expression of inflammatory cytokines in the liver by real-time reverse transcriptionepolymerase chain reaction. Mice underwent PH with sham operation or splenectomy. Total mRNA was extracted pre-PH and at 1 and 2 h after PH. The mRNA expression of TNF-a and IL-6 in the liver was determined by real-time reverse

transcriptionepolymerase chain reaction as described in detail in the Materials and methods. (A) mRNA expression of TNF-a and (B) mRNA expression of IL-6. Black bars, WT mice; white bars, KO mice; gray bars, WT mice that underwent splenectomy; and plaid bars, KO mice that underwent splenectomy. Data represent mean ± standard error. *P < 0.05 compared with the WT mice by analysis of variance with Bonferronipost hoc test.

IL-6 also plays a key role in liver regeneration after PH[20]. Although the mRNA expression of IL-6 was greater in the KO mice than in the WT mice, there were no significant differ-ences between the two groups. The result is related to the

development of Th17 cells. Both Th17 and T-reg (regulatory T cell) cells can develop from naive CD4þ T cell precursors in the presence of TGF-b[21]. In addition to TGF-b, differentiation to Th17 from these cells also requires IL-6[22]. It was reported that once the T-reg cell differentiation pathway gains ascen-dancy, T-reg cells inhibited the generation of Th17 cells at mucosal surfaces [23]. Based on these results, greater expression of IL-6 in the liver in the KO mice may be due to negative feedback due to lack of IL-17A[24].

4.2. Role of the splenic in liver regeneration after PH

Th17 cells are distributed in several organs in the body, such as the liver, the small intestine, and the spleen[25]. Further-more, in addition to the Th17 cell, IL-17A production is detected in non-Th17 cells, such as gd-T cells, CD8-positive T cells, monocytes, and intestinal epithelial cells [26,27]. Organs-involved production of IL-17A during liver regenera-tion after PH was not clear. It has been reported that sple-nectomy improves liver regeneration after PH [15e18]. The splenectomy affects platelet activation, portal blood flow, and oxygen supply[15,28,29]. Indeed, Ren et al.[15]reported that splenectomy improved liver regeneration in the rat. However, the present study demonstrated that the splenectomy impaired regeneration of healthy normal liver after PH. Indeed, in the WT mice that underwent splenectomy, liver regeneration was significantly impaired compared with animals that underwent the sham operation after PH (Figs. 1e3). In contrast, in the KO mice that underwent splenec-tomy, liver regeneration was not different compared with animals that underwent the sham operation. Importantly, serum IL-17A levels decreased significantly in the WT mice that underwent splenectomy compared with the WT mice that underwent the sham operation after PH (Fig. 5). The

IL -1 7A l e v e l i n p or tal v e in s e ru m ( p g/ m l) 0 10 20 30

Sham Spx Sham Spx Sham Spx

Before-PH 24h 72h

*

*

Fig. 5e Serum IL-17A levels in portal blood. Serum IL-17A level in the portal blood was evaluated as described in detail in the Materials and methods (n [ 8 in each group). Black bars, WT mice and gray bars, WT mice that

underwent splenectomy. Data represent mean ± standard error. *P < 0.05 compared with the WT mice by analysis of variance with Bonferronipost hoc test.

Fig. 6e Immunohistochemical staining for IL-17A in the spleen. Immunohistochemical staining for IL-17A was performed as described in detail in the Materials and methods. Representative photomicrographs are shown. Original magnification, 3400. (A) WT mice pre-PH; (B) WT mice at 72 h after PH; (C) KO mice pre-PH; and (D) KO mice at 72 h after PH.

percentage of IL-17Aepositive cells was significantly increased in CD4þlymphocytes, but not gd-T cells lympho-cytes, in the spleen after PH compared with the animals pre-PH (Fig. 7B). These results suggested that the spleen plays an important role in the liver regeneration after PH by producing IL-17A. Taken together, these results indicate that IL-17A produced by CD4þlymphocytes in the spleen plays a pivotal role in liver regeneration after PH.

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expression in blood and CSF mononuclear cells is augmented in multiple sclerosis. Mult Scler 1999;5:101.

[17] Rosu A, Margaritescu C, Stepan A, Musetescu A, Ene M. IL-17 patterns in synovium, serum and synovial fluid from treatment-naive, early rheumatoid arthritis patients. Rom J Morphol Embryol 2012;53:73.

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72h after PH 5.48 94.52 1.38 98.62 CD4 _CD4 IL -17A SSC(side scatter) Gate Gate

A

B

*

Fig. 7e Intracellular cytokine staining for IL-17A in CD4D_{lymphocytes. (A) Lymphocytes were isolated from the spleen, and}

IL-17Aeproducing CD4D_{lymphocytes were determined by intracellular cytokine staining as described in detail in the}

Materials and methods (n [ 4 in each group). Above, the WT mice pre-PH and below, the WT mice 72 h after PH. (B) Analysis of CD4Dand IL-17Aeproducing CD4Dlymphocytes in the spleen. CD4Dand IL-17Aepositive CD4Dlymphocytes were determined as described in detail in the Materials and methods. Data represent mean ± standard error (n [ 4 in each group). *P < 0.05 compared with ratio of IL-17Aeproducing CD4D_{lymphocytes/CD4}D_{lymphocytes in the spleen from normal}

animals by analysis of variance using Bonferronipost hoc test.

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