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약학석사 학위논문

T M4S F5- dependent cross- talks

between hepatocytes and

macrophages during liver fibrosis.

T M4S F5-의존적 간 상피세포와 대식세포의

상호관계에 의한 간 섬유화 연구

2020년 02월

서울대학교 대학원 약학대학 의약생명과학전공

박 진 수

CONT ENT S

A BS T RA CT ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1

INT RODUC T ION ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙6

MA T ERIA LS A ND MET HODS ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙9

RES ULT S ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15

Figure 1. Hepatic C C L 20 and C X C L 10 ex pression depends on T M 4S F 5 level

and IL 6 treatment.

Figure 2. T M 4S F 5 binds IL - 6 receptor α on the plasma membrane of

hepatocytes.

Figure 3. IL - 6 was expressed by M 1 (C lassical), but not by M 2 (alternative)

macrophages.

Figure 4. C C L 20/C X C L 10 suppressed M 1 (classical) but induced M 2

(alternative) macrophag es differentiation.

Figure 5. T M 4S F 5 is highly expressed during liver fibrosis. A nd T S A HC

alleviate liver fibrosis.

Figure 6. C cl20 siRNA alleviate liver fibrosis.

Figure 7. T he scheme to summarize the observation.

DIS CUS S ION ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙39

REFERENCES ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙43

국문초록∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙47

LIS T OF FIGURES

Figure 1. Hepatic C C L20 and C X C L10 expression depends on T M 4S F5

level and IL6 treatment.

Figure 2. T M 4S F 5 binds IL - 6 receptor α on the plasma membrane of

hepatocytes.

Figure 3. IL- 6 was expressed by M 1 (C lassical), but not by M 2 (alternative)

macrophages.

Figure 4. C C L20/C X C L10 suppressed M 1 (classical) but induced M 2

(alternative) macrophages differentiation.

Figure 5. T M 4S F 5 is highly expressed during liver fibrosis. A nd T S A HC

alleviates liver fibrosis.

Figure 6. C cl20 siRNA alleviates liver fibrosis.

Figure 7. T he scheme to summarize the observation.

1

A BS T RA CT

T M4S F5- dependent cross- talks

between hepatocytes and

macrophages during liver fibrosis.

J insoo Park

C ollege of Pharmacy

T he G raduate S chool

S eoul National U niversity

C hronic liver disease includes fatty liver, hepatitis, fibrosis/cirrhosis in

which the inflammatory environment is known to play an important role.

In particular, hepatic fibrosis is a consequence of chronic liver damage and

is caused by the accumulation of extracellular matrix (EC M ) following

chronic damage of hepatocytes and thereafter inflammatory reactions.

Exacerbation of liver fibrosis leads to cirrhosis and eventually cancer.

T herefore, earlier diagnosis and cure of liver fibrosis would be clinically

beneficial. T he previous researches have shown that expression level of

transmembrane 4 L6 superfamily 5 (T M 4S F 5) in hepatocytes, a membrane

2

protein belonging to the tetraspanin superfamily, increased in fibrotic

human and mouse liver tissues. However, the roles of the

microenvironment of the T M 4S F 5- positive hepatocytes in the

development of fibrosis has not been examined. T herefore, the aim of this

study was to investigate the effect of T M 4S F 5 on the cross- talk between

hepatocytes and macrophag es as well as the immune environment which

would be involved in the progression of liver fibrosis.

RNA - S equencing of S NU 449 cells and mice liver tissues without or with

T M 4S F 5 expression showed that the expression of certain

cytokine/chemokine genes down- stream of IL- 6 increased by the

expression of T M 4S F5. T he open database also showed a positive

correlation between T M 4S F 5 and C C L20/C X C L10, indicating that C C L20

and C X C L10 could be regulated by T M 4S F 5 expression. T hus, this study

has been examined the roles of C C L20 and C X C L10 in the T M 4S F 5-

dependent fibrosis in the liver.

F irst, when IL- 6, a representative pro- inflammatory cytokine, was

treated to hepatocytes, the phosphorylation of S T A T 3 was increased in

cells expressing T M 4S F 5 as compared to cells not expressing T M 4S F5.

Expression of C C L20 and C X C L10, as known as down- stream genes of

IL- 6, were upregulated by T M 4S F 5 expression. T he physical binding of

IL- 6 receptor α with T M 4S F 5 was confirmed by co-

3

immunoprecipitation, indicating that T M 4S F 5- positive hepatocytes could

be affected by IL- 6 signaling, presumably for C C L20 and C X C L10

expression. U nder these conditions, treatment of T S A HC [4’ - (p-

toluenesulfonylamido)- 4- hydroxychalcone], a specific inhibitor of

T M 4S F 5, disrupted the physical binding of IL - 6 receptor α and T M 4S F5

in hepatocytes. In addition, expression of C C L20 and C X C L10 were also

gradually reduced by T S A HC treatment in a dose- dependent manner.

M acrophages, on the other hand, are polarized into M 1 or M 2 depending

on their surrounding microenvironment. W hen the conditioned- media (C M )

from M 1- macrophages or M 2- macrophages differentiated from T HP- 1

monocytes was treated to hepatocytes without or with IL - 6 receptor

antibody, the expression of C C L20 and C X C L10 in hepatocytes increased

via the presumable secretion of IL - 6 in M 1- macrophages. In other words,

IL- 6 was secreted from M 1- macrophages, and T M 4S F 5 in hepatocytes

were involved in the signaling to synthesize C C L20 and C X C L10 by

macrophage IL- 6. F urthermore, C C L20 and C X C L10 secreted from

T M 4S F 5- positive hepatocytes promoted the polarization of M 2-

macrophage, which was confirmed by increases in M 2 markers (A rg- 1,

Ppar-γ, and C D206) in T HP- 1 and bone- marrow derived macrophage.

T he C M from M 2- macrophages then activated hepatic stellate cells to

produce C ol1α1 (collagen 1 chain α1) mRNA and stimulated hepatocytes

4

to induce the expression of L amc2 (laminin γ2), suggesting their roles in

fibrosis development.

T o confirm these results using in v iv o animal model, a hepatic fibrosis

mouse model was induced by Intraperitoneal injection of C C l4. Injection of

C C l4 resulted in an increase in T M 4S F 5, plasma A LT and liver weight to

body weight ratio, which were signs of liver damage. However, the

injection of C C l4 together with T S HA C resulted in reduction of plasma

A LT and liver weight to body weight ratio. Immunohistochemistry (IHC )

and qPC R analyses confirmed that the expression of α- S M A , collagen1

α1 were higher in the group injected with C C l4, which were declined by

additional T S A HC treatment. In addition, it was found that the expression

of C C L20 and C X C L10 in the C C l4- induced fibrotic livers were reduced by

T S A HC treatment, and importantly the macrophages infiltration into the

liver was also the case, too.

Because the expression of C C L20 in hepatocytes depended on T M 4S F 5

expression and M 1- macrophages, I wanted to know the significance of

C C L20 in liver fibrosis. C cl20 siRNA was intravenously injected during IP

injections with C C l4. A s a result, IHC and qPC R showed that the group

treated with C C l4 and C cl20 siRNA reduced degree of inflammation,

accumulation of EC M , and infiltration of macrophages, although the group

treated with C C l4 alone showed higher levels of them. T hus, secretion of

5

C C L20 in hepatocytes depending on T M 4S F 5 expression can be important

for T M 4S F 5- dependent fibrosis.

A ltogether, this study showed that the importance of T M 4S F 5-

dependent expression of C C L20 and C X C L10 and the effect of M 2

macrophage polarization in the process of liver fibrosis. It is reasonable

that liver fibrosis can be alleviated by regulating the cross- talks of

hepatocytes expressing T M 4S F 5 with macrophages.

Key words: IL- 6, Liver fibrosis, T S A HC , CC l4, CCL20, CX CL10 and

macrophage.

S tudent number: 2018- 24790.

6

Introduction

Hepatic fibrosis is caused by the failure of hepatocyte regeneration

because of chronic inflammation and of accumulation of extracellular

matrix (EC M ) (1). T here are many causes of liver fibrosis. Depending on

the cause, it is divided into nonalcoholic fatty liver disease (NA F LD),

alcoholic liver disease (A LD), nonalcoholic steatohepatitis (NA S H), and

autoimmune liver disease, chronic viral hepatitis (2). But they have

something in common: inflammation and accumulation of EC M .

Multiple cell types, such as hepatocytes, hepatic macrophages consisting

of resident kupffer cells, infiltrating monocyte- derived macrophages and

hepatic stellate cells, are critically involved in developments of hepatic

fibrosis (3). F irst, macrophages are differentiated into M 1 (classical) and

M 2 (alternative)- macrophage depending on their surrounding

environment (4). M 1- macrophages were described as the pro-

inflammatory and anti- fibrotic type and secretion of pro- inflammatory

cytokines (e.g . IL - 6, C C L2). M 2- macrophages were reported to have the

opposite function: regulation of the resolution phase of inflammation and

the repair of damaged tissues (5). Because macrophages play a critical

role in causing and maintaining inflammation, it is important to prev ent

macrophage inflow into liver.

7

In particular, it is known that hepatic stellate cells (HS C s) are

activated by T G F -β, IL- 10 secreted by M 2- macrophage, leading to

EC M formation such as collagen1 (6). HS C s are contributing to

approximately 90% of EC M production (7). Hepatocytes occupy 70- 85%

of liver volume and secrete various mediators. A s a result, hepatocytes

are known to be involved in the innate- immunity of liver and recruit

macrophages into liver (8). It is also known that hepatocytes produce

EC M by stimulation such as T G F -β, IL- 10 and contribute to the

progression of liver fibrosis (9). T herefore, it is necessary to approach

liver fibrosis therapy through understanding and regulating the

relationship of various cells.

On the other hand, T ransmembrane4 L6 family member 5 (T M 4S F 5) is

a liver- specific protein that has been reported to increase expression in

C C l4- M ediated hepatic fibrosis mouse model. T M 4S F 5 was correlated

with a- smooth muscle actin (α- S M A ) expression, collagen I deposition,

and T G Fβ1 and epidermal growth factor receptor signaling activation in

fibrotic septa regions. However, the cross- talks between hepatocytes

expressing T M 4S F 5 and macrophages are unknown (10).

I found that hepatocytes, which express T M 4S F5 through IL - 6

secreted by M 1- macrophages in inflammation, secrete C C L20 and

C X C L10. C C L20 and C X C L10 are known to be involved in the hepatic

8

fibrosis and recruited monocytes into inflamed liver. I demonstrated that the

two chemokines recruited monocytes into hepatic liver and induced M 2-

polarization. M 2- macrophages activated HS C and produced EC M . In

other words, I confirmed that micro- environment of the liver altered by

T M 4S F 5 deteriorates the liver fibrosis.

9

MA T ERIA LS A ND MET HODS

C ell culture

Parental Hep3B, S NU 449 cells were cultured in Roswell Park M emorial

Institute medium (RPM I 1640, Hyclone) supplemented with 10% F BS and

1% penicillin/streptomycin (G enDEPOT Inc.) at 37°C in 5% C O2. Huh7,

HepG 2 cell was cultured in DM EM (Hyclone) under the same condition.

T o achieve T M 4S F 5 knock- down, shRNA targeting the human T M 4S F 5

sequence (5’- C C A T C T C A G C T T G C A A G T C - 3’) were cloned into

lentiviral vector pLK O.1 (A ddgene). Human monocyte T HP- 1 were

treated with 150 nM phorbol- 12- myristate- 13- acetate (PM A ) for 24

hours, and then classically differentiated into M 1 macrophages using 100

ng/ml lipopolysaccharide (Invivogen, #T lrl- eblps) and 20 ng/ml IFN -γ

(R& D system, #285- IF ) for 24 hours or alternatively differentiated into

M 2 macrophages using 20 ng/ml IL - 4 (R& D system, #204- IL) and 20

ng/ml IL- 13 (R& D system, #213- ILB) for 24 hours. Primary hepatocytes

were seeded into collagen (10 μg/ml) coated plates and cultured in

M edium 199 supplemented with 10% F BS , 23 mM HEPES (G ibco,

#15630080), and 10 nM Dexamethasone (Invitrogen, #A 61900).

G eneration of BM DM s & C ell s timulation

M acrophage precursors were obtained from the bone marrow of C 57BL/6

10

mouse. M acrophage precursors were cultured in RPM I- 1640

supplemented with 10% F BS , 1% penicillin/streptomycin and 20 ng/ml

M - C S F (R& D system, #416- M L) during 3 days. T he cells were

incubated at 37 °C with 5% C O2. On day 4, BM DM s were plated in 12-

well plates at a concentration of 1 million cells/well or in 24- well plates

at a concentration of 500 K cells/well and allowed to adhere overnight.

T he following day, then differentiated into M 1 macrophages using 100

ng/ml lipopolysaccharide (Invivogen, #T lrl- eblps) and 20 ng/ml IFN -γ

(BioLegend, #575304) for 24 hours or alternatively differentiated into M 2

macrophages using 20 ng/ml IL - 4 (BioLegend, #574304) and 20 ng/ml

IL- 13 (BioLegend, #575904) for 24 hours. C ells were processed for RNA

extraction.

A nimal ex periments

W ild- type (W T ) and T m4sf5- /- - K O C 57BL/6 mice were housed in a

specific pathogen- free room with controlled temperature and humidity.

T o induce hepatic fibrosis in mice, C C l4 injection was used. In first

experiment, W T mice aged 12 weeks (n ≥ 5) were injected

intraperitoneally with or without C C l4 (trc canada, C 176905, 5 mg/kg) in

40% olive oil twice a week for 6 weeks. A nd T S A HC (5 mg/kg in 40%

DM S O, 60% PBS ) or vehicle were also injected intraperitoneally twice a

11

week for 6 weeks. In second experiments, W T mice aged 12 weeks (n ≥

5) were injected intraperitoneally with or without C C l4 (trc canada,

C 176905, 10 mg/kg) in 40% olive oil twice a week for 4 weeks. C cl20

siRNA (sense 5’- G G A G G A A A U G A U C A C A G C U tt- 3’, antisense 5’-

A G C U G U G A U C A U U U C C U C C tt- 3’) purchased from ambion (A ustin

T exas, U S A ) and diluted in PBS . C cl20 siRNA or vehicle were injected

intravenously (1.66 mg/kg) twice a week. A t the end of experiments,

mice were sacrificed, liver and plasma collected.

Immunoblotting

T he cells were grown in 6- well culture plates and harvested at 80% of

confluency, before preparation of whole cell lysates with RIPA buffer

(150 mM NaC l, 1% NP- 40, 50mM T ris- HC l, and 0.25% sodium

deoxycholate, pH 7.4) with a protease inhibitor cocktail (G enDEPOT ,

U S A ). Primary antibodies for immunoblot were used as follows: β- actin

(santa cruz, #sc- 47778), C C L20 (santa cruz, #sc- 517441), C X C L10

(A bcam, #ab8098), S T A T 3 (santa cruz, #sc- 482), pY 705- S T A T 3 (C ell

signaling, #9145), IL - 6 receptor α (R& D system, #M A B227).

C o- immunoprecipitation

W hole- cell lysates were prepared using Brij58 lysis buffer (20 mM

12

HEPES , pH 7.4, 150 mM NaC l, 2 mM M gC l2, 2 mM C aC l2, and 1%

Brij58) and precipitated with High- C apacity S treptavidin Resin (T hermo

F isher S cientific) 6 hr at 4°C . Precipitates were washed three times

with ice- cold Brij58 lysis buffer and three times with ice- cold PBS ,

after which they were boiled in S DS - PA G E sample buffer before

immunoblotting assay.

Immunofluorescence

C ells were seeded onto glass coverslips pre- coated with fibronectin (10

μg/ml; BD Bioscience, S an J ose, C A , U S A ) and allowed to attach

overnight. T he next day, cells were transfected HA tagged T M 4S F 5

cDNA using Lipofectamine 3000 reagent (T hermo Fisher S cientific,

W altham, M A , U S A ) according to the manufacturer’s protocol. A fter 48

hr, C ell were fixed with ice- cold methanol and stained with the following

primary antibodies: HA (C ovance; 1:300 dilution, #16B12), and IL - 6

receptor α (S anta cruz; 1:50 dilution, #sc- 661). C onfocal images were

analyzed using NIS software (Nikon).

Q uantitativ e rev erse- transcription (qR T ) PC R

T otal RNA was extracted from cells or animal tissues using Qiazol

reagent (Qiagen, #79306), according to the manufacture’s protocol. T otal

13

RNA (500 ng) was reverse transcribed into cDNA using ReverT ra A ce

qPC R RT master mix (T oyobo, Osaka, J apan), according to the

manufacture’s protocol. Quantitative PC R (qPC R) was performed using

specific primers and EvaG reen Q master mix (C osmogenetech, S eoul,

S outh K orea) and the C F X 96 real- time system (Bio- Rad, Hercules, C A ,

U S A ).

Immunohistochemistry and staining

Paraffin blocks and liver tissue sections were prepared by A bion Inc.

(S eoul, K orea). T he sections were analyzed by immunohistochemistry.

H& E staining and M asson’s trichrome was performed by A bion Inc.

(S eoul, K orea). Primary antibodies were used as follows: C cl20 (santa

cruz; 1:50 dilution, #sc- 517441), C xcl10 (A bcam, 1:50 dilution, #ab8098),

α- S M A (sigma, 1:200 dilution, A 2547), F 4/80 (C ell signaling, 1:100

dilution, #70076), Laminin-γ2 (santa cruz; 1:100 dilution, sc- 28330). T he

vectastain A BC - HRP kit (V ector Laboratories, C A , U S A ) was used to

visualize the stained samples. M ayer’s hematoxylin (S igma- A ldrich)

was used for counter- staining the nuclei. S tained tissues were scanned

using M oticEasyS can (M otic, C anada).

14

S tatis tics .

Nonparametric analyses were conducted using a M ann- W hitney U test

for qRT - PC R analysis. Otherwise, S tudent’s t test was performed for

statistical comparisons of mean values to determine significance.

15

RES ULT S

1. T M4S F5 regulates expression of CCL20 and CX CL10.

A ccording to the RNA - S equencing data in human HC C derived from

hepatocytes cell lines and mouse liver tissues, I found that positive-

correlation between T M 4S F 5 and IL - 6 down- stream genes (e.g . C X C L1,

C X C L 6, C X C L 10, C C L 20) (F ig. 1A ) (11) (12). T hus, I hypothesized that

IL- 6 signaling pathway was regulated by T M 4S F 5. T he open database

shows that T M 4S F 5 has a positive- correlation with C X C L10 (Pearson’s

r = 0.279, weak- correlation) and C C L20 (Pearson’s r = 0.930, strong-

correlation) (F ig 1B). A nd also, the two chemokines are related to

progression of liver fibrosis. Expression of C C L20 and C X C L10 was

elevated by IL - 6 treatment as expected. But stable T M 4S F 5 knock- down

cell lines and T M 4S F 5 knock- out murine hepatocytes do not express

C C L20 and C X C L10 by IL - 6 treatments (F ig 1C ~1D). T hus, expression of

C C L20 and C X C L10 was controlled by T M 4S F 5 with IL- 6. IL- 6 signaling

was mediated by phosphorylation of S T A T 3. In stable T M 4S F 5 knock-

down Huh7 cell lines and stable T M 4S F 5 over- expression S NU - 449 cell

lines, phosphorylation of S T A T 3 regulated by T M 4S F 5 with IL- 6 (F ig 1E).

Inhibition of T M 4S F 5 function, using T S A HC (13), reduced expression of

C C L20 and C X C L10 as dose- dependent manner (F ig 1F ). But stable

T M 4S F 5 knock- down Huh7 cell lines were not changed expression of the

two chemokines mRNA levels, when T S A HC treatment (F ig 1G ).

16

A

B

C

17

D

E F

G

18

Figure 1. Hepatic CCL20 and CX CL10 expression depends on T M4S F5 level

and IL6 treatment.

(A ) S catter plot of RNA - S equencing S NU - 449 pC M V - N- T M 4S F 5- HA

vs S NU - 449 pC M V - N- HA . (B) C orrelation of T M 4S F 5 with IL - 6 down-

stream genes (C X C L 1, C X C L 6, C X C L 10, C C L 20) from open database

(DepM ap, https://depmap.org). (C ) C C L 20 and C X C L 10 mRNA level from

stable T M 4S F 5 Huh7 knock- down cell lines in the treatment of IL - 6 (100

ng/ml, 24 hr) or not were measured by qRT - PC R. (D) C C L 20 and C X C L10

mRNA level from W ild- type hepatocytes (n=4) or T M 4S F 5 knock- out

hepatocytes (n=3) after treatment of IL - 6 (50 ng/ml, 24 hr) or not were

measured by qRT - PC R. (E) Immunoblotting of stable T M 4S F 5 Huh7

knock- down cell lines and stable S NU - 449 T M 4S F 5 over- expression cell

lines. T reatment of IL - 6 (100 ng/ml) for 24 hr. (F ) mRNA level of C C L 20

and C X C L 10 in Huh7 cells when treatment of IL - 6 (100 ng/ml) together

with T S A HC (0.1 or 0.5 μM ) for 24 hr were measured by qRT - PC R. (G )

stable T M 4S F 5 Huh7 knock- down cell lines were treated T S A HC (0.2 μ

M ) or not for 24 hr were measured by qRT - PC R. T he data are presented

as the mean ± S EM . *P< 0.05, **p< 0.01, ***P< 0.001.

19

2. T M4S F5 is co- localized with IL- 6 receptor α in

Hepatocytes.

Hepatocyte- derived carcinoma cell line (HepG 2 and Huh7) transiently

transfected with streptavidin- tagged T M 4S F 5. Next, break down Brij 58

buffer and pull down with streptavidin beads. Result of co-

immunoprecipitation, I know that T M 4S F 5 physically binds to IL - 6

receptor α (F ig 2A ). In addition, to confirm this binding was a cellular

system, immunofluorescence assay performed in S NU 449 and the

interaction between T M 4S F 5 and IL - 6 receptor α was identified in the

cellular system (Fig 2B). T S A HC is specific inhibitor of T M 4S F 5 (F ig 2C ).

T reatment of T S A HC dissociated with T M 4S F 5 and IL - 6 receptor α

(F ig 2D). T hus, T M 4S F5 functions are important for interaction with IL -

6 receptor α. A nd, physically binding of two proteins was important for

IL- 6 signaling.

20

A

B

C D

21

Figure 2. T M4S F5 binds IL- 6 receptor α on the plasma membrane of

hepatocytes.

(A ) HepG 2 and Huh7 cells transiently transfected with pEX PR - 103- S trep

(EV ) or pEX PR- 103- T M 4S F 5- S trep (T M 4S F5) for 48 hr. next, C o-

immunoprecipitation and immunoblotting were performed to show

interaction between T M 4S F 5 and IL - 6 receptor α. (B) S NU - 449 cells

transfected with pC M V - N- T M 4S F 5- HA for 48 hr. Immunofluorescence

assay was performed to identify co- localization (white arrowheads) of

T M 4S F 5- HA (red) and IL - 6 receptor α (green). (C ) S tructure of

T M 4S F 5 inhibitor, T S A HC [4 ’ - (p- toluenesulfonylamido)- 4-

hydroxychalcone]. (D) HepG 2 cells transiently transfected with pEX PR -

103- S trep (EV ) or pEX PR- 103- T M 4S F 5- S trep (T M 4S F 5). A fter 24 hr,

T S A HC (5 μM ) was treated to transfected HepG 2 cells. A fter 24 hr, C o-

immunoprecipitation and immunoblotting were performed.

22

3. IL- 6 expressed by M1 (classical)- macrophages.

M acrophages are classified into two phenotypes, M 1 (classical) or M 2

(alternative) macrophage. T hey have opposite functions and

characteristics. M 1- macrophages are known as role of tissue damage

and anti- fibrotic. But, M 2- macrophages are known as role of tissue

remodeling, matrix deposition and pro- fibrotic. Expression of IL - 6 in

human monocytic T HP- 1 cells and murine bone- marrow derived

macrophages were highly elevated when differentiated into M 1-

macrphages (F ig 3A ). M 1- macrophages secrete various species of

cytokines and chemokines (14). S o, I wanted to know that the differences

in the expression of C C L20/C X C L10 caused by T M 4S F 5 were not only

IL- 6 but also macrophage’s C onditioned media.

M ouse BM DM was separated from W ild- type (W T ) C 57BL/6 mouse

and polarized into M 1- or M 2- macrophage. Next, I obtained M 1 and M 2

conditioned media (C .M .). It was treated to W T or T m4sf5- /-

hepatocytes. Only M 1 C .M . elevated C cl20, C x cl10 mRNA level. W T

hepatocytes expressed C cl20, C xcl10 more than T M 4S F 5- /- hepatocytes

(F ig 3B). C o- culture with kupffer cells from W T mouse. W T or

T M 4S F 5- /- hepatocytes also showed same results (F ig 3C ). LPS induced

kupffer cells into M 1- phenotype (15). T reatment with M 1 C .M . and IL - 6

receptor antibody gradually reduced expression of C cl20 and C xcl10 by

quantity of IL - 6 receptor antibody (F ig 3D).

23

A

B

C

D

24

Figure 3. IL- 6 was expressed by M1 (C lassical), but not by M2 (alternative)

macrophages.

(A ) T HP- 1 and BM DM polarized M 1 (classical) or M 2 (alternative) for 24

hr. M 0 was un- polarized macrophages. Il- 6 mRNA was measured by

qRT - PC R. (B) BM DM was separated from W T mouse and differentiated

into M 1 or M 2. A fter 24 hr, C .M . were obtained. C .M . were treated into

W T or T M 4S F 5- /- hepatocytes for 24 hr and C cl20 and C x cl10 measured

by qRT - PC R (n=4). (C ) K upffer cells separated from W T mouse and

polarized by LPS (100 ng/ml) for 6 hr. and then, insert was inserted in well

plate incubation O/N (n=3). (D) C ontrol media or M 1- conditioned media

were treated into stable T M 4S F 5 Hep3B knock- down cell line for 24 hr.

IL- 6 receptor antibody incubate with media for 24 hr. mRNA level of

C C L 20 and C X C L 10 measured by qRT - PC R. T he data are presented as

the mean ± S EM . *P< 0.05, **p< 0.01, ***P< 0.001.

25

4. CCL20/CX CL10 induce M2- polarizaion and M2-

macrophages highly produced ECM when were exposed to

CCL20/CX CL10.

I wanted to find out the role of C C L20/C X C L10 in macrophages

polarization. T HP- 1 cells, human monocytic cell line, were polarized into

M 1- or M 2- macrophage. C C L20 with or without C X C L10 was treated

when macrophages differentiated into M 1- or M 2- macrophage. A fter 24

hr incubation, total RNA isolated from polarized macrophages was

analyzed by qRT - PC R. Not only T HP- 1 cells but murine BM DM were

conducted similar experiments. M ouse bone- marrow derived macrophages

polarized into M 1- or M 2- polarization and were treated with or without

C cl20 50 ng/ml and C xcl10 50 ng/ml, 24 hr. A fter 24 hr incubation, total

RNA isolated from polarized macrophages were analyzed by qRT - PC R.

M 1 markers (IL - 6, IL - 1β and T N F -α in T HP- 1/T nf-α, Il- 1β and

N os2 in BM DM ) were down- regulated by C C L20/C X C L10. But, M 2

markers (C D163, C D206 and F ibronectin in T HP- 1/C d206, A rg- 1 and

Ppar-γ in BM DM ) were up- regulated by C C L20/C X C L10 (16)(17). In

other words, C C L20/C X C L10 suppressed M 1 (classical) polarization but,

induced M 2 (alternative) polarization (F ig 4A ~D).

I obtained conditioned media (C .M .) from M 1- macrophges and M 2-

macrophages with or without C C L20/C X C L10 in T HP- 1 cells. LX - 2

(human hepatic stellate cells) were treated with C .M . A s results of, M 1-

macrophages’s C .M . reduced α- S M A and C O L 1α1 mRNA level in LX -

26

2 cells. However, M 2- macrophages’s C .M . induced α- S M A and C O L 1

α1 mRNA level in LX - 2 cells, even more, when M 2- macrophages were

exposed to C C L20/C X C L10 (F ig 4E). Hepatocyte’s L A M C 2 mRNA was

also the case, too (F ig 4F ). In other words, expression of C C L20/C X C L10

was regulated by T M 4S F 5 and IL - 6. T he two chemokines induced M 2-

polarization as known as pro- fibrotic character. T hus, M 2- macrophages

activated HS C and induced EC M in HS C .

27

A

B

C

D

28

E

F

29

Figure 4. CCL20/CX CL10 suppressed M1 (classical) but induced M2

(alternative) macrophages differentiation.

(A and B) T HP- 1 cells were treated PM A 150 ng/ml for 24 hr. Next, the

adherent T HP- 1 cells were polarized by LPS 100 ng/ml together with

IF N-γ 20 ng/ml during 24 hr for M 1- polarization and C C L20 100 ng/ml

with or without C X C L10 100 ng/ml. On the other hand, the adherent T HP-

1 cells were polarized by IL - 4 20 ng/ml together with IL - 13 20 ng/ml

during 24 hr for M 2- polarization and was treated C C L20 100 ng/ml with

or without C X C L10 100 ng/ml. A fter 24 hr incubation, total RNA isolated

from polarized macrophages were analyzed by qRT - PC R. (C and D)

M ouse bone- marrow cells were separated from wild- type C 57BL/6. Bone-

marrow cells were incubated for 3 days in M - C S F 20 ng/ml containing

media. Next, macrophages polarized into M 1- polarization (by LPS 100

ng/ml together with IFN -γ 20 ng/ml, 24 hr) or M 2- polarization (by IL- 4

20 ng/ml together with IL - 13 20 ng/ml, 24 hr) and were treated with or

without C cl20 50 ng/ml and C xcl10 50 ng/ml, 24 hr. A fter 24 hr incubation,

total RNA isolated from polarized macrophages were analyzed by qRT -

PC R. (E and F ) C onditioned media (C .M .) were collected 24 hours after M 1

or M 2 polarization which were exposed to the two chemokines or not and

filtered through a 0.22-μm syringe filter. LX - 2 cells or Huh7 cells were

treated with C .M . (final concentration 20% v⁄v), for 6 hr. T he data are

presented as the mean ± S EM . *P< 0.05, **p< 0.01, ***P< 0.001.

30

5. T M4S F5 expressed in CCl4- induced liver fibrosis mouse

model and T S A HC inhibits accumulation of macrophages

and ECM.

T o investigate the role of T M 4S F 5 in in- v iv o, C 57BL/6 female mouse

induced liver fibrosis by C C l4 injection (18). W ild- type mice (12 weeks)

were randomly grouped. M ice were injected C C l4 twice a week (i.p. 5

mg/kg) for 6 weeks and injected with T S A HC (i.p 5 mg/kg) or vehicle (i.p

40% DM S O + 60%PBS ). C C l4 injected group elevated T m4sf5 mRNA level,

plasma alanine aminotransferase (A LT ) and liver weight to body weight

ratio. It suggesting liver damage. But, C C l4 together with T S A HC injected

group reduced liver damage (F ig 5A ). Hematoxylin and eosin (H& E) stains

and M asson’s trichrome stains show that T S A HC reduced inflammation

and collagen1α1 (F ig 5B). C C l4 injected group elevated T m4sf5 level. A nd

T m4sf5 induced C cl20 and C xcl10 according to histology and mRNA levels.

But, C C l4 together with T S A HC injected group reduced C cl20 and C xcl10

to approximately the only T S A HC injected group (F ig 5C and 5D).

C cl20 and C xcl10 are well known as chemotaxis (19). T he chemokine

receptor 6 (C C R6) for C C L20 is expressed by various cell subsets, such

as monocytes, T h17 and regulatory T cells (20). A nd the C X C chemokine

receptor 3 (C X C R3) for C X C L10 is expressed on peripheral blood

monocyte surface (21). T m4sf5- mediated C C l4- induced liver fibrosis

model induced C cl20 and C xcl10. T hus, macrophages influx into liver.

However, T S A HC decreased the two chemokines. T herefore,

31

macrophage reduced in liver, as assessed by F 4/80 liver mRNA level and

histology (F ig 5E). S everal studies have shown that the quantity of liver

macrophages are important for hepatic inflammation (22- 23).

32

A

B

C

D

33

E

34

Figure 5. T M4S F5 is highly expressed during liver fibrosis. A nd T S A HC

alleviates liver fibrosis.

(A ) Liver fibrosis was induced in C 57BL/6 wild- type mice (12 weeks) by

injecting C C l4 (5 mg/kg, twice a week) over 6 weeks; Olive oil–injected

mice served as controls. T S A HC (5 mg/kg) or vehicle (40% DM S O + 60%

PBS ) treatment was started with C C l4 injection. Plasma A LT levels are

shown as a marker of hepatocyte necrosis. Plasma A LT and liver weight

to body weight ratio were reduced by T S A HC . (B) Hematoxylin and eosin

staining and M asson’s trichrome of representative liver sections. (C ~ E)

Immunohistochemistry of representative liver sections and liver mRNA

levels were analyzed by qRT - PC R. T he data are presented as the mean

± S EM (n = 5 for control group, n= 6 for experimental groups). *P< 0.05,

**p< 0.01, ***P< 0.001.

35

6. Ccl20 siRNA alleviate CCl4- induced liver fibrosis.

Expression of C C L20 was regulated by T M 4S F5. A nd to investigate the

role of C C L20 in liver fibrosis, expression of C C L20 was reduced by C cl20

siRNA during C C l4 injection. W ild- type female mice (12 weeks) were

injected C C l4 twice a week (i.p. 10 mg/kg) for 4 weeks and injected with

C cl20 siRNA (i.v. 1.66 mg/kg) or PBS (i.v.) twice a week. Reduction of

C cl20 level was confirmed by staining liver tissue and measuring liver

mRNA level (Fig 6B). C cl20 siRNA reduced inflammation and collagen1α

1, as assessed by H& E and M asson’s trichrome (F ig 6A ). M acrophages

and Laminin γ- 2 in liver were reduced by siRNA , according to histology

and liver mRNA levels (F ig 6C and 6D).

36

A

B

C

D

-

-

+

-

+

+

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

2 . 5

Re

lativ

e m

RN

A e

xp

re

ss

ion

(C

ol1

1

)

C C l4

C c l 2 0 s i R N A

* * **

37

Figure 6. C cl20 siRNA alleviates liver fibrosis.

(A ) W T mice aged 12 weeks were intraperitoneal injected with or

without C C l4 (10 mg/kg) in olive oil twice a week for 4 weeks. C cl20

siRNA or PBS were injected intravenously (1.66 mg/kg) twice a week.

Hematoxylin and eosin staining and M asson’s trichrome of

representative liver sections. A nd C ol1α1 mRNA level measured by

qRT - PC R. (B ~ D) mRNA levels of C cl20 and macrophages (F 4/80)

measured by qRT - PC R. IHC assay of C cl20, F 4/80 and Laminin γ2.

T he data are presented as the mean ± S EM (n = 7 for control group, n=

7 or 5 for experimental groups). *P< 0.05, **p< 0.01, ***P< 0.001.

38

7. T he scheme to summarize the observation

T M 4S F 5 in hepatocytes expressed during liver fibrosis. Inflammatory

molecules like LPS induce secretion of IL - 6 that mediated by M 1- polarized

liver resident macrophage, kupffer cells. T M 4S F 5 in hepatocytes regulated

IL- 6 signaling. A nd, the two chemokines, C C L20 and C X C L10, were

expressed by T M 4S F 5 together with IL6. M onocytes in blood recruited

into inflamed liver. Because, C C L20 and C X C L10 exhibit a chemotactic

activity. C C L20 and C X C L10 suppressed M 1- polarization, and induced

M 2- polarization. M 1- macrophage is well known as anti- fibrotic but, M 2-

macrophage is well known as pro- fibrotic. M 2- macrophage activated

hepatic stellate cells that produced C ol1α1 and induced laminin γ2 in

hepatocytes. In other words, liver micro- environments induced by

T M 4S F 5 in hepatocytes aggravate liver fibrosis.

Figure 7. T he scheme to summarize the observation.

39

DIS S CUT ION

In the past decade, numerous studies have shown that macrophages

are closely related to liver fibrosis. In addition, various studies have

demonstrated that liver diseases are improved or deteriorated depending

on the number of macrophages and phenotypes of macrophages. Because,

Inflammation and extra-cellular matrix (ECM) deposition by hepatic stellate

cells (HS C ) are one of the major causes of hepatic fibrosis to progress to

cirrhosis and cancer. A nd macrophages are key players in tissue injury and

repair.

T M 4S F 5 was also known as an important factor in liver/pulmonary

fibrosis through previous studies. However, further studies have been

needed on the correlation between T M 4S F 5-expressing hepatocytes and

macrophages.

I found that T M 4S F 5 interacts with IL - 6 receptor α. And interaction of

these two proteins induce expression of chemokines, such as C C L20 and

C X C L10. C C L20, also known as macrophage inflammatory protein (M IP-

3α), serves as a ligand for its only receptor, C C chemokine receptor 6

(C C R6). C X C L10, also known as Interferon gamma- induced protein 10

(IP- 10), serves as a ligand for C X C chemokine receptor 3 (C X C R3).

T hese two chemokines contributed to the exacerbation of the liver

disease such as liver fibrosis, NA F LD and alcohol hepatitis in mice and

humans (24- 27).

In order to address our research, we employed mouse model of hepatic

fibrosis, the C C l4 injection. C arbon tetrachloride (C C l4) has long been

40

used to produce experimental liver fibrosis (28). C C l4 in olive oil induce

increased liver weight/body weight ratio, liver enzyme activities, and

clear histopathological evidence of liver damage with single cell necrosis;

whereas the long- term exposure to C C l4 causes marked hepatotoxicity

with resulting fibrosis, bile duct proliferation, cirrhosis and even

hepatocellular carcinoma (29). A nd also T M 4S F5 level was elevated by

C C l4 injection. T S A HC , a selective inhibitor of T M 4S F 5, was injected

together with C C l4 to inhibit T M 4S F 5’s function.

F irst, T S A HC treatment significantly reduced inflammation and

macrophages of the liver as seen by reduced plasma alanine

aminotransferase (A LT ) level, liver weight to body weight ratio and

histology. T his could be attributed to the reduction of C C L20 and

C X C L10 levels in liver by T S A HC . Also, liver fibrosis model which reduced

Ccl20 using Ccl20 siRNA showed decreased macrophages in liver. S o, the

Infiltration of C C R6+ and C X C R3+ monocytes into inflamed liver were

decreased by TSAHC and Ccl20 siRNA. It is known that reduced intrahepatic

inflow of macrophages reduces inflammation (30- 31).

However, C C R6+ and C X C R3+ immune cells have various cells such as

T cells and neutrophils as well as macrophages. F urther studies are

needed to investigate the effects of fibrosis on these immune cells. In

addition, C C R6+ and C X C R3+ immune cells play an important role in viral

fibrosis or HC C , so it is necessary to study the role of T M 4S F 5 in these

diseases.

S econd, extra- cellular matrix replaces liver cells that are killed by

41

various inflammatory substances. EC M maintains the liver structure. In

acute liver disease, EC M is reversibly produced and eliminated by M atrix

metalloproteinases (M M Ps). However, in chronic liver disease, EC M

accumulates with an increase of T issue inhibitor of metalloproteinases

(T IM Ps)/M M Ps ratio. T he accumulation of matrix is associated with an

impairment of hepatic function and predisposition to the development of

hepatocellular cancer. T his architectural disruption of liver anatomy

results in the well- known complications of chronic liver injury,

particularly the development of portal hypertension, which is a major

cause of death in patients with chronic liver injury (32). T herefore,

inhibiting the accumulation of EC M is important in preventing the

deterioration of liver disease.

I found that C C L20/C X C L10 suppressed monocytes differentiation into

M 1- macrophage and promoted into M 2- macrophage in T HP- 1 and

murine BM DM . Besides, macrophages exposed to C C L20/C X C L10 during

differentiation into M 2- mcarophage promoted the production of EC M

such as α- S M A and collagen1α1 in LX - 2 cells and laminin γ2 in

Huh7 cells, compared to M 2- macrophage without exposure to the two

chemokines. In in- v iv o experiment, C C l4- induced hepatic fibrosis model

showed that T S A HC treatment group reduced EC M accumulation such

as collagen1α1. In the second mouse experiment, C cl20 siRNA treatment

group also reduced EC M accumulation such as collagene1α1 and laminin

γ2. In other words, as shown in- v itro results, the C C L20/C X C L10,

which expressed dependently on T M 4S F 5, contributed to the production

42

of EC M in mouse experiments.

However, it remains unclear what proportion of C C R6+ and C X C R3+

macrophages in liver fibrosis. T herefore, if the accurate analysis of the

number of C C R6+ and C X C R3+ macrophages is performed through flow-

cytometric analysis, the influence of T S A HC and C cl20 siRNA can be

accurately determined. A nd also, if we can distinguish the characteristic

and role of liver resident macrophage, kupffer cell, vs C C R6+ macrophage

vs C X C R3+ macrophage, it would be of significance.

In my study, I confirmed the relevance of chemokine- dependent

macrophage accumulation in T M 4S F 5- dependent liver fibrosis via C C l4-

administration, via functional associations between T M 4S F 5- positive

hepatocytes and M 2- polarized macrophages involving IL6, C C L20, and

C X C L10. C onsequently, inhibition of macrophage infiltration by T S A HC

reduced inflammation and EC M accumulation in experimental mouse

models. A nd also, it suggests that inhibition of T M 4S F 5 is useful for

treating liver fibrosis through regulating M 1/M 2- polarization.

43

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47

국문초록

T M 4S F 5-의존적 간 상피세포와

대식세포의 상호작용에 의한

간 섬유화 연구

박 진수

약학대학 의약생명과학전공

서울대학교 대학원

만성적 간질환은 지방간, 간염 및 섬유화/경화 그리고 간암을

포함하며 만성적인 염증 환경이 중요한 역할을 하는 것으로 알려져

있다. 특히, 간 섬유화는 만성 간 손상에 의한 결과물로 비가역적인 간

세포의 손상과 염증반응으로 인한 세포외기질(Extracellular matrix ,

EC M )의 축적에 의해 일어난다. 간 섬유화의 악화는 간경화와 간암으로

이어지며, 현재까지 간 섬유화의 정확한 진단 방법과 치료 약물에

대해서는 잘 알려진 바가 없다. 이전 연구에 따르면 간 섬유화가 진행된

마우스 간 조직과 인간 간 세포에서 T etraspanin superfamily에 속하는

48

막 단백질인 T ransmembrane 4L 6 superfamily 5(T M 4S F 5)의 유전자와

단백질의 발현이 증가되어 있는 것으로 알려진 바 있다. 따라서 이를

토대로 본 연구에서는 T M 4S F 5가 간 섬유화를 유발하는 과정에서 간

상피세포와 대식세포 사이의 상호작용 및 면역적 환경에 미치는 영향에

대해 알아보고자 하였다.

S NU - 449 세포와 마우스 간 조직의 RNA - S equencing을 통해,

T M 4S F 5의 발현이 증가하면 IL- 6의 하위 유전자의 발현 또한 함께

증가하는 것을 알 수 있었다. 그리고 Open database를 통해서

T M 4S F 5와 C C L20과 C X C L10 양의 상관관계를 알 수 있었다. 이를

바탕으로 C C L20과 C X C L10이 T M 4S F 5 발현에 의해 조절됨을 알 수

있었다.

다음으로, C C L20과 C X C L10의 발현을 조절하는 IL- 6를 간 상피세포에

처리하였다. 이때, T M 4S F 5가 발현되지 않는 세포에 비해 T M 4S F 5가

발현되는 세포에서 S T A T 3의 인산화가 증가되었을 뿐만 아니라 C C L20,

C X C L10의 발현이 증가된다는 것을 확인하였다. IL- 6의 수용체와

T M 4S F 5의 물리적인 결합을 위한 면역침강법을 통해 IL- 6의 작용과

T M 4S F 5의 발현이 상관성 있음을 확인하였다. 이러한 조건에서

T M 4S F 5의 선택적 억제제인 T S A HC [4′- (p- toluenesulfonylamido)- 4-

hydroxychalcone]를 처리하였을 경우 간 세포 내 IL- 6 수용체와

T M 4S F 5의 물리적 결합이 소실 되고, C C L20, C X C L10의 발현 역시

T S A HC의 농도에 의존적으로 감소함을 확인하였다.

한편, 대식세포는 주변 환경에 따라 M 1(classical) 또는

M 2(alternative)로 분극화된다. M 1-대식세포와 M 2-대식세포의

C onditioned M edia(C .M .)을 얻은 뒤 간 상피세포에 처리해 C C L20,

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C X C L10의 발현을 측정하거나 IL- 6 수용체의 항체를 함께 처리한

실험을 통해 M 1-대식세포에서 IL- 6가 분비됨을 알 수 있었다. 즉, IL-

6는 M 1-대식세포에서 분비되며, 간 상피세포의 T M 4S F 5가 IL- 6의

신호전달에 관여해 간 상피세포에서 C C L20, C X C L10을 분비하는 것으로

파악되었다. 이때 T M 4S F 5가 발현되는 간 상피세포로부터 분비된

C C L20, C X C L10은 M 2-대식세포로 분화가 유도되는 것을 더욱 촉진하는

것을 T HP- 1과 Bone- marrow derived macrophage의 M 2 marker(A rg- 1,

Ppar-γ, C D206 등)가 증가한 것을 통해 알 수 있었다. M 2-대식세포는

Hepatic stellate cell을 활성화 시켜 C ol1α1을 생성하며, 간 상피세포를

자극해 Laminin γ2의 형성을 유도하였다.

세포 수준의 실험 결과를 동물 수준에서 확인하기 위해, C C l4 처리에

의해 간 섬유화가 유도된 마우스 모델을 구축하여 분석하였다. C C l4의

처리는 T M 4S F 5의 증가와 Plasma A LT , 간 무게/몸무게 비율의 증가를

일으켰으며, 이는 간 손상의 지표이다. 그러나 C C l4와 T S HA C를 함께

처리하면 Plasma A LT , 간 무게/몸무게 비율이 덜 증가 하거나

궁극적으로 간 손상이 덜 일어난 것을 확인하였다. 그리고 조직 염색과

mRNA level을 통해 α- S M A , C ollagen1α1의 발현이 T S A HC를 함께

처리한 그룹에서 감소한 것을 확인하였다. 또한, 간의 C cl20, C xcl10의

발현이 감소되고 이로 인해 간으로 유입되는 대식세포가 감소한 것을 알

수 있었다.

한편, 간 상피세포의 T M 4S F 5와 M 1-대식세포에 의존적으로 증가하는

C cl20의 간 섬유화에서의 역할을 알기 위해 C cl20 siRNA를

정맥주사하고 C C l4로 간 섬유화를 유도하였다. 그 결과, C cl20 siRNA을

함께 처리한 그룹이 C C l4만 처리한 그룹에 비해 염증의 정도와 EC M의

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생성, 대식세포의 유입이 감소한 것을 조직 염색과 mRNA level을 통해

확인하였다. 결과적으로, T M 4S F 5를 발현하는 상피세포로부터 C cl20의

분비가 T M 4S F 5-의존적 섬유화에 중요함을 확인할 수 있었다.

따라서, 이 연구를 통해 간 섬유화 과정에서 T M 4S F 5 의존적인 C C L20,

C X C L10의 발현, 그에 따른 대식세포 분극화의 중요성을 알 수

있었으며, 간 섬유화 과정에서 T M 4S F 5를 발현하는 간 상피세포와 대식

세포의 상호작용을 조절하여 간 섬유화를 치료할 수 있는 가능성을

제시하였다.

주요어 : IL- 6, 간 섬유화, T S A HC , C C l4, C C L20, C X C L10, 대식세포

학번 : 2018- 24790