[2016] pathogenesis of liver fibrosis

1

Ayman Alsebaey, MD.

Lecturer of Hepatology,

National Liver Institute.

2

It is a reversible wound-healing response to either acute or chronic

cellular injury that reflects a balance between liver repair and scar

formation.

Acute conditions e.g. hepatitis is associated with transient and

reversible changes in liver architecture.

Chronic conditions unfortunately is characterized by progressive

substitution of the liver parenchyma by scar tissue.

The continuous hepatocyte regeneration stand for the progressive

scar formation.

Liver fibrosis is a dynamic process in which increased synthesis of

matrix components and a failure of physiological mechanisms of matrix

turnover.

What is liver fibrosis?

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Hepatitis, NASH

ASH, Hemochromatosis

DILI, Wilson, Autoimmune, PSC

PBC

Hepatocyte Injury

HSCs activation Fibrosis

Cirrhosis

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Fibrosis provides mechanical stability.

Inflammatory cells contribute to the removal of cellular

debris.

Inflammatory signals also exert important functions in

the promotion of liver regeneration.

Is liver fibrosis beneficial?

6

It’s a constellation of extracellular molecules secreted by the cells to do

structural and biochemical support.

it is present between the hepatocytes and liver sinusoidal endothelial cells

“LSECs”

ECM is formed of interlocking mesh of fibrous proteins and

glycosaminoglycan.

SIMPLY it is the road and the streets around the home “cells”. If

blocked, not paved the home begins troubles.

The homeostasis of ECM is dependent on the balance of:

matrix metalloproteinases (MMPs) ECM

tissue inhibitors of matrix metalloproteinases (TIMPs) ECM.

What is extracellular matrix [ECM]?

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How is altered?

Marked increase of TIMP 1 and 2.

TIMP-1 is anti-apoptotic effects on HSCs survival with progressive

fibrosis.

Accumulation of other matrix proteins e.g. elastin, hyaluronan,

proteoglycans and fibronectin.

What is the impact of ECM alternation:

Endothelial cells alternation “capillarization” impaired transport of

solutes from the sinusoid to the hepatocytes hepatocyte dysfunction

“altered cell behaviour”.

Altered ECM is a signal for more fibrosis.

What is the impact of ECM alternation?

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What are the implicated or accused cells of liver fibrosis?

Liver fibrosis

Hepatic stellate cells

(HSCs)

Portal fibroblasts

Fibrocytes

Bone marrow-derived

mesenchymal stem cells

(MSCs)

Epithelial to Mesenchymal

Transition (EMT)

Myofibrocyte

“MFs”

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Portal fibroblasts

They are implication in liver fibrosis due to biliary and cholestatic liver

injuries.

They are activated to MFs.

They have different genetic profiles and signaling responses than the HSCs.

Fibrocytes

Fibrocytes originate from hematopoietic stem cells and migrate to the liver,

proliferate and secrete growth factors that promote deposition of the ECM.

Bone marrow-derived MFs

Bone marrow-derived mesenchymal stem cells (MSCs) myofibrocytes.

Epithelial to Mesenchymal Transition (EMT)

Hepatocytes and cholangiocytes undergo EMT acquire mesenchymal

features “MFs”.

A lot of debate is present especially against it.

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We are cells that live in the space of Disse as perisinusoidal cells in the

subendothelial space between hepatocytes and sinusoidal endothelial

cells.

We are derived from mesodermal-derived multipotent mesenchymal

progenitor cells (MMPC) so we express neural and mesenchymal lineage

markers.

Our functions are:

Activation of the immune system by through secretion of cytokines and

chemokines and interacting with immune cells.

Angiogenesis.

Regulation of oxidant stress.

How HSCs introduce themselves?

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HSC is the primary effector cell, orchestrating the deposition of

ECM in normal and fibrotic liver.

They lay fibrous tissue and have receptors for inflammatory signals.

Inactive HSCs are characterized by:

A lot of retenoids “Vitamin retinyl esters” storage.

Lipid droplets so its old name was Ito and lipocytes.

Synthesis of glial fibrillary acidic protein (GFAP).

On activation to myofibrocytes

There are loss of the above, de novo expression of receptors for

fibrogenic, chemotactic, and mitogenic factors and expression of α-

smooth muscle actin so being contractile.

Let us navigate with HSCs

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Life cycle of activated HSCs

Quiescent

inactive HSCs Active HSCs Resolution

HSCs

Initiation Active HSCs

Senescence

Apoptosis

Proliferation, chemotaxis,

fibrogenesis, contractility,

matrix degradation, retinoid

loss, and WBC chemo-

attractant/cytokine release

Removal of injurious agent

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Initiation (pre-inflammatory stage):

It is the early changes in gene expression and phenotype.

The HSCs is activated in auto and paracrine manner from the

neighboring injured cells.

Perpetuation:

The continuous presence of inflammatory milieu cause prolonged

survival of the HSCS “perpetuation”.

It lays down the fibrous tissue with accumulation of extracellular

matrix.

Proliferation, chemotaxis, fibrogenesis, contractility, matrix

degradation, retinoid loss, and WBC chemo-attractant/cytokine release.

Resolution phase if the injury subsides:

HSCs go to apoptosis, senescence, or quiescence.

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Who and how do activate the HSCs?

Hepatocytes Reactive oxygen species

Natural killer cells

Platelets

Leucocytes

Lymphocytes

Kupffer Cells

Liver sinusoidal

endothelial cells Toll-like receptors

Epigenetics

Genes

Nuclear Receptors

MicroRNAs

DNA demethylation

Growth factor signaling

Neuroendocrine

signaling

SIGNALING

Adipokine signaling

Chemokine signaling

Fibrogenic signaling

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Hepatocytes: The injured hepatocytes undergo apoptosis (Fas and TRAIL mediated) with release of apoptotic

bodies, DNA, fibrogenic lipid peroxides and DAMPs.

All of them activate the HSCs.

Liver sinusoidal endothelial cells (LSECs): The endothelial injury causes loss of the fenestrations which activate the HSCs.

Kuppfer cells (KCs): They are indwelling liver macrophages.

Together with the recruited monocytes, secrete cytokines that cause fibrosis progression,

They express CCR1, CCR2, CCR6, CCR8, and CCR9 that are essential for conversion of

monocytes to proinflammatory macrophages that augment the fibrosis milieu.

There is recruitment of high Gr1hi (Ly6chi) expression macrophages.

Antifibrogenesis monocytes: Dendritic cells promote fibrosis resolution via the release of MMP-9, a gelatinase active on

collagens I, III, IV, and elastin.

The Gr1lo monocyte subset expresses chemokine receptors with anti-inflammatory and

antifibrogenic functions, such as CXCR1 and CX3CR1.

Cellular activation of HSCs

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Lymphocytes:

CD4 T-helper lymphocytes secrete cytokines the activate the HSCs.

Th2 is more fibrogenic than Th1.

On the hand they also aid in the resolution of liver fibrosis

Th1 INF-γ apoptosis of the myofibrocytes.

Th17 IL17 mainly, IL21, IL22

It activates myofibrocytes and KCs

It shifts macrophages from M2 "repair" to M1 "killer" macrophages.

Despite this IL22 is hepatoprotective and antifibrogenic cytokine.

B lymphocytes

They fibrogenic but the mechanism is unknown.

Treg:

It is antifibrotic.

Innate lymphoid cells (ILCs):

A recently discovered cell population, which express many TH cell-associated cytokines, but no

cell-surface markers of known lymphoid lineage.

Hepatocytes IL33 ILC2 expansion IL13 and activation of (STAT) 6 pathway HSCs

activation.


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Natural killer cells (NK)

NK cells inhibit and kill activated HSCs antifibrotic effects.

Retinoid [retinoic acid early inducible 1 (RAE1)] activate NK cells secrete INF-γ plus

“FasL and TRAIL” HSCs apoptosis.

This effect is present on early activated HSCs as containing the retinoic acid but fully

active cells are devoid of it.

NK T-cells:

has diverse effects on liver fibrosis depending on the stage of the disease

Leukocytes:

Neutrophils reactive oxygen species (ROS) and NO counteract the effect of superoxide on

collagen production.

Platelets:

They produce TGF-β1, PDGF and epidermal growth factor (EGF) paracrine stimuli in HSC

activation and fibrogenesis.

On the other hand several studies have described antifibrotic effects of platelets [the more

the platelet count, the less the liver fibrosis].


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Origin Cell Type Target Cell/Mechanisms of Action

BM

Macrophages

Promote HSC survival by cytokines - Up-regulate TIMP-1 by cytokines

Promote liver fibrosis as demonstrated by genetic or pharmacological ablation

Promote fibrosis regression through MMP-12- and MMP-13-mediated ECM degradation and through killing of

HSCs by TRAIL

NK cells

Active killing of HSCs by FasL and TRAIL

Reduction of liver fibrosis in mice treated with poly I:C–activated NK cells

Increased fibrosis in SCID-BEIGE mice lacking NK cells

NKT cells Promote liver fibrosis through CXCL16-CXCR6- αGalCer treatment enhances NKT cell-mediated liver fibrosis

B lymphocytes Promotion of collagen, but not α-SMA, expression in HSCs - Effects independent of Ig

T lymphocytes No decrease in fibrosis observed in mice lacking CD4+ T cells, CD8+ T cells, and γ-δ T cells

Neutrophils Recruited in a TLR2-CXCL2-S100A9–dependent manner

Play minor roles for liver fibrosis

ILC2 In response to IL-33, ILC2 expand and promote HSC activation and liver fibrosis through IL-13

Platelets

Increased fibrosis in thrombocytopenic mice

Decreased fibrosis in SCID mice receiving human platelets

Repression of HSC activation by platelets through Met

Hepatocytes

Promotion of HSC activation and liver fibrosis through heptocyte-secreted IL-33

Apoptotic hepatocytes promote HSC activation through phagocytosis.

Apoptotic hepatocytes increase inflammatory and profibrogenic cytokines in macrophages.

Liver

resident

Cholangiocytes Promotion of portal fibroblast activation by cholangiocyte-secreted CCL2

Promotion of cholangiocyte proliferation through hyaluronan from myofibroblasts

LSECs

LSECs from normal liver suppress HSC activation.

LSECs from fibrotic liver lose the ability to suppress HSC activation.

LSECs from fibrotic liver promote HSC activation depending on CXCR4 and FGFR-1 expression on LSECs.

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ROS:

Hepatocytes, macrophages, cholangiocytes and inflammatory cells lipid

peroxidation especially in the presence of PDGF, TGF-β leptin and Angiotensin II

ROS release HSCs activation.

NADPH oxidase is implicated in ROS formation Homologs of NADPH oxidase

(NOX) HSCs and KCs activation.

Antioxidants and CYP2E1 inhibitors liver fibrosis.

Toll-like receptors (TLRs):

CLD intestinal permeability portal flow of gut-derived microbial products

e.g. lipopolysaccharides (LPS) activate TLR4, TLR9 and TLR2 activate HSCs

and KCs BAMBI TGF-β liver fibrogenesis.

High mobility group box 1 protein (HMGB1) is endogenous ligand that may activate

the above cascade without the need of LPS.

Molecular activation of HSCs

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Gene transcription:

There are increase gene transcription of factors of fibrogenesis and their downstream targets.

Type 1 collagen, α-SMA, TGF-β-1, TGF-β receptors, MMP-2, TIMPs 1 and 2.

downstream targets; Ets-1, Mef2, CREB, Egr-1, Vitamin D receptor, Foxf1, JunD and

C/EBPβ.

Nuclear receptors:

PPARγ down-regulates HSC activation fibrogenesis.

RXR an FXP collagen production.

Steroids and antibiotics PXR dimerizes RXR to induce cytochrome p450

fibrogenesis.

MicroRNAs:

TGF-β and LPS in cultured HSCs mi-R29 ECM proteins, including collagens.

miR-221/222 fibrogenesis.

MiR-19b TGF-β signaling. It was found to that its expression is decreased in patients with

advanced fibrosis.

Gene regulations in activated HSCs

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DNA methylation and histone modifications:

DNA methylation of genes expressed in quiescent HSCs contributes to

the maintenance of the quiescent phenotype.

During activation HSCs express DNA-methyl binding proteins

(MeCP2) silencing of antifibrogenic genes and increase the

expression of histone methyl transferases, leading to enhanced

transcription of collagen, TIMP-1 and TGF-β.

Epigenetics

Epigenetic changes modulate fibrosis susceptibility.

Offspring from the progeny of male fibrotic rat ancestors are found

to be more resistant to liver fibrosis than their counterparts with no

previous history of fibrosis.

Gene regulations in activated HSCs

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Detailed Life cycle of activated HSCs

Quiescent

inactive HSCs Active HSCs Resolution

HSCs

Initiation Active HSCs

Senescence

Apoptosis

Proliferation, chemotaxis,

fibrogenesis, contractility,

matrix degradation, retinoid

loss, and WBC chemo-

attractant/cytokine release

Removal of injurious agent

27

HSCs proliferation:

PDGF is the most potent mitogen.

PDGF also stimulates Na+/H+ exchange.

Other mitogens: VEGF, thrombin and its receptors, transforming growth factor α

(TGFα), epidermal growth factor (EGF), keratinocyte growth factor, and bFGF.

Downstream pathways:

PI3 kinase and ERK/MAP kinase

PDGF antagonism potential anti-fibrotic strategy.

Sorafenib a multiple receptor tyrosine kinase inhibitor targeting the PDGF

receptor and the Raf/ERK signaling pathway is effective in advanced HCC

patients.

Sorafenib displays anti-fibrotic activity.

HSC remodels the ECM into one rich in fibril-forming collagens, particularly types I

and III.

The ECM components in turn act in a positive feedback loop by releasing

additional matrix-bound growth factors resulting from increased protease activity, as well

as increasing liver stiffness, both of which propagate HSC migration and contraction.

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HSCs chemotaxis:

Usually there is chemotaxis of the HSCs to the site of injury.

The usual chemokines are including VEGF, PDGF, MCP-1, CXCR4,

CXCR3 and CCR5 and its ligand RANTES.

Hypoxia:

Is another activator of HSC migration via VEGF and ROS.

ECM:

Cellular fibronectin containing an alternatively spliced domain A

(EIIA) has been shown to induce motility of HSCs.

Adenosine:

Enhanced adenosine signaling stimulates HSC fibrogenesis.

Caffeine adenosine signaling antifibrotic effect.

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HSCs in fibrogenesis: ECM:

In the normal liver ECM is composed of collagens IV and VI.

Fibrosis ECM is composed of collagens I and III and cellular fibronectin.

HSCs secrete collagen type I fibrosis.

It is mediated by TGF-β, retinoids and angiotensin II.

TGF-β:

TGF-β is the most profibrogenic cytokine in the liver.

It stimulates the production of collagen type I, cellular fibronectin and proteoglycan.

TGF-β is produced by Kupffer cells, liver sinusoidal endothelial cells, hepatocytes and HSCs

and has paracrine/autocrine effects on HSCs.

It stimulates SMADs signaling.

TGF-β1 may also contribute to liver homeostasis during regeneration, therapeutic

antagonization of TGF-β1 is challenging.

Connective tissue growth factor (CTGF/CCN2):

It is released in response to hyperglycemia, hyperinsulinemia and alcohol-induced cellular

injury.

It stimulates the HSCs independent of TGF-β unlike the hepatocyte.

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Adipokines:

There is similarity between the adipocyte and the HSCs as containing LDs.

Leptin:

Leptin leptin receptor (OB-R) JAK 2 and STAT 3 pathway HSC fibrogenesis and

activates Kupffer cells, macrophages and endothelial cells to produce TGF-β1.

Leptin partially suppresses PPARγ, which can reverse HSC activation and maintain

senescence.

Leptin norepinephrine activity fibrogenesis.

Adiponectin:

Is a counter-regulatory hormone of leptin, inhibits hepatic fibrogenesis both in vivo and in

vitro.

Neurochemical and neurotrophic factors:

Activated HSCs express specific endocannabinoid receptors CB1 and CB2 with 2

opposing effects.

CB1 stimulation fibrogenesis

CB2 stimulation fibrogenesis hepatoprotective.

Serotonin and thyroid hormones are also involved in fibrogenesis.

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HSCs contractility:

HSCs acquire contractility with formation of the cytoskeletal protein α-smooth muscle actin

(α-SMA). This contractility perisinusoidal constriction and portal hypertension. Endothelin-1 is

an agonist of contractility.

Retinoid loss of HSCS:

Retinoid is stored as retinyl esters "perinuclear droplets".

Lecithin retinol acetyl transferase (LRAT) catalyzes the esterification of retinol into retinyl

ester in liver. Activated HSCs retinyl esters hydrolysis retinol release outside HSCs.

PPARs:

Their expression decreases with the activation of HSCs. Forced expression of PPARγ

TGF-β1 signaling collagen expression.

Adipose differentiation related protein (ADRP):

an intracellular lipid storage protein. Its expression is reduced during HSC activation.

Autophagy:

It produces energy for activated HSCs by digestion of intracellular lipids "LD".

HSCs in inflammation and WBCs chemoattraction

HSCs may produce chemokines that amplify inflammatory responses by inducing migration of

inflammatory cells. HSCs promote ICAM-1- and VCAM-1-dependent adhesion and migration of

lymphocytes.

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The Gut Microbiota/TLR Pathway:

Bacterial translocation is a cardinal feature of chronic liver disease. It produces

chronic inflammatory status.

Microbiota pathogen-associated molecular patterns (PAMPs) e.g. LPS

stimulate TLR e.g. TLR4.

TLR4 signaling TGF-β decoy receptor, BMP and activin membrane bound

inhibitor (BAMBI) TGF-β-mediated HSC activation.

The HSCs activation expression of chemokines and adhesion molecules

recruitment of macrophages to sites of fibrogenesis

Microbiota and HSCs activation stimulate LSECs directly and indirectly through

fibronectin produced from HSCs.

TLR9 is also profibrogenic.

TLR3 and 7 prevent liver fibrosis by stimulation of the NK cells but it was found to

downregulated in liver fibrosis.

Triggers of Inflammatory Signaling

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Class Mediator Target Cell and Mechanisms of Action G

ut

mic

ro

bio

ta a

xis

/TL

R

pa

thw

ay

TLR4

Directly stimulates HSC to down-regulate BAMBI and produce

chemokines in BDL and CCl4-induced liver fibrosis

Stimulates KCs to produce proinflammatory and fibrogenic cytokines

that activate HSCs in ALD and NASH

Stimulates LSECs to induce angiogenesis that promotes HSC

activation and fibrosis

TLR2

Stimulates KCs to produce cytokines that activate HSCs in NASH-

Stimulates macrophages in intestine, which promote bacterial

translocation

TLR9 Stimulates KCs to produce cytokines that activate HSCs in NASH-

Stimulates HSCs by host DNA released from apoptotic hepatocytes

TLR3 Stimulates NK cells to produce IFN-γ that induces antifibrotic effect by

killing HSCs

TLR7 Stimulates DCs to produce type I IFN that inhibits liver fibrosis

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Inflammatory Cytokines: IL-1β:

IL-1β is secreted by the macrophages. It is powerful inflammatory cytokine.

IL-1β TIMP1 and BAMI fibrosis

TNF-α:

Main functions are hepatocyte apoptosis, immune cell activation, and HSC activation.

TNF-α TIMP1 and BAMI fibrosis

IL-17:

CD4+ Th17 T cells secrete IL-17 activation of NF-κB and STAT3 17A stimulates both

KCs and HSCs to produce IL-6, TNF-α, and TGF-β.

IL-20:

It acts on both the hepatocytes and the HSCs.

IL-20 promotes the activation, proliferation, and migration of HSCs.


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Inflammatory Cytokines: IL-22:

IL-22 is implicated in the defense against bacterial infections by inducing antimicrobial

proteins, including β-defensin, as well as in cell proliferation, tissue repair, and wound healing.

IL-22 HSC senescence.

Liver cirrhosis is associated with IL-22, especially complicated with ascites, hepatorenal

syndrome, spontaneous bacterial peritonitis, and reduced survival.

IL-33:

Injured hepatocytes IL-33 secretion ILC2 IL-13 HSCs activation.

TGF-β:

Macrophages TGF-β Smad pathway HSCs activation type I and III collagen

production.

It represses NK cells prevent HSCs apoptosis.

TGF-β is secreted in inactive form that is activated with MMPs, pH, trombospondin-1, reactive

oxygen species (ROS), or αv integrins.


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IFNs Types:

Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ).

Antifibrotic effects:

IFN-γ proliferation and α-SMA expression. It also NK cell activation.

IFN-α basal and TGF-β-induced collagen gene transcription in HSCs.


40

Class Mediator Target Cell and Mechanisms of Action

Infl

am

ma

tory

cy

tok

ine

s

IL-1β

Up-regulates TIMP-1 and down-regulates BAMBI in HSCs

Promotes HSC survival

Promotes lipid accumulation and cell death in hepatocytes during NASH and ALD

IL-33 Secreted from damaged hepatocyte

Stimulating ILC2 to produce IL-13 that, in turn, activates HSCs

TNF-α

Induces hepatocyte apoptosis

Up-regulates TIMP-1 and down-regulates BAMBI in HSCs

Promotes HSC survival and proliferation

Activates liver macrophages

IL-17

Stimulates KCs and HSC to produce IL-6, TNF-α, and TGF-β

Activates NF-κB and STAT3 in KCs and HSCs

HSC activation through STAT3

IL-20 Promotes activation, proliferation, and migration of HSCs

Prevents hepatocyte injury

IL-22 Induces HSC senescence through STAT3-p53

HSC senescence inhibits liver fibrosis.

IFN-γ Suppresses HSC proliferation and activation

Activates NK cells to promote HSC killing

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Chemokines: The main function is the recruitment of immune and nonimmune cells into the inflamed sites.

CCL2:

Is produced by the HSCs and the NK cells recruitment of macrophages and the monocytes to

the liver.

It induces also HSCs activation.

CCL5:

Activates its receptor (CCR1 and CCR5) on macrophages and HSCs activation of HSCs.

Chemokine (C-X3-C motif) ligand (CX3CL)1/chemokine (C-X3-C motif) receptor 1

interaction on liver macrophages negatively regulates liver inflammation

Other cytokines:

CXCL16 and its receptor, CXCR6, CCL20, and its receptor, CCR6, and CXCL9 and CXCL10

and their receptor, CXCR3,


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Class Mediator Target Cell and Mechanisms of Action C

hem

ok

ines

CCL2 (MCP-1) Macrophage and HSC recruitment

HSC activation

CCL5 Macrophage and HSC recruitment

HSC activation

CXCL9 Suppresses HSC activation

Inhibits angiogenesis that inhibits liver fibrosis

CXCL10 Promotes hepatocyte death and HSC activation

Inhibits NK cell-mediated HSC inactivation

CX3CL1 Prolongs KC survival

Promotes anti-inflammatory property in KCs

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Downstream Signaling Pathways Linking Inflammation and Fibrosis

NF-κB

NF-κB is a transcription factor that acts as a key regulator of inflammation and cell death.

It is stimulated by TLRs, IL-1β, and TNF-α.

Effect of activation

Physiological prevents hepatocyte apoptosis.

Pathological over-activation liver inflammation by increased production of

proinflammatory, NF-κB-regulated cytokines, such as TNF-α, IL-1β, and IL-6.

Effect on the HSCs:

NF-κB HSC survival fibrogenesis.

NF-κB HSC survival TLR4- and TNF-α-mediated down-regulation of BAMBI

TGF-β fibrogenesis.


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Downstream Signaling Pathways Linking Inflammation and Fibrosis

c-Jun N-Terminal Kinases

c-Jun N-terminal kinases (JNKs) are mitogen-activated protein kinases.

They are activated by TLRs, IL-1β, TNF-α, ROS, and saturated free fatty acids (FFAs).

In HSCs, JNK exerts a direct profibrogenic role by promoting PDGF, TGF-β, and angiotensin

II–induced proliferation, α-SMA expression, and/or collagen production.

JNK plays important roles in TGF-β- and PDGF-mediated Smad2 and Smad3

phosphorylation.

JNK also regulates hepatic steatosis, cell death in hepatocytes, and inflammatory gene

expression in immune cells, all of which modulate liver fibrosis.


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Ayman Alsebaey, MD.

Lecturer of Hepatology,

National Liver Institute.

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Health & Medicine

[2016] pathogenesis of liver fibrosis