Study of the fate of resident macrophages and
monocytes upon partial liver resection and their impact
on hepatocarcinoma outgrowth
Thesis submitted by Jean-François Hastir
in fulfilment of the requirements of the PhD Degree in Biomedical and
Pharmaceutical sciences (“Docteur en Sciences Biomédicales et
Pharmaceutiques”)
Academic year 2019-2020
Supervisor: Professor Véronique Flamand
…men, who flatly despise reason, who reject and turn away from understanding as naturally corrupt, these, I say, these of all men, are thought to, lie most horrible! To possess light from on High. A Theologico-Political Treatise, Baruch de Spinoza (1632 - 1677), translated by R. H. M.
Acknowledgments
The entirety of this work was realized with the complete support of my family. I wish to thank my mom, dad and sister for their help during the years necessary for its completion.
I secondly wish to thank the professor Véronique Flamand, as well as the entirety of her research team, Sandrine Delbauve, Arnaud Khöler, Justine Smout, Clara Valentin and David Torres. I can proudly say that it was an honor working in a team with such competent and brilliant searcher.
I wish to thank all the people working at the Institute for Medical Immunology for the great working conditions as well as my friends and all the people I love. I hope they will be indulgent but the complete list would be too long to stand here. I’m sure they will recognize themselves. Thank you guys, without you things would have probably gone harder.
I also want to thank Yves Zorza and Clarisse Locoge whose contribution in making the man that I am today is probably greater than what they think.
Table of contents Table of contents ... 4 List of abbreviations ... 6 I. Introduction ... 8 1. Immunity ... 8 1.1. Innate immunity ... 8
1.2. Innate immune cells ... 9
1.2.1. Granulocytes ... 10
1.2.2. Dendritic cells ... 10
1.2.3. Innate lymphoid cells ... 11
1.2.4. Monocytes/Macrophages ... 12
1.3. Adaptive immunity ... 12
2. The liver ... 14
2.1. Cellular components of the liver ... 15
2.1.1. Hepatocytes ... 15
2.1.2. Liver sinusoidal endothelial cells ... 16
2.1.3. Hepatic Stellate cells ... 17
2.1.4. Kupffer cells ... 19
2.1.5. Natural killer cells (NK cells) ... 23
2.1.6. NKT cells ... 25
2.1.7. Dendritic cells ... 26
2.1.8. Conventional T lymphocytes ... 28
2.1.9. γδ T Lymphocytes ... 29
3. Primary liver cancers ... 30
3.1. Cholangiocarcinoma ... 31
3.2. Hepatocellular Carcinoma ... 32
4. Liver regeneration ... 34
4.1. Clinical implications of liver regeneration ... 38
5. Cytokines ... 40
5.1. Tumor Necrosis Factor-α ... 40
5.1.1. Complex I ... 41
5.1.2. ComplexIIa/b ... 42
5.1.4. Inflammation and tumorigenesis ... 45
5.2. Interleukin-6 ... 46
5.3. Interleukin-1β ... 50
5.3.1. Inflammasome and pyroptosis ... 52
II. Research objectives ... 55
III. Results ... 56
1. Experimental set-up and models ... 56
1.1. Model of hepatectomy ... 56
1.2. Choice of tumoral model ... 57
2. Hepatocarcinoma induces a tumor necrosis factor-dependent Kupffer cell death pathway that favors its proliferation upon partial hepatectomy ... 59
3. Complementary observations ... 61
3.1. Liver regeneration in IL-6 KO mice ... 61
3.2. Rejection of the tumor is impaired in CD3 KO mice ... 62
3.3. Liver regeneration in Batf3 KO mice ... 62
3.4. Positive control staining of p-MLKL (Ser345) ... 63
IV. Discussion ... 64
1. Tumor development model ... 64
2. Impact of liver regeneration on tumoral growth/tumor rejection ... 66
3. Nature of Kupffer cells disappearance following PH ... 69
4. IL-6 importance for Kupffer cells survival and tumor rejection ... 72
5. Tumor growth modifies partial hepatectomy-induced death of KC ... 74
6. Differential recruitment of monocyte-derived cells is dictated by death pathway activation in KC ... 77
7. Possible clinical applications ... 79
8. Dendritic cells and liver regeneration ... 81
9. Kupffer cells potential interactions with other cells ... 83
V. Bibliography ... 85
A
ALR: Absent in melanoma 2-Like Receptor
APAP: N-acetyl-p-aminophenol / Acetaminophen APC: Antigen Presenting Cell
ARF: ADP-Ribosylation Factors
B
Batf3: Basic leucine zipper transcriptional factor ATF-like 3
BCR: B Cell Receptor
C
c-IAP: cellular Inhibitor of Apoptosis CD: Cluster of Differentiation
CCL: Chemokine Ligand
cDC: conventional Dendritic Cells CDC37: Cell Division Cycle 37
CRIg: Complement Receptor of the immunoglobulin superfamily
CTNNB1: Catenin Beta 1
D
DAMP: Dammage Associated Molecular Pattern DC: Dendritic Cell
DEN: Diethylnitrosamine DNA: Deoxyribonucleic acid DRP1: Dynamin-Related Protein 1
E
ECM: Extracellular Matrix EGF: Epidermal Growth Factor
ERK: Extracellular Signal-Related Kinase
F
FADD: Fas-associated death domain FcγRIIb2: Fc-gamma receptor IIb2
FERM: 4.1 protein, Ezrin, Radixin, Moesin
FLS2: LRR receptor-like serine/threonine-protein kinase FLIP: FLICE inhibitory proteins
G
Gab1: GRB2-associated-binding protein 1 gp130: Glycoprotein 130
GSDMD: Gasdermin D
H
HBEGF: Heparin-Binding EGF-like Growth Factor HBV: Hepatitis B Virus
HCC: Hepatocellular Carcinoma HCV: Hepatitis C Virus
HGF: Hepatocyte Growth Factor HMGB1: High–Mobility Group Box 1 HSC: Hepatic Stellate Cell
HSP: Heat Shock Protein
I
IAP: Inhibitor of apoptosis ID3: Inhibitor of DNA3 IFN: Interferon
IGF: Insulin-like Growth Factor
IGFBP: Insulin-like Growth Factor Binding Protein Ig: Immunoglibulin
IκBα: Inhibitor of the κB alpha IKK: Inhibitor of the κB Kinase IL: Interleukin
IL-xR: Interleukin-x Receptor ILC: Innate Lymphoid Cells
iNOS: inducible Nitric Oxide Synthase IRAK: IL-1 receptor-associated kinases IRI: Ischemia Reperfusion Injury
J
JAK: Janus Kinase/Just Another Kinase JNK: cJun N-terminal kinases
K
KC: Kupffer cell
L
LPS: Lipopolysaccharide
LSEC: Liver Sinusoidal Endothelial Cell
LUBAC: Linear Ubiquitin Chain Assembly Complex LXRα: Liver X receptor α
M
MAPK: Mitogen-Associated Protein Kinase Mdr2: Multidrug resistance 2
MHC: Major Histocompatibility Complex MKK: Mitogen-activated protein Kinase Kinase MLKL: Mixed Lineage Kinase-Like
MMP: Matrix Metalloproteinase
MyD88: Myeloid Differentiation primary response 88
N
NE: Norepinephrine
NEMO: NF-kB Essential Modulator NF-κB: Nuclear Factor κB
NK: Natural Killer NKT: Natural Killer T NLR: NOD-Like Receptor
NLRP: Nucleotide-binding domain and Leucine-rich Repeat pyrin containing Protein
NOD: Nucleotid-binding Oligomerization Domain
P
PAMP: Pathogen Associated Molecular Pattern
PGAM5: Phosphoglycerate Mutase Family Member 5 PI3-K: Phosphatidylinositol-3-Kinase
Poly I:C: Polyinosinic:polycytidylic acid PRR: Pattern Recognition Receptor
K
KRAS: Kirsten Rat Sarcoma
R
RAF: Rapid Accelerated Fibrosarcoma RHIM: RIP homotypic interaction motif RIG: Retinoic acid-Inducible Gene
RIPK: Receptor-Interacting Protein Kinase RNA: Ribonucleic Acid
ROS: Reactive Oxygen Species
S
SFSS: Small-For-Size-Syndrome
SHP2: Src Homology region 2 domain-containing Phosphatase-2
STAT: Signal Transducer and Activator of Transcription
SOCS: Suppressors Of Cytokine Signaling SODD: Silencer of Death Domain
T
TAB: TAK1 binding protein
TACE: Tumor Necrosis factor-α Converting Enzyme
TAK: Transforming growth factor β-Activated Kinase
TCR: T Cell Receptor
TGF-β: Transforming Growth Factor-β Th: T helper
TIR: Toll/IL-1 Receptor TLR: Toll Like Receptor
TNF-α: Tumor Necrosis Factor-α
TNFR: Tumor Necrosis Factor Receptor
TRADD: TNF Receptor Associated Death Domain TRAF: TNF-R–Associated Factor
TP53: Tumor Protein 53 Treg: Regulatory T cell
U
uPA: urokinase-type Plasminogen Activator
V
VEGF: Vascular Endothelial Growth Factor
W
WT: Wild type
WNT: Wingless-related integration site
Y
YAP: Yes-associated protein
Z
Dendritic cells Monocytes Granulocytes B cells T cells NK/ ILC Megakaryocyte Erythrocyte
Figure 1
Global model of hematopoiesis. Trajectories of differentiation of hematopoietic stem cells (HSC) are represented on the top graph. The continuum spectrum of differentiation is represented on the bottom graph. Red circles indicate Flt3 expression by the corresponding cell type. The fate choices that are available to hematopoietic stem cells are a continuum as shown by the short central arc below the yellow arrow. The fates choices of each of the indicated progenitors are shown as a shorter arc that spans the end cell types each progenitor cell population can give rise to. HSC: Hematopoietic Stem Cell; MPP: Multi-Potent Progenitor; LMPP: Lymphoid-primed Multi-potent Progenitor; MEP: Megakaryocyte-Erythrocyte Progenitor; CMP: Common Myeloid Progenitor; GMP: Granulocyte-Macrophage Progenitor; CLP: Common Lymphoid Progenitor; EPLM: Early Progenitors with Lymphoid and Myeloid potential; ILC: Innate Lymphoid Cell; DC: Dendritic Cell; Eo: Eosinophil; CFU: Colony Forming Unit; Mon: Monocyte; M-CSFR: Macrophage–Colony Stimulating Factor Receptor; EpoR: Erythropoietin Receptor; GM: Granulocyte-Macrophage; ProB: progenitor B-lymphocyte; B: B-lymphocyte; T: T-lymphocyte. Adapted from Tsapogas, P., Mooney, C. J.,
I. Introduction
1. Immunity
The immune system is a vast group of diverse components present in an organism that aims at protecting it from any harmful exterior elements such as pathogenic bacteria, viruses, parasites or toxins. It is also an important protection against the development of tumoral cells. The ability to protect the organism relies on the capacity of the components to make the difference between healthy, non-harmful self-elements and harmful non-self-elements. This distinction allows the organism to eradicate or control other dangerous organisms, cells or compounds and to tolerate self-cells and innocuous factors. The immune system is traditionally divided in two major branches: innate and adaptive immunity. Both branches are in constant contact with each other and rely mainly on leukocytes (“white blood cells”) to mediate their effects. Just as red blood cells and platelets, leukocytes are derived from progenitors present in the bone marrow. The broadly accepted consensus is that hematopoietic stem cells (a population which itself contains different subtypes) are the multipotent progenitor cell type. These cells give rise to the rest of the hematopoietic lineage through the step-wise generation of oligo-potent progentors (like the common lymphoid progenitor/CLP and the common myeloid progenitor/ CMP) (Figure1). These progenitors exist within a continuum of differentiation and give rise to fully differentiated cell types like T lymphocytes, dendritic cells or monocytes (Figure 1).
1.1. Innate immunity
Global mechanisms of the innate and immune responses. Innate immune response uses barriers, phagocytosis of pathogens, or direct cell killing. Adaptive responses uses either antibodies produced by B cells, cytokines produced by CD4+ T lymphocytes and cytotoxic CD8+ T lymphocytes to protect the organism. Adapted from A. K. Abbas, A. H. Lichtman, Basic Immunology. 4th edition
Elsevier, 2012.
alert other leukocyte populations of the invasion. Its action is rapid and unspecific to the type of pathogen (Figure 2). Pathogen detection is mediated via pattern recognition receptors (PRR) a group of receptors that recognize pathogen associated molecular patterns (PAMP). PAMPs are highly conserved structural patterns across microorganisms, absent from the host and essential for the survival of the pathogens. From a functional standpoint, PRRs are divided into 3 groups:
• Signalization PRRs, which induce the expression of genes implicated in the inflammatory response. This group of receptors encompasses the membrane expressed Toll-like Receptor (TLR) family, the DNA-dependent activator of IFN regulatory factors, the NOD-like receptors (NLR), the double-stranded RNA-dependent protein kinase and other cytosolic receptors like the RIG-1-like helikases. • Secreted PRRs, which bind to the microbial membrane and facilitate its phagocytosis
or its recognition by elements of the complement like Mannan-binding lectin.
• Endocytic PRRs, which mediate capture and degradation of pathogens in lysosomes (e.g. flagellin receptor FLS2 or the Macrophage Mannose Receptor).
PRRs are also able to recognize an important amount of different endogenous molecules and factors that are released upon inflammation process, after tissue injury or cellular stress. These molecules are grouped under the generic term “damage associated molecular patterns” (DAMPs). DAMPs can come from various cell compartments like the extracellular matrix (heparan sulfate), the cytosol (Heat Shock Proteins/HSP), the nucleus (HMGB1), the mitochondria (Formyl peptide) or the endoplasmic reticulum (Calreticulin).
1.2.1. Granulocytes
Granulocytes constitute one of the first groups of leukocytes pathogens encounter during invasion. As such they constitute major actors of inflammation process. Granulocytes are subdivided in 3 groups:
• The neutrophils, which clear pathogens either via phagocytosis, degranulation of bactericidal molecules (e.g. cathepsin or lysozyme) or secretion of extracellular trap (made of DNA and histones) 1.
• The eosinophils, implicated in the protection against helminths and in some allergic diseases such as asthma 2.
• The basophils, implicated in the protection against multicellular parasites and also in the pathogenesis of asthma and allergic reactions 3.
1.2.2. Dendritic cells
• The conventional DC (cDC /further subdivided in cDC1 and cDC2), which express high levels of MHC molecules and are potent activators of T lymphocytes. This subset is able of cross-presentation of antigens (presentation of internalized antigens on MHC I molecules).
• The plasmacytoïd DC, which constitute major producers of type I interferon (IFN) and express relatively lower levels of MHCII with relatively limited capacity to capture and present antigens.
• The inflammatory DC generated after the recruitment and differentiation of monocytes under inflammatory conditions.
Of note, this classification has been recently challenged by a publication showing that cDC2 acquire a hybrid phenotype, sharing gene expression, and function with cDC1 and monocyte-derived cells upon inflammation6. Nevertheless, all these groups have different morphologies and produce different cytokines.
1.2.3. Innate lymphoid cells
Innate lymphoid cells are a heterogeneous group of cells that has the peculiar ability to secrete cytokines classically produced by activated T CD4+ lymphocytes without requiring the same conditions of activation. This group is further classified into 3 subpopulations mainly based on their cytokine productions:
• The ILC2 group, which secretes important quantity of IL-5 and Il-13 upon stimulation. This group is implicated in the resistance against parasites (e.g. N. brasiliensis) 8, tissue repair following influenza virus infection 9 and development of asthma.
• The ILC3 group, which secretes IL-17A/F and or IL-22 upon activation 10,11. This group encompasses cells necessary for the secondary lymphoid organ organogenesis and tissue repair 12. ILC3 also have an important role in the maintenance of intestinal homeostasis 13.
1.2.4. Monocytes/Macrophages
Monocytes constitute a multipotent population circulating in the blood. They have the ability to migrate into the various tissue of the organism and to differentiate there depending on the cytokines (immunity signaling molecules) and microenvironment signals. Differentiation of monocytes can either give rise to macrophages or certain types of dendritic cells 14. Their role is to phagocyte pathogens, or debris, produce various types of cytokines such as interleukins (IL)-6, IL1-β and tumor necrosis factor (TNF)-α and promote the activation of adaptive immunity. They are also implicated in the termination of immune responses and return to homeostasis due to their ability to produce an anti-inflammatory cytokine, IL-10 14. Pro-inflammatory macrophages are regrouped under the label M1 (classically activated macrophages) while macrophages with a restorative, anti-inflammatory profile are branded M2 (alternatively activated macrophages) 15.
1.3. Adaptive immunity
branch of immunity has also the ability to generate immune memory, which will allow for a faster and more efficient elimination of previously encountered pathogens (Figure 2). Its functions mainly rely on B lymphocytes, which finish their maturation in the bone marrow and T lymphocytes, which finish their maturation in the thymus. Both group clonally express an extremely variable antigen receptor (B cell receptor/BCR and T cell receptor/TCR) generated by somatic mutation and recombination in their DNA. This receptor will interact with antigens in order to activate the naïve lymphocytes. In the case of T cells, this activation will require the encounter with a professional APC presenting a peptide-derived antigen in a MHC molecule.
B lymphocytes secrete immunoglobulin (Ig) in order to counter extracellular pathogens. These Ig bind specifically to the corresponding antigen, neutralize the pathogen and facilitate its elimination via phagocytosis by innate immune cells. Naïve B cells require direct encounter of the antigen with their BCR in order to produce Ig. Upon activation, B cells will proliferate and differentiate into Ig producing plasmocytes.. T lymphocytes are subdivided in two functionally and phenotypically distinct groups based on the expression of a TCR co-receptor (CD4 or CD8):
• The CD4+ T lymphocytes (also called T helper/Th cells), which modulate the various immune responses via the production of cytokines.
Figure 3
2. The liver
The liver is a crucial organ in multiple important functions for the organism. It is vital for the homeostasis of carbohydrates due to its ability to store the glycogen generated from glucose and to reintroduce it into blood circulation after glycogenolysis. It can convert amino acids, produce albumin and other various proteins such as acute phase proteins, important for inflammation, or blood coagulation factors. It is also essential for cholesterol synthesis to produce triglycerides and to create the lipoproteins, a fatty acids and steroid hormones carrier. Besides these metabolic aspects, the liver is also producing bile and is a crucial detoxification organ because it can neutralize numerous toxic metabolites via the cytochrome p450 enzymes. 16
Figure 4
Adpated from BrainKart.com The liver - Structure and Function of Digestive System
Figure 5
TJ: tight junctions Adapted from Chapter 7 - Paracellular Channel in Organ
of a very efficient immunological filter able to remove these elements from the blood circulation. The remaining 25% of blood comes from the systemic circulation via the hepatic artery and is rich in oxygen 17. This mix of blood circulates from the artery and the portal vein through hepatic sinusoid in direction of the central vein (Figure 4). From the central vein, the blood will reach the hepatic veins and later the inferior cava vein.
Hepatic sinusoids are fenestrated capillary vessel with reduced diameter constituted by the LSECs. This reduced diameter allows for a slowed down blood flux inside the sinusoids that only reach around half the speed of other capillary tracts in the organism. These peculiar conditions favor exchange between hepatocytes and the sinusoidal spaces. A perisinusoidal space, known as space of Disse, exists between hepatocytes and LSECs (Figure 5). It contains hepatic stellate cells, and plasma components of the sinusoid, which can be absorbed by hepatocytes. It is thus an important space for the exchanges between circulating blood and hepatocytes. Kupffer cells were initially thought to be present mainly on the outside of the space of Disse, patrolling the sinusoids with only some protrusions present inside of it. However, a recent publication has revealed that a significant part of the cells were actually inside the space of Disse and maintained a very close relation with stellate cells 18 .
2.1. Cellular components of the liver
2.1.1. Hepatocytes
Figure 6
Figure 7
(A) Structure of mouse liver sinusoidal endothelial cells under scanning electron microscope. (B) Higher magnification picture of a liver sieve in LSEC illustrating the lack of basal lamina between hepatocytes and LSEC. (C) In ultra-thin sections, the sinusoid is delimited by endothelial cell thin cytoplasmic processes [e] which contain fenestrations (small arrows). They appear as open connections between the sinusoidal lumen [S] and the space of Disse [sD]. In the space of Disse between the LSEC and the hepatocytes, hepatic stellate cells [HSC, marked with an asterix] processes may be observed. Adapted from Warren, A. et al., T lymphocytes interact with hepatocytes through fenestrations in murine liver
sinusoidal endothelial cell Hepatology (2006).
TJ: tight junctions adapted from Decaens et al., Which in vitro models could be best used to study hepatocyte polarity? Biol. Cell 100, 387–398 (2008).
basal membrane of hepatocytes faces LSECs while their apical poles contribute to the formation of the bile canaliculli 19. In normal physiological conditions, hepatocytes have a very slow turnover rate that can be reverted by cell loss.
Under inflammatory conditions (e.g. liver injury or pathogen invasion), hepatocytes cross-talk with multiple immunity cellular subsets. They can acquire the expression of class II MHC molecules in addition to the ubiquitous class I expression and stimulate CD4+ T lymphocytes20. Overall, these interactions will result in the release of pro-inflammatory signals and proliferation-associated cytokines, which will in turn lead to the activation of the repair pathways.
2.1.2. Liver sinusoidal endothelial cells
LSEC constitute the largest population of liver non-parenchymal cells (50%) 21. Although bone marrow derived cells can help replenish the LSEC population when necessary, they are usually derived from liver resident progenitors 22. These highly specialized endothelial cells
form the thin wall of the hepatic sinusoid. Their unique morphological feature resides in their numerous fenestrations (100 to 150 nm diameter pores) associated with the lack of basal lamina propria, which creates an access between the space of Disse and the blood flow 23,24, facilitating the bidirectional transfer of substrates between blood and parenchymal cells 24,25 (Figure 7). This fenestration is notably regulated by VEGF derived from stellate cells and hepatocytes 26,27.
Figure 8
Electron microscopic photographs of hepatic stellate cells. Lipid droplets (arrows) in HSCs. Adapted from Murakami,
K. et al., Therapeutic effects of vitamin A on experimental cholestatic rats with hepatic fibrosis. Pediatr. Surg. Int. 27, 863–870 (2011).
Maximal intensity projection of mouse liver sections stained for Clec4F (Kupffer cells /red), Desmin (Hepatic stellate cells/green), CD31 (LSEC/ blue), and DAPI (Nucleus/gray). Scale bar 20µm Adapted from Bonnardel et al., Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell
Identity on Monocytes Colonizing the Liver Macrophage Niche, Immunity (2019).
substance by swelling or contracting, therefore reducing the vessel lumen 31, and also have immune functions. They express the endocytic Fc-gamma receptor IIb2 (FcγRIIb2)32, various PRRs, such as scavenger receptors 33 and several TLRs 34 and can produce cytokines (e.g., TNF- α, IL-6, and IL-1β). Although several studies have reported their role in the generation of hepatic immune tolerance 35,36 with an the ability to present antigens in class II MHC
molecules these observations were never made with in-vivo set-up and this feature of LSECs is controversial at present time 37–39.
Considering liver pathologies, change in the LSEC barrier with loss of fenestration has been observed in fibrosis induced by alcoholic liver disease 40. Under this condition, HSC were
normally activated and fibrosis could progress. Since HSC revert back to quiescent state when normal LSECs are present 41, their role as determining elements of fibrosis has been proposed. LSECs are also participating in the liver regeneration and they favor hepatocyte proliferation by secreting HGF and angiopoietin-2 42,43. They are also known to improve colorectal cancer metastasis invasion of the liver since these cells have the ability to bind to the lectin expressed on LSEC surface 44.
2.1.3. Hepatic Stellate cells
Figure 10
Panel 1 is the maximal intensity projection of Clec4fCre/+-Rosa26TdT/+ mouse liver sections with CD31 (LSEC/blue) and TdTomato (Kupffer cells/ red). In panel 2 is the overlay of confocal microscopy and electron microscopy allowing identification of the Kupffer cells and delimiting of the blood vessels. In panel 3 is a 2D electron microscopy slice showing the Kupffer cells (red) interacting with an HSC (green) outside of blood vessels (blue) and located in between hepatocytes (yellow). In panel 4 is the 3D reconstruction of the electron microscopy. Scale bar, 10µm. Adapted from
that HSC derive from bone marrow cells, these cells encompass mesenchymal stem cells as well as hematopoietic stem cells and conflicting results as to which progenitor is responsible for the adult HSC pool exist49,50.
Under physiological conditions, hepatic stellate cells are in close contact with Kupffer cells in the space of Disse and with hepatocytes 18 with no signs of proliferation (Figure 9 and 10). They adapt their responses to physiological fluctuations in the liver tissue and notably control the sinusoidal diameter and blood flow 31,51. Following liver injury, a wide variety of immune cells secreted factors (such as KC secreted TNF-α and IL-1β) or damaged epithelial cells released factors (e.g. ROS) are able to activate HSC. Once activated, they transdifferentiate into a myofibroblastic state. These activated HSCs lose their vitamin A reserve, are proliferative, contractile, and produce abundant extracellular matrix components such as types I and III collagens, inhibitors of matrix metalloproteinases and inflammatory mediators 46,52,53. As such, perpetuation of the activated phenotype can lead to the development of liver fibrosis and the role of HSC in this pathology is of major importance. Indeed, HSCs give rise to the vast majority of myofibroblasts in parenchymal and cholestatic liver fibrosis 54 and various studies have linked apoptosis or senescence of HSC with the resolution of the disease 55,56.
2.1.4. Kupffer cells
Kupffer cells (KCs) are the liver resident macrophages and constitute the biggest macrophage population of the organism. Their main role is to be “gatekeepers” of the organ and as such, they express a wide array of TLRs, scavenger receptors, complement receptors and antibody receptors that notably allow them to detect, bind, capture and kill potentially armful pathogens. For a long time viewed as patrolling inside the sinusoids, recent paper published by Bonnardeel and colleagues 18 have revealed their intricate localization with stellate cells within the space of Disse as well as interactions with hepatocytes (Figure 9 and 10). From there, KCs send protrusions within the sinusoid allowing them to scan the blood flow components. One of the major bacteria clearance mechanism used by KCs relies on the complement receptor of the immunoglobulin superfamily (CRIg)60. Pathogens are not the sole target of KCs as they are also of major importance to bind and clear apoptotic neutrophils61. KCs are also potent APCs since they express MHC-I, MHC-II and other co-stimulatory molecules essentials for complete T lymphocyte activation. In basal state, continuous LPS exposure limits KCs ability to trigger efficient adaptive immune responses 62,
which allows for a limitation of inflammation in the liver. This tolerogenic state is reverted when exposed to other TLR-ligands (i.e. detection of other pathogen components) or pro-inflammatory cytokines 62,63.
development of hepatocellular carcinoma (HCC). However, the implication of KCs in alcoholic liver disease (a possible step toward the development of cirrhosis and therefore HCC) is crucial since TNF-α is a major element driving alcoholic liver disease lesions and activated KCs are the major source of TNF-α production and secretion after activation of the LPS/TLR4 signaling pathway. KCs have also been implied in the regulation of hepatocytes metabolism. Hepatocytes cultured with KCs deficient for the peroxisome proliferator activated receptor δ (PPARδ), a receptor required for the full expression of the immune phenotype of activated KCs, showed a decreased rate of fatty acid oxidation (a metabolic pathway allowing for the generation of energy from fat). This was furthermore strengthened by the observation that livers containing PPARδ KO KCs showed the presence of steatosis and an increased rate of extractable triglycerides 65. KCs are also important elements regarding tumor surveillance and can eliminate circulating neoplastic cells reaching the liver. Due to its position and anatomical structure, this organ constitutes a site easily reached by various types of gastro-intestinal cancer cells. Experiments in rodent models have shown that in early stages of colorectal cancer liver metastasis, KCs displayed tumoricidal activity by recruiting and activating NK cells. These activated cells would produce pro-inflammatory cytokines that, in turn, activate KCs by enhancing their phagocytic capacity and sensitize tumor cells to cytotoxic effect 66. The concept of an early anti-tumoral role is furthermore supported by the observation made in models where early depletion of KCs with gadolinium chloride favored tumor induction and tumor growth 67. Interestingly, depletion at later stages
Different models of The first models of fetal macrophages ontogeny. Red arrow indicates the proposed major path of ontogeny and differentiation in each model. Cell colors are matched to their proposed origins. Model 1 considers the contribution of fetal liver monocytes unlikely, models 2 and 3 propose that these cells represent the main precursor of fetal macrophage populations, with the exception of microglia, which arise predominantly from c-Myb-independent yolk sac macrophages. (FL) Fetal liver, (YS) Yolk sac, (EMPs) erythro-myeloid precursors, (HSC) Hematopoietic stem cells (Mo) Monocytes, (Mac) Macrophages, (Ex) Embryonic day x.Adapted from Ginhoux, F. & Guilliams, M. Tissue-Resident Macrophage Ontogeny and Homeostasis. Immunity 44, 439–449 (2016).
(Programmed Death-Ligand 1). They can also secrete trophic factors for the tumor cells such as TGF-β (transforming growth factor-β) or VEGF (vascular endothelial growth factor) (reviewed in 68).
KCs mostly originate from fetal liver progenitors and self-maintain during life, a feature shared by the lung alveolar macrophages 69. Macrophages ontogeny is a complex topic due to the fact that embryonic hematopoiesis in mammalian occurs in sequential successive waves. Due to the limitations in the fate-mapping techniques used in various studies, different interpretations and therefore different models of hematopoiesis in rodent exist (Figure 11/ reviewed in 70). The consensus is that 3 successive waves occur during development. The first
the para-aortic splanchnopleura with the generation of immature hematopoietic stem cells. Mature cells will be obtained at embryonic day 10.5 and these precursors will then colonize the fetal liver and establish definitive hematopoiesis. They will also colonize the fetal bone marrow and ultimately generate adult hematopoietic stem cells. In sharp opposition with the model proposing a role for erythro-myeloid precursors as the main precursors of tissue-resident macrophages, it was proposed that this third wave of fetal hematopoietic stem cells was responsible for the generation of the macrophage pool in various adult tissues with the exception of microglia and, partially, epidermal Langerhans cells. Yet, the experiments supporting this concept have been criticized for the inability of the fate-mapping technique used to correctly distinguish between hematopoietic stem cells and erythromyeloid progenitors.
KCs alongside the transcription factor ID3 (inhibitor of DNA 3) 18,72. Interestingly, KCs do not seem to be an homogeneous population of cells and two distinct groups have been delineated based on the cell marker CD11c even though no differences were seen either in distribution or ability to capture bacteria 73.
2.1.5. Natural killer cells (NK cells)
Two populations of NK cells are found in the liver under physiological conditions: • The circulating conventional NK cells.
• The liver-resident NK cells, also named “pit cells” in some studies.
Figure 12
Electron microscopy of liver NK cells in a normal rat liver. Organelles are situated on one side of the central nucleus. The other side of the cell is composed of hyaloplasm without organellesNK cells make contact with sinusoidal endothelial cells, and microvilli sometimes protrude into the space of Disse (top left). NK cells possess characteristic electron-dense granules. The pit cell in this figure measures approximately 8 µm in length. Adapted
Liver-resident NK cells have a diameter between these of lymphocytes and granulocytes, an irregular shape reminiscent of these of KC and contain numerous granules (lysosomes) that remain together on one side of the cytoplasm 80. They are localised outside of the space of Disse, attached to the endothelium from where they send microvilli toward parenchymal cell microvilli as if they were anchoring their position (Figure 12). They can often be seen neighboring or making contact with KC, but unlike these cells they lack endocytic or phagocytic abilities 80. Interestingly, elimination of KC from the liver in experimental models showed a concomitant decrease in the number of liver-resident NK cells and in in vitro assays, NK cells showed significant signs of activation during culture in KC conditioned medium 81. These striking evidences suggest a parallel between KC and liver resident NK cell
reactions.
The role of liver-resident NK cells in the various pathologies or challenges faced by the organ is of dual nature. On one hand, they can drive the liver toward pro-inflammatory and immunocompetent responses. This is well demonstrated during liver regeneration since suppression of NK cell activity or their depletion promotes liver regeneration 82,83 whereas the poly I:C or viral infection-induced activation negatively regulates liver regeneration in an IFN-γ-dependent manner 83. In fulminant hepatitis, the interactions between KC and NK cells result in severe injury of the organ 84 and upon response to viral challenge NK cells favors hepatocyte necrosis and hepatic failure 85. These cells are also highly cytotoxic against tumor
Figure 13
Model depicting the opposing roles of type I and type II NKT cells in inflammatory diseases in the liver. Type I NKT cells are rapidly activated following liver injuries induced by alcohol, high-fat diet, ischemia and/or gut-derived microbial products. Liver-resident antigen-presenting cells, such as KCs, and TLRs/cytokines mediate their activation, which results in the cytokine/chemokine- dependent recruitment of myeloid cells (CD11b+Gr-1+) and neutrophils, and the activation of HSC and NK cells. In contrast, type II NKT cells are activated following the presentation of self-lipids, such as sulfatide and LPC, which results in the induction of a cross-regulatory pathway that blocks inflammation. cDCs, conventional DCs; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; KC, Kupffer cell; LPC, lysophosphatidylcholine; NK, natural killer; NKT, natural killer T cells; OPN, osteopontin; TLR, toll-like receptor. Adapted from Bandyopadhyay, K.et al., NKT cell subsets
2.1.6. NKT cells
NKT cells are innate-like T cells that co-express TCR-αβ chains and typical NK cell markers. Due to this unique phenotype, they are perceived as an intermediate cell type between innate and adaptive immunity. They are found in higher proportion in the liver sinusoid where they participate in the control of the induction and/or the prevention of inflammation in the organ
91,92. They recognize either exogenous and endogenous lipid antigens bound to the MHC-like
molecule CD1d 93. NKT cells are classified in two main subsets:
• Type I (invariant) NKT cells, which express a conserved semi-invariant αβ-TCR. • Type II (diverse) NKT cells, with variable αβ-TCR
Proportion of both types is variable across species with a higher proportion of type I in mouse while human has a higher proportion of type II. While the exact nature of all the ligands recognized by both type is still under investigation, multiple studies suggest that type I and II NKT cells recognize distinct molecular motifs 94–96.
Table 1
Adapted from Rahman, A. H. & Aloman, C. Dendritic cells and liver fibrosis. Biochim. Biophys. Acta - Mol. Basis Dis. (2013).
Figure 14
Conventional and non-conventional dendritic cell subsets and ontogeny. Subsets of conventional DCs (cDCs) and non-cDCs derive from a common DC precursor (CDP), which itself derive from the macrophage DC progenitor (MDP). They require the transcription factor FMS-like tyrosine kinase 3 ligand (FLT3L) for development. The genetic signature of cDCs from different tissues is similar but differs from that of plasmacytoid DCs (pDCs), monocytes and macrophages. Although some pDCs share a common developmental precursor with cDCs, they are mostly derived from the common lymphoid progenitor (CLP). Under specific conditions, monocytes can differentiate into Monocyte-derived DCs (MoDCs). This group encompass various cells including including TNF/iNOS-producing (TIP)-DCs as well as a difficult-to-define, heterogeneous group of cells collectively termed inflammatory (TIP)-DCs (i(TIP)-DCs). Like classical monocytes, MoDCs are dependent on the macrophage colony-stimulating factor (M-CSF), Specific surface markers are indicated below each cell type , with those in parentheses indicating heterogeneous or tissue-restricted expression. Below each population, the dominant (but not sole) function of each population is indicated. BDCA , blood DC antigen; CLEC9A , C-type lectin domain-containing 9A ; CX3CR1, CX3C-chemokine receptor 1;EPCAM, epithelial cell adhesion molecule; *Human-specific marker. Adapted from
Type I NKT cells are prone to induce immuno-competent responses (Figure 13). This is notably the case in HCV and HBV infections and is most likely due to their IFN-γ secretion
103,104. Yet, this ability can also reveal itself as harmful and lead to the lack of hepatocytes
proliferation, death and utterly, liver damage 105,106. Moreover type I NKT cells are important in the generation of ischemia-reperfusion injury and limit liver regeneration 107,108.
Type II NKT cells are reactive to pollen-derived lipids, self-glycolipid sulfatide, some other β-linked glycolipids and self-phospholipids 109. This population acts as an immune-modulator and therefore participates in the protection against liver damage, notably through tolerization of cDC (Figure 13). This is especially well demonstrated in ischemia-reperfusion injury 107
and is also supported by their lack of ability to activate T, B or NK cells following their stimulation with sulfatides 110. Due to these observations, type II NKT cells can be considered as the regulatory counterpart of type I NKT cells.
2.1.7. Dendritic cells
As previously described, DC constitute a heterogenic cellular group originated from hematopoietic stem cells broadly divided in cDC and pDC. The cDC group is further divided in cDC1 and cDC2 subgroups based on the expression of cellular markers, which may vary between species (Table 1 and Figure 14). At steady state in mice, pDC constitute the most prevalent population of DC, followed by cDC2 and cDC1 111. In human, the amount of pDC
is much lower (with similar levels as in the blood) and DCs are sparsely distributed through the liver. They are mainly found in the portal regions and less frequently in the parenchyma
112. The composition of DC populations is dynamic and may vary depending on the liver
TNFα and inducible nitric oxide synthase (iNOS) in the setting of bacterial and parasitic infection 113,114.
Most of the actual knowledge on liver DC population is based on observations made in other lymphoid and non-lymphoid tissues 115,116. Yet, they are seen as participating in the
maintenance of a tolerogenic environment in the liver since DC in this organ produce higher levels of IL-10117,118. DC ability to produce IL-10 was notably shown in ischemia/reperfusion injury mouse models119. Exposure to IL-10 can suppress the activity of effector T-cells and can also turn DC into tolerogenic DC. The liver also seems to display a unique role in DC traffic as observed in rodent model where DC undergo a blood-lymph node translocation via the hepatic sinusoids 120. This translocation may act as a biological concentrator of circulating DC into the regional hepatic nodes and therefore facilitate the presentation of antigens present in the liver.
Figure 15
Following immunization, cDC1 (XCR1+ DC), activate naive CD4+ T cells via the interaction between MHCII (containing
a specific antigen) and TCR. CD4+ T cell license cDC1 (XCR1+ DC) through a CD40-dependent process. Licensed
DCs express higher levels of MHC and co-stimulatory molecules and can recruit CD8+T cells to their cognate DC through the secretion of CC-chemokine ligand 3 (CCL3), CCL4 and CCL5. Activation of CD8+ lymphocyte is mediated via the interaction between MHCI and TCR. Licensed DCs also secrete IL-12 and IL-15, which increase the expression of CD25 on CD8+ T cells and promote cell survival, respectively. Enhanced expression of CD25 facilitates the response of CD8+ T cells to IL-2 and promotes CD8+ T cell survival and their ability to proliferate on secondary antigen encounter. Both CD4+ T cells and CD8+ T cells act as sources of IL-2 and, in response to cellular antigens, are also capable of directly interacting through CD40–CD40 ligand (CD40L). Adapted from Laidlaw et al.,The multifaceted role
to increase DC IL-10 and TNF-α production 128. DCs also enhance estrogen receptor expression and inhibit IFN-γ production of T cells in this setup resulting in liver regeneration promotion128.
2.1.8. Conventional T lymphocytes
Conventional T lymphocytes encompass two major populations scattered throughout the parenchyma, as well as in the portal tracts: the CD4+ T cells and the CD8+ T cells. Both populations express various TCR αβ chains, used for antigen recognition in the context of MHCI (for CD8+ T cells) or MHCII (for CD4+ T cells) molecules 129 (Figure 15). Typically, CD8+ T cells outnumber CD4+T cells in the liver 21,130. While the frequency of
effector/memory cells is higher in this organ than in the blood, the liver contains also a significant amount of naïve cells 130. Under physiological conditions, the liver contains a significant amount of lymphocytes that undergo cell death and also some cells that effectively participate in the maintenance of homeostasis of the organ and in local or global immune responses 130. The tolerogenic status of the liver is further supported by the induction of
tolerance in CD8+ T cells rather than effector function under LSEC-mediated presentation of antigens in non-inflammatory conditions 36.
Figure 16
2.1.9. γδ T Lymphocytes
Hepatic γδ T cells are a highly localized population of lymphocytes found throughout the parenchyma and portal tracts. They account for 15-25% of total liver T cells whereas peripheral blood γδ T cells only account for 2-10%133. They possess a TCR composed of γδ chains instead of the conventional αβ chains. Although γδ T cells can recognize antigens presented on MHC molecules like other conventional T cells, their TCR repertoire is more restricted 134. They are also able to recognize numerous nonpeptide antigens and stress-induced ligands without the need for TCR engagement 134,135. Hepatic γδ T cells exhibit mixed phenotypes (Vγ1, Vγ4, and Vγ6 in mice and Vδ1 and Vδ3 in humans) and in humans they are more mature than their counterparts in peripheral blood 136.
Two main distinct subsets of γδ T cells are described based on the cytokine they produce: • The IL-17A-producing γδ T cells
• The IFN-γ producing γδ T cells
recurrence of HCC 140, their role in the development of fibrosis is still unclear. This is due to relatively low amount of data available and the contradiction between results and their interpretation 141–143.
3. Primary liver cancers
Figure 17
Approach to management of (A) intrahepatic and (B) perihilar cholangiocarcinoma HCC=hepatocellular carcinoma. CA 19-9=carbohydrate antigen 19-9. Razumilava, N. & Gores, G. J.
3.1. Cholangiocarcinoma
Cholangiocarcinoma is an aggressive cancer of the biliary tract. A vast majority of the cases of cholangiocarcinoma (90%) have the characteristics (both molecular and histologic) of adenocarcinoma 145. While the development process of the disease is complex, the actual knowledge points out that the pluripotent hepatic stem cell is the most likely progenitor cell line for cholangiocarcinoma with a tendency for the disease to develop in the setting of cholestasis and chronic inflammation. At present time the disease is divided in two subtypes depending purely on anatomic criteria: the intrahepatic type (accounting for 25% of the cases) and the extrahepatic type (accounting for the remaining 75%)146. Some studies of the somatic mutations present in the tumor have shown that among the usual cancer-associated genes, mutations in the tumor protein 53, a transcription factor in responsible for the maintenance of genome integrity, was the most common. Several genetic changes have also been described in the literature including the cell survival signaling pathways (with copy number variation in KRAS, BRAF, RAS, MAPK, and MET), cell fate and differentiation (Notch signaling) and also epigenetic changes147. Activation of inflammatory pathways causing overexpression of cytokines and STAT3 have also been described 147.
Figure 18
Barcelona Clinic Liver Cancer (BCLC) staging and treatment strategy The BCLC system establishes a prognosis in accordance with the five stages that are linked to first-line treatment recommendation. The expected outcome is expressed as median survival of each tumour stage according to the available scientific evidence. Note that liver function should be evaluated beyond the conventional Child-Pugh classification or the Model of End-stage Liver Disease (MELD) score. For most treatment options, compensated liver disease is required to obtain optimal outcomes. The sole option that could be applied irrespective of liver function is liver transplantation. ECOG PS=Eastern Cooperative Oncology Group Performance Status. *Patients with end-stage cirrhosis due to heavily impaired liver function should be considered for liver transplantation. In these patients, hepatocellular carcinoma might become a contraindication if it exceeds enlistment criteria. †Currently, sorafenib followed by regorafenib has been shown to be effective. Lenvatinib has been shown to be non-inferior to sorafenib, but no second-line option after lenvatinib has been explored. Forner, A., Reig, M. & Bruix, J. Hepatocellular carcinoma. The
not surgical candidates for curative resection, chemotherapy and targeted radiation are considered (Figure 17).
3.2. Hepatocellular Carcinoma
Development of hepatocellular carcinoma is a complex multistep process that involves genetic changes through the progressive accumulation of mutations in the context of increased cell death and division associated with fibrotic deposition, persistent inflammation and oxidative stress 144,148. From a pragmatic standpoint, one of the most common mechanism
leading to HCC is the development of cirrhosis and therefore chronic liver disease associated with persistent inflammation and oxidative stress 149. This is especially true in the context of alcohol consumption and HCV infection and explains the development of most HCV related HCC 144,148. Surprisingly, the same explanation is not necessarily valid in HBV infection and a direct oncogenic effect of HBV has explained the occurrence of HCC in non-fibrotic liver
150. Regarding specific mutations, while a great heterogeneity exists between patients
suffering from HCC, several cancer driver genes have been identified using next generation sequencing 148. Aberrant telomerase reverse transcriptase activation is the most common somatic alteration observed in HCC (70%). The next most found mutations affect the TP53 (30%) and CTNNB1 (30%), a gene coding for β-catenin. Other mutations found in a lower fraction of HCC samples affect members of the WNT pathways. Besides these somatic alterations, deregulation in chromatin remodeling has also emerged as a major feature in HCC. Unfortunately, the molecular classifications of HCC that have been proposed so far were unable to predict progression or recurrence of the disease.
Figure 19
(Left panel) Schema of TACE under portal vein occlusion for HCC with significant arterioportal shunts. T: tumor; PV: portal vein. Murata, S. et al. Transcatheter Arterial Chemoembolization Based on Hepatic Hemodynamics for
Hepatocellular Carcinoma. ScientificWorldJournal (2013). (Right panel) Schematic illustration of design of
transarterial chemoembolization (TACE) agents, in terms of polymeric beads or hydrogels, to occulate the blood supply of liver tumor. Adapted from Chen, Yet al., Recent advances on polymeric beads or hydrogels as
chemoembolization, surgical resection or transplantation and tumor ablation. The choice of treatment will mainly be dependent on the development stage of the disease and specific patient related aspects such as portal vein hypertension, compensated or uncompensated cirrhosis etc 144. If a line of treatment is not suited for a specific case the next most suitable option will be envisaged. Advanced stage patients (as determined by the Barcelona Clinic Liver Cancer, see Figure 18) receive tyrosine-kinase inhibitor but their effectiveness is still limited 151. Intermediate stage patients follow chemoembolization procedure, a treatment that associates the injection of chemotherapy with blockade of the arterial blood supply (Figure 19). More than 50% of the patients following this procedure achieve extensive tumor necrosis, which improves their survival 152,153. Yet, tumor vascularization increases following
Figure 20
B
(A) Cryosurgery of hepatocellular carcinoma with corresponding ultrasound imaging of the probe within the tumor. (B) Radio frequency ablation of hepatocellular carcinoma. (C) Liver transplantation before and after surgical procedure. (D) Partial hepatectomy of hepatocellular carcinoma. Adapted from Gurakar A. et al., Hepatocellular
Carcinoma (Liver Cancer) open web access source: hopkinsmedicine.org
A
tumoral progression which in turn limits transplantation success. Finally, hepatic resection constitutes the treatment of choice for hepatocellular carcinoma in patients without cirrhosis (Figure 20D). In this case, important resections are possible without inducing life-threatening complications. It is important to highlight here that for patients with decompensated cirrhosis hepatic resection is formally contraindicated, and liver transplantation should be considered. While resection gives great overall survival chance for patients with up to 70% survival at 5 years, tumor recurrence constitute a major problem for this approach with complications in 70% of the cases at 5 years 155,156. Recurrence includes true recurrence due to dissemination and “de novo” tumor development within the oncogenic liver. At the present time, the only option that has demonstrated to prevent recursion effectively is liver transplantation. This underlies the fact that the development of strategies aiming at reducing the risk of recurrence is a paramount element of the surgical resection approach. Both technique of liver transplantation and liver resection relies heavily on the ability of the liver to regenerate, which allows for large resection and transplantation of only a fraction of the donor organ.
4. Liver regeneration
decipher the complex and multiple interconnected mechanisms occurring during liver regeneration as well as the cellular types involved in it. It is worth noting that following partial hepatectomy, the important metabolic needs of the liver must be fulfilled since no growth factor nor cytokines alone is enough to provide the energy and amino acids required for a correct cell-division. This concept is supported by studies where amino acids administration leaded to hepatocyte proliferation, whereas protein restriction limited the regenerative process and the discovery that amino acid regulate hepatocyte proliferation through modulation of cyclin D1 expression 157,158. Moreover, the regenerative process is also dependent on circadian rhythms. While DNA synthesis in hepatocytes peaks 36 hours following resection, the transition from G2 to mitosis occurs at the same time of the day whatever the timing of the resection is 159.
Induction of liver regeneration itself is a complex phenomenon and the delayed regeneration observed in various experimental set-up has lead to the concept that there is no unique single signaling pathway at the origin of liver regeneration and that there is an important redundancy and overlap between signals, which in the end provide for the missing activation of one experimental set-up blocked pathway. Modifications of the arterial blood-flow 160, circulating LPS 161 and DAMPs, elements of the complement system 162 as well as circulating glucose 163 have been proposed as elements participating in the induction of liver regeneration. The importance of the adaptor protein MyD88 has also been demonstrated in the initiation of regeneration, yet the exact activating molecule responsible for this activation is yet to be determined 164.
Figure 21
Downstream signaling pathway of HGF-Met signal. HGF, hepatocyte growth factor; shp2, Src homology region 2 domain-containing phosphatase-2; Grb2, growth factor receptor-bound protein 2; Stat3, signal transducer and activation of transcription-3; PI3K, phosphoinositide 3-kinase; Cdc42, cell division control protein 42 homolog; Rac1, Rac family small GTPase 1; MEK, mitogen-activated protein kinase kinase; mTOR, mechanistic target of rapamycin; ERK, extracellular signal-regulated kinases; Akt, protein kinase B. Kato, T. Biological roles of
pathways are presented in further chapters). The production of these cytokines depends mainly on KCs 167 and therefore innate immune cells are key player in driving the regenerative process 168. After partial hepatectomy, TNF-α binds to the TNF receptor 1 (TNFR1) on Kupffer cells. Other ligands are objectively suspected to be part of the TNFR1 signaling since mouse model using TNF-knockout mice showed no impairment of liver regeneration whereas TNFR1 knockout mice had multiple defects 169,170. Nevertheless, TNFR1 activation leads to the activation of NF-kB and production of IL-6 165. IL-6 acts on hepatocytes via the IL-6 receptor, activating STAT3 166,171. In turn STAT3 activation in hepatocytes leads to the activation of early gene expression 172 hepatocyte protection 173 inhibition of apotosis and proliferation. Importantly, all of the IL-6 effects are not solely dependent on STAT3 and extracellular signal-related kinase 1 and 2 (ERK1/2) pathways is also activated and plays an important role in cell proliferation 166.
Alongside cytokine signaling, a variety of mitogenic growth factors are required for hepatocytes to progress through cell cycle, override the G1 restriction point and allow hepatocytes to pass into the S phase. The major growth factors for liver regeneration includes: • The hepatocyte growth factor (HGF), produced by stellate cells and acting in a
paracrine and endocrine fashion on hepatocytes 174.
• The epidermal growth factor (EGF), produced by Brunner’s gland in the duodenum175.
insulin-Epidermal growth factor receptor (EGFR) and its downstream signaling proteins. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; JAK, Janus kinase; STAT, signal transducer and activator of transcription; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; Akt, protein kinase B; P, phosphorylation. Arrows indicate activation/induction. Eitsuka, T., et al., Synergistic Anticancer Effect of Tocotrienol
Combined with Chemotherapeutic Agents or Dietary Components: A Review. Int. J. Mol. Sci. (2016).
like growth factor (a mitogenic and hepatoprotective protein important for cell-cycle regulation) binding protein (IGFBP) which can be activated by IL-6 and HGF 179,180.
As for EGF signaling, multiple ligands are able to bind and activate EGF receptor and induce notably the activation of STAT5, ERK or PI3-K downstream of the receptor 175 (Figure 22).
These ligands encompass various molecules that have overlapping functions. EGF, transforming growth factor (TGF)-α, heparin-binding EGF-like growth factor (HBEGF), and amphiregulin are all EGF receptor ligands; all of which can compensate the lack of each other during liver regeneration. This is notably demonstrated with TGF-α which is able to compensate HBEGF absence 181. Redundancy between HGF and EGF signaling has also been
proposed. These molecules are also assisted by a variety of auxiliary mitogens whose absence may delay liver regeneration. They encompass a wide array of molecules such as bile acids
182, norepinephrine (NE) 183, endothelial growth factor (VEGF) 184, insulin-like growth factor
(IGF) 185, estrogen 186 and serotonin 187 (See Table 2).
While the mechanisms inducing and controlling liver regeneration have been a major focus in the field, the termination of the regeneration process upon liver having attained a size sufficient to cover the functional needs of the organism is still a subject of debate.
The elements described at present time encompass:
• The suppressors of cytokine signaling (SOCS) 3. SOCS 3 is a major element for the control of hepatocytes during regeneration 188. It acts as a negative feedback loop 189 after its induction by IL-6 by preventing STAT3 phosphorylation and activation. • The transforming growth factor-β (TGF-β). TGF-β role in the termination process is
Tang, Hong Tao, Y. et al., Liver Regeneration: Analysis of the Main Relevant Signaling Molecules. Mediators Inflamm. (2017).
there is a lack of clear evidence that TGF-β on its own constitute the primary stimulus required for the termination process.
• The mitogen-activated protein kinase kinase 4 (MKK4). MKK4 is a ubiquitously expressed component of the stress activated MAP kinase signaling, with a structure closely related to other MKK family members 192. It constitutes a master regulator of hepatocytes proliferation and control their entry as well as their progression into the cell cycle 193.
4.1. Clinical implications of liver regeneration
The main implication in medicine for liver regeneration is i) liver transplantation and ii) partial ablation of a fragment of the liver in order to remove a tumoral site as stated previously. This can be used either in the case of primary liver cancer or in the case of metastasis development in the liver. The main challenge faced when running transplantation protocol remains the overwhelming demand for transplantable organ compared to the low donated organ supply. Partial liver transplantation from a living donor is increasingly used in order to alleviate this problem. This approach requires liver regeneration and the key element for its success relies on transplanting an adequate-size, functioning graft while still maintaining a high safety profile for the donor. Failure to do so would lead to the development of small- for-size-syndrome (SFSS). SFSS is the result of an inability of a small graft to regenerate, and it limits the role of partial liver transplant 194. From a clinical stand
While resection and transplantation constitute viable therapeutic options, especially for patients with early diagnosis, the major problem faced with the surgical approach remains the recurrence rate of the disease155. Development and progression of liver cancer is a highly complex molecular process and it is linked with major alterations in cellular signaling pathways, namely, the RAS-MAPK pathway, the PI3K pathway, the WNT/ β-catenin pathway, the IGF pathway, the HGF/c-MET pathway and the growth factor-regulated angiogenic pathway are implicated in cancer development 196–198. Since most of these signaling pathways are activated during liver regeneration and based on the observations made in various preclinical models, the concept of regeneration as well as post-operative stress and ischemia-reperfusion injury responses helping the recurrence phenomenon has been developed 199–202. This seems very likely since regeneration generates a microenvironment favorable for tumorgenesis and tumor propagation through cell activation, proliferation, migration and angiogenesis 199,202–204. The cytokines (TNF-α and IL-6) and growth factors (HGF, EGF) produced during liver regeneration have been associated with tumor aggressiveness and metastasis 196,197,205. Moreover, the activity of MMP during liver regeneration induces the breakdown of ECM molecules that usually help maintain the dormant state of the tumor 206,207. Regeneration might also directly facilitate hepatic carcinogenesis through the activation of progenitor cells 208.
tested in the context of relapse (including chemoembolization). Repeated hepatectomy in carefully selected patients appeared has giving the best survival chances (with resectability rate ranging from 10% to 77%) 209–211. Another important factor to take into account when considering liver regeneration and tumorigenesis is the clinical context in which the regeneration takes place. While animal models focus mainly on regeneration in healthy liver in order to decipher the complex mechanisms taking place, most patients suffer from viral infection or excessive alcohol consumption. These additional environmental cues might as well influence regeneration and the risk of tumor recurrence and more investigations are required to delineate their proper impact.
5. Cytokines
5.1. Tumor Necrosis Factor-α
Varfolomeev, E. & Vucic, D. Intracellular regulation of TNF activity in health and disease. Cytokine (2018).
Figure 23
LUBAC
