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Hepatic stem cells
Hepatic stem cells have been hypothesized to exist in the liver, taking part in its regeneration after injury. In rat, “oval cells” were observed in injury where hepatocytes did not proliferate. It was proposed that, in certain contexts, these stem cells would differentiate into new hepatocytes when hepatocyte proliferation was not sufficient. But their existence has not been formally proven, and some extensive studies failed to find any evidence of their presence (Grompe, 2014; Yanger et al., 2014). In the mouse, it is now demonstrated by lineage tracing that the hepatocyte epithelium is capable of robust regeneration. When hepatocyte proliferation is experimentally impaired during regeneration, cells originating from the biliary epithelium (cholangiocytes) can act as Hepatic stem cells and take over hepatocyte regeneration, in particular in chronic liver injury (Raven et al., 2017).
Liver Capsular Macrophages (LCMs)
In the late 80s, a population of MHCII positive cells was described and ascribed to the dendritic cell family (Prickett et al., 1988). In the following years, different papers described these populations under different names from KCs to stellate cells, and their real identity remained elusive. Recently, they have been described as a population of macrophages distinct from the KC pool (S. A. Park et al., 2018; Sierro et al., 2017).The authors propose that they are paramount to the defence of the liver against pathogens derived from the peritoneum as opposed to KCs that take charge of blood borne intrusions
They express key macrophage markers (F4-80, CX3CR1, CSF1R, MHCII) but express low levels of CD11b and the DC CD11c marker and lack TIM4. Irradiation followed by bone marrow transplantation, together with liver transplants and parabiosis, showed that these LCMs received significant contribution from the circulation in the absence of inflammation. Intravital microscopy also showed LCM ability to sense bacterial antigens. LCM depletion in the context of infection evidenced their role in controlling bacterial influx through the recruitment of neutrophils (Sierro et al., 2017). These intriguing findings are important for our comprehension of liver macrophages heterogeneity but only a couple of papers have characterised these macrophages so far.
Natural Killer (NK) cells and other innate lymphoid cells(?)
The liver harbours the largest population of NK cells in the body. They are lymphocytes with a cytotoxic activity that, contrary to CD8 T cells, do not need prior activation to perform their functions. As such, NK cells are now considered as the cytotoxic effector branch of the family of innate lymphoid cells (ILCs). NK cells were first identified in the liver by electron microscopy observation of rat liver, in the mid 1970s (Wisse et al., 1976). They were described as “pit cells” of irregular shape, found attached to the endothelium. They were found in the sinusoidal space, in close vicinity with the Kupffer cells, with which they often interacted. Their cytoplasm presented numerous small granules. These cells did not appear phagocytic and were not proliferating, except in certain pathological contexts, such as regeneration after partial hepatectomy (Peng et al., 2016). Although NK cells are rare in circulation, they represent up to half of the liver lymphocytes. In the liver, it has been shown that two lineages coexist: one that is dependent on the transcription factor Eomes and can be found in circulation, and another that is under the control of T-bet and is restricted to the liver sinusoid (Daussy et al., 2014). Together, they take part in the maintenance of a tolerogenic environment in the liver environment and in the response to microbial infections.
Transcriptional control of KC identity
Resident macrophages throughout the body share a core transcriptional programme that defines the macrophage identity. This transcriptional signature is acquired as soon as macrophages start to differentiate from EMP derived progenitors during development (Mass et al., 2016). It includes transcription factors like Maf and Batf3, but also receptors like CSF1R and CX3CR1. This core programme further includes the expression of a large array of cytokine receptors and antibody (Fc) receptors (T’Jonck et al., 2018).
The well-studied PU.1. is the master regulator of macrophage development. It is upregulated during macrophage differentiation and controls the expression of many genes, notably the CSF1R receptor. (Ross et al., 1998; Zhang et al., 1994). After tissue seeding, macrophages start to express transcriptional programs specific to their tissue of residency. DNA binding protein inhibitor (Id3) and LXRa have been specifically associated with liver macrophages. LXRa expression is itself under the control of ZEB2, a transcription factor involved in the maintenance of tissue specific macrophage identity (Scott et al., 2018). A deficiency in Id3 resulted in a reduction in Kupffer cell numbers while other macrophage populations appeared unaffected. In these mice an upregulation of Id1, which is co-expressed with Id3 was observed (Mass et al., 2016). As Id3 and Id1 are under the control of TGFß, at least in tumour contexts, this cytokine could play a role in establishing KC identity (Strong et al., 2013).
Interestingly, most of the KC-specific signature was only observed once the premacrophages had colonised the liver and differentiated into macrophages, and not at the precursor stage, suggesting that their tissue specialisation was not predetermined but emerged in contact with the tissue environment (Figure 9). This is reinforced by the important transcriptomic changes detected in macrophages after birth and even during weaning, when major modifications of the organ function and environment occurs. Analysis of the chromatin landscape of different adult tissue macrophage populations captured these tissue specific regulatory signatures and correlated with the functions they entail. CLEC4F expression was notably specific of KC while SpiC, a transcription factor involved in red blood cell efferocytosis was highly active in spleen red pulp macrophages and to a lesser extend KCs. Irradiation followed by bone marrow transplant experiments further supported a role of the microenvironment in shaping the chromatin landscape and the transcriptome (Lavin et al., 2014).
Clearance of dead and senescent cells
KCs routinely perform the removal of aged cells from the liver and the circulation. They are notably involved in the removal of aged and/or damaged erythrocytes and platelets. The mechanisms of platelet removal have recently been elucidated, shedding light on the essential role of Macrophage Galactose Lectin (MGL) and the Ashwell Morell Receptor (AMR). Platelets continuously circulate through the liver sinusoids, and transiently contact KCs before exiting. But as they age, they lose their sialic residues, and tend to get stuck on KCs. They are then phagocyted. This process seems to be of importance for clotting efficiency throughout the body, as impairment of this removal leads to an accumulation of desilyated platelets and an increase duration of bleeding. (Deppermann et al., 2020).
In the liver itself, senescent cells, especially hepatocytes, may accumulate, in physiological ageing or over the course of chronic diseases. Senescent cells are in irreversible cell cycle arrest, and they express proteins such as p16, p21 and p53, that prevent cell cycle progression while also inhibiting programmed cell death (apoptosis). Senescence can be triggered by DNA damages, ROS induced lesions, or extensive DNA damage (Stahl et al., 2018). It has been proposed to be a defence mechanism to prevent the proliferation of damaged cells that could turn into cancer. Senescent cells excrete a variety of molecules called Senescence Associated Secretory Phenotype (SASP). Senescent cells help initiate tissue remodelling : produced during development, they help regulate embryogenesis (Davaapil et al., 2017; Muñoz-Espín et al., 2013) and in injury contexts they promote tissue repair (DiRocco et al., 2014). But their abnormal accumulation has been involved in the onset of age-related diseases (Muñoz-Espín & Serrano, 2014). Their timely removal is thus required to maintain tissue homeostasis and KC have been shown to participate in this process after senescent cells where targeted by NK cells in the context of liver cancer (Xue et al., 2007).
Csf1rMeriCreMer Rosa26Tomato
To pulse label our cells of interest, we used inducible Cre lines : Csf1rMeriCreMer Rosa26Tomato (Qian et al., 2011), and CCR2CreERT2 Rosa26Tomato (Croxford et al., 2015; Lacerda Mariano et al., 2020). In these mice, the Cre is only active for a short period of time after the injection of tamoxifen. Thus, the cells that retain the labelling in the long term after the injection are the very same cells (or the descendants) that were pulsed. Mer-iCre-Mer is a double fusion protein between the murine mutant estrogen receptor (mer) and the improved Cre recombinase (Shimshek et al., 2002). CRE-ERT2 is a fusion protein that combines the cre recombinase with a mutated human estrogen receptor (Qian et al., 2011; Zhao et al., 2006).
In the absence of the estrogen analog Tamoxifen, these fusion proteins are restricted to the cytoplasm by the HSP90 chaperon protein. Upon injection of tamoxifen, the interaction between them is disrupted, and the cre recombinase can enter the nucleus to recognize the floxed regions it can act on.
CSF1R is the receptor for Colony Stimulating Factor 1 (CSF1) also known as MCSF (Macrophage Colony Stimulating Factor. It is expressed in macrophages. By injecting tamoxifen in steady state, when Kupffer cells are the only macrophages in the liver, we selectively label them. Monocytes are also labelled, but their labelling efficiency decreases rapidly several days after pulse, due to their short half-life. This model can then be used to look at the maintenance of Kupffer cells throughout time or injury as any dilution of the proportion of KC labelled by the reporter would suggest the contribution of newly arrived, non-labelled cells.
CCR2CreERT2 Rosa26Tomato
In these mice, the Cre recombinase is under the control of the CCR2 promoter. Upon hydroxytamoxifen injection, monocytes are efficiently labelled (around 80-90% are tomato positive). This labelling then rapidly decreases, as these cells are short lived. Pulse-labelling these cells before a challenge (KC depletion or liver injury) allows to track their contribution to the maintenance or recovery of the KC pool. Monocyte contribution will translate into an increase proportion of labelled cells among KCs.
Table of contents :
ACKNOWLEDGEMENTS
ABBREVIATIONS
INTRODUCTION
1. Macrophages, swiss army knifes of the innate immune system
1.1. The innate immune system
1.2. The origins of Macrophages
1.3. Inflammation
1.4. Ageing and the innate immune system
2. The liver
2.1. General considerations
2.1.1. Development
2.1.2. General anatomy
2.1.3. Functions
2.2. Liver cell types
2.2.1. The hepatocytes
2.2.2. The hepatic stellate cells
2.2.5. Innate immune cells
2.2.5.2. Liver Capsular Macrophages (LCMs)
2.2.5.3. Neutrophils
2.2.5.4. Dendritic Cells (DCs)
2.2.5.5. Natural Killer (NK) cells and other innate lymphoid cells(?)
2.2.5.6. Monocytes
2.2.5.7. Infiltrating Macrophages
2.3. Liver diseases
2.3.1. Acute injury and liver failure
2.3.2. Fibrosis and cirrhosis
2.4. Liver Ageing
3. Kupffer Cells
3.1. First and foremost, Phagocytes
3.2. Transcriptional control of KC identity
3.3. The Kupffer Cell Niche
3.4. CSF1 signalling
3.5. Non-immune functions
3.5.1. Clearance of dead and senescent cells
3.5.2. Iron recycling
3.5.3. Lipid removal
3.6. Functions in tissue repair
3.7. Tools to study KC maintenance
3.7.1. Fate mapping
3.7.2. Flt3Cre Rosa26YFP
3.7.3. Csf1rMeriCreMer Rosa26Tomato
3.7.4. CCR2CreERT2 Rosa26Tomato
3.7.5. Selective depletion
3.7.5.1. Plexxikon
3.7.5.2. Genetic models
AIMS AND OBJECTIVES
RESULTS
1. Kupffer Cell Maintenance in Ageing
2. Preliminary data: Kupffer cell maintenance after PLX depletion
3. Preliminary data: Maintenance of Kupffer Cells in Acetaminophen Induced Liver Injury
3.1. Acute Acetaminophen Induced Liver Injury
3.2. Repeated Acetaminophen Induced Liver Injury
4. Methods (Maintenance after PLX5622 and Injury)
CONCLUSION
DISCUSSION
1. Effects of inflammation on Kupffer Cell viability
1.1. Apoptosis
1.2. Pyroptosis
1.3. Necroptosis
2. Determinants of IMs persistence and differentiation into KCs
2.1. Absence of competition
2.2. An adequate niche
2.3. Inhibition of infiltrating macrophage clearance
2.3.1. The role of CSF1/CSF1R
2.3.2. The role of CX3CL1/CX3CR1
3. Age-associated ultrastructural changes in liver
3.1. LSEC fenestrations
3.2. Liver fibrosis
3.3. Ageing is a dynamic and multi-factorial process
4. KC loss in ageing: impact in age related diseases
4.1. Lipid accumulation
4.2. Senescent cells and fibrosis
4.3. Relevance in age related human diseases
5. Challenges in studying KC specific functions in injury
5.1. Inflammation and depletion
5.2. Heterogeneity beyond ontogeny
6. Perspectives
6.1. In situ observation of KCs
6.2. Macrophage transplant
6.3. Phagocytosis and cellular longevity
6.4. Repeated Injury
REFERENCES
RESUME DÉTAILLÉ EN FRANCAIS
ABSTRACT
RESUME
