The complex function of macrophages and their subpopulations in metabolic injury associated fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD), recently also defined as metabolic dysfunction‐associated fatty liver disease (MAFLD), is a major health problem, as it affects ∼25% of the population globally and is a major cause of hepatic cirrhosis and thereby liver failure, as well as hepatocellular carcinoma. MALFD comprises a broad range of pathological conditions in the liver, including simple fat accumulation (steatosis) and the more progressive non‐alcoholic steatohepatitis (NASH) that can lead to fibrosis development. Cells of innate immunity, and particularly macrophages, comprising the liver resident Kupffer cells and the recruited monocyte‐derived macrophages, play complex roles in NASH‐related inflammation and disease progression to fibrosis. Here, we discuss the recent developments with regards to the function of liver macrophage subpopulations during MAFLD development and progression.


Introduction
Non-alcoholic fatty liver disease (NAFLD) was recently also defined as metabolic dysfunction-associated fatty liver disease (MAFLD). It affects approximately 25% of the population worldwide and is rapidly and continuously increasing; the highest prevalence of the disease is found in South America and the Middle East and the lowest in Africa (Eslam et al., 2020;Younossi et al., 2016bYounossi et al., , 2018. In addition to the health problem, the socioeconomic burden of MAFLD is enormous with annual medical costs summing to about $103 billion in the United States and €35 billion in four European countries (Germany, Italy, France and the United Kingdom), as reported in 2016 (Younossi et al., 2016a). MAFLD encompasses multiple liver pathological conditions spanning from simple hepatic fat accumulation (defined as hepatic steatosis) to the more advanced non-alcoholic steatohepatitis (NASH) (Barreby et al., 2022). NASH, a disease with currently no approved treatment, displays multiple histomorphological features, including hepatocyte death and inflammation, and may further progress to fibrosis; the latter can lead to cirrhosis and liver failure and can also drive hepatocellular carcinoma development (Barreby et al., 2022). NASH is regarded as the manifestation of the metabolic syndrome in the liver because >80% of NASH patients are overweight, >70% are dyslipidaemic and >40% have type 2 diabetes mellitus (Diehl & Day, 2017).
In the context of obesity-related NASH, the adipose tissue exhibits chronic inflammation and releases adipokines and inflammatory cytokines, for instance, leptin and tumor necrosis factor α (TNFα) (Nati et al., 2022). Furthermore, the obese adipose tissue releases free fatty acids into the circulation that may promote lipid accumulation in the liver (Nati et al., 2022). Excess fat accumulation in hepatocytes leads to lipotoxicity, endoplasmic reticulum stress, mitochondrial dysfunction and the production of reactive oxygen species (ROS) (Nati et al., 2022). Lipotoxicity, enhanced inflammatory cytokine release, and additionally, gut-derived bacterial pathogens associated with an altered gut barrier are some factors that act together to promote inflammation, a process believed to be initially orchestrated by the liver resident macrophages, designated Kupffer cells (KC) (Daemen, Gainullina et al., 2021;Nati et al., 2022;Thibaut et al., 2022). Activated KCs may in turn support the recruitment of monocyte-derived macrophages (MoMf) and other immune cells, thereby exacerbating hepatic inflammation and damage (Nati et al., 2022;Thibaut et al., 2022). However, the entire array of mechanisms involved in the initiation of inflammation during NASH progression is not fully understood. Moreover, a recent study has shown that the resident KCs are not pro-inflammatory during MAFLD (Remmerie et al., 2020). Therefore, whether KCs are indeed initiators of inflammation during MAFLD is not entirely clear and warrants further investigation.
The aggravated inflammation can drive the fibrogenic activation of hepatic stellate cells (HSC) and their transdifferentiation into highly proliferative myofibroblasts that also produce high amounts of extracellular matrix, thereby leading to hepatic fibrosis development (Nati et al., 2022). Fibrosis severity is the main factor associated with mortality during NASH (Angulo et al., 2015;Diehl & Day, 2017;Vilar-Gomez et al., 2018). Consistently, NASH patients with an F3 or F4 stage fibrosis display a significantly enhanced risk of liver-related death than those with little or no fibrosis (Diehl & Day, 2017). Liver macrophages are integrally involved in NASH development and progression, playing complex roles; various signalling cascades and pathways regulate their differentiation, proliferation and activation (Thibaut et al., 2022;Wen et al., 2021;Xu et al., 2021). In the present review, we will summarize the functions and heterogeneity of liver macrophage subpopulations and discuss their role in MAFLD development.

Physiological functions of liver macrophages
Macrophages are the most abundant immune cell population in the liver and play crucial roles in both maintaining hepatic homeostasis and promoting liver diseases (Wen et al., 2021). In mice under healthy conditions, 20−40 macrophages accompany every 100 hepatocytes (Krenkel & Tacke, 2017). Liver macrophages can be divided into two subtypes, the tissue resident, self-renewing KCs and the MoMfs; the two subsets can be distinguished based on their cell surface markers (Barreby et al., 2022;Wen et al., 2021). In mice, KCs are identified as CD11b low , F4/80 high , and express C-type lectin domain family 4 member F (CLEC4F) (Scott et al., 2016;Wen et al., 2021). In addition, T-cell immunoglobulin and mucin domain containing 4 (TIM4) and C-type lectin-like receptor 2 (CLEC2) have been identified as KC-specific markers in mice (Tran et al., 2020). KC markers, such as TIM4, are helpful for distinguishing between resident KCs and MoMfs Daemen, Gainullina et al., 2021). For instance, TIM4 is highly expressed in resident KCs of the liver but not in MoMfs; the latter are identified as F4/80 high TIM4 neg Daemen, Gainullina et al., 2021). In NAFLD, a sub-population of MoMfs was defined as monocyte-derived KCs; these cells are identified, at least at the early stages of their differentiation, as CLEC4F pos , TIM4 neg (Daemen, Gainullina et al., 2021). Furthermore, CLEC2 has been identified as an early marker of macrophages that are in the process of becoming monocyte-derived KCs, as shown in Clec4f-DTR mice bearing depletion of resident KCs (Remmerie et al., 2020). Therefore, identification of additional markers specific to only resident KCs would be beneficial to further distinguish these cells from recruited macrophages during NASH.
It is now accepted that KCs originate from yolk sac derived precursors that infiltrate the fetal liver during early embryogenesis, while MoMfs originate from circulating monocytes deriving from the bone marrow that infiltrate the liver (Gomez Perdiguero et al., 2015;Wen et al., 2021). Under steady state conditions, KCs make up the majority of the liver macrophage population, are located in the hepatic sinusoids close to the endothelial cells and are capable of self-renewal, which is regulated by the repressive transcription factors MafB and cMaf (Nati et al., 2022;Soucie et al., 2016). However, when KCs are depleted and the niche is available, circulating monocytes engraft the liver and become self-renewing, long-lived cells (Beattie et al., 2016;Scott et al., 2016). Recently, the transcription factor zinc finger E box binding homeobox 2 (ZEB2) was found to be important for maintaining the identity of KCs, via regulating the expression of the transcription factor liver X receptor α (LXRα) . During NASH, the self-renewing capacity of KCs is impaired; this results in monocyte-derived cells gradually seeding the KC pool (Tran et al., 2020). However, the exact mechanisms and signalling pathways that may lead to reduced KC self-renewal during NASH are unclear. Recently, a further population of liver resident macrophages named liver capsular macrophages (LCM), which are distinct from KCs, was described and found to be located in the hepatic capsule (Sierro et al., 2017). The LCM subset is replenished at steady state by blood-derived monocytes and is defined as MHCII + CD45 + F4/80 + CD11c low CSF1R + TIM4 − cells (Sierro et al., 2017).
Hepatic macrophages promote hepatic homeostasis by performing several functions, including clearance of cellular debris, iron and cholesterol metabolism, removal of aged platelets and damaged red blood cells, defence against gut-derived pathogens and maintenance of immunological tolerance in the liver (Barreby et al., 2022;Papachristoforou & Ramachandran, 2022;Wen et al., 2021). For example, macrophage galactose lectin present on KCs is essential for removing aged platelets from the circulation (Deppermann et al., 2020). Additionally, CLEC4F on KCs functions in the removal of desialylated platelets in the liver via phagocytosis (Jiang et al., 2021). In mice, KCs play a major role in the removal of oxidatively damaged red blood cells from the circulation, via phosphatidylserine-and polyinosinic acid-sensitive scavenger receptors (Terpstra & van Berkel, 2000). A substantial portion of 51 Cr-sodium chromate labelled oxidized red blood cells injected into mice are removed from the circulation by liver KCs (Terpstra & van Berkel, 2000). In rats, red blood cell-derived hemoglobin containing vesicles are rapidly removed by KCs from the circulation (Willekens et al., 2005). Accordingly, KCs express a wide range of scavenger receptors such as CD36, CD68, TIM4, CD163 and CD206, which promote clearance of a variety of ligands (Papachristoforou & Ramachandran, 2022). An essential outcome of the clearance of red blood cells and red blood cell-derived vesicles by KCs is the recycling of iron and the maintenance of iron homeostasis Wen et al., 2021). Furthermore, circulating Gram-positive bacteria are captured and cleared by KCs, a process mediated by the complement receptor CRIg, which binds to bacterial lipoteichoic acid (Zeng et al., 2016). CRIg-positive KCs are also responsible for clearing gut-derived extracellular vesicles that contain microbial DNA, via a C3-dependent opsonization mechanism (Luo et al., 2021). Of note, high fat diet feeding of mice reduces the CRIg-positive macrophage population in the liver, suggesting that bacterial clearance may be diminished during obesity (Luo et al., 2021). Moreover, KCs regulate cholesterol metabolism by producing the protein cholesteryl ester transfer protein (CETP) (Wang et al., 2015). KCs maintain immunological tolerance in the liver by activating regulatory T cells and concomitantly suppressing the activation of effector T cells by other antigen presenting cells (Heymann et al., 2015;You et al., 2008).

Heterogeneity of liver macrophages
Emerging single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) technologies have revolutionized the analysis of the heterogeneity of the cellular composition and have increased the understanding of the diversity of liver macrophages both under healthy conditions and during MAFLD. It should be stated that although both scRNA-seq and snRNA-seq techniques produce high quality data delineating the major cellular classifications, J Physiol 601.7 care is required when directly comparing data sets derived from the two techniques due to differences in the frequencies of the cell types analysed by each technique (Andrews et al., 2022). Particularly, comparison of the transcriptomes identified by scRNA-seq and snRNA-seq techniques performed in healthy human liver samples revealed that scRNA-seq captures a higher percentage of immune cells as compared to snRNA-seq, and in contrast, snRNA-seq captures a higher proportion of hepatic mesenchymal cells and cholangiocytes than scRNA-seq (Andrews et al., 2022).
Healthy liver -mouse. In healthy mice, in addition to the presence of KCs, non-KC macrophages have been identified as being present around the bile ducts and defined as bile duct lipid-associated macrophages (BD-LAMs); these BD-LAMs are Gpnmb + (glycoprotein nmb), as shown by combining scRNA-seq, snRNA-seq and molecular cartography techniques (Guilliams et al., 2022). Gene Ontology (GO) term analysis shows that the KCs are involved in regulating humoral responses while the BD-LAMs are rather broadly associated with immune responses (Guilliams et al., 2022). In accordance to the GO analysis, the BD-LAMs express higher levels of Il1b mRNA as compared to KCs at steady state; despite this, following in vivo TLR4 activation, the BD-LAMs are less responsive compared to KCs with regards to production of both pro-inflammatory and anti-inflammatory molecules (Guilliams et al., 2022).
Healthy liver -human. ScRNA-seq analysis performed in healthy human livers revealed the presence of two distinct CD68 + macrophage populations, specifically, a pro-inflammatory population and an immunoregulatory population (MacParland et al., 2018). The inflammatory macrophage population is characterized by high expression of genes such as CSTA (cystatin A), LYZ (lysozyme), CD74, S100A8 and S100A9 (S100 calcium binding protein A8 and A9, respectively). On the contrary, the tolerogenic macrophage population is characterized by enriched expression of genes such as HMOX1 (haem oxygenase 1), CD5L (CD5 molecule like), VSIG4 (V-set and immunoglobulin domain containing 4), encoding the aforementioned complement receptor CRIg, CPVL (carboxypeptidase vitellogenic like), MARCO (macrophage receptor with collagenous structure), and CD163 (MacParland et al., 2018). Accordingly, pathway enrichment analysis revealed that the tolerogenic macrophages show enrichment of pathways, such as negative regulation of cytokine production and inhibiting leukocyte differentiation, whereas the inflammatory macrophage population displays enrichment of pathways, such as innate immune response, neutrophil activation, bacterial defence, and nuclear factor κB (NF-κB) pathway, among others (MacParland et al., 2018). Similar to the study by MacParland et al., another recent study shows the presence of distinct sub-populations of KCs/macrophages in non-diseased human livers, using scRNA-seq technology (Aizarani et al., 2019). Analysis of the VSIG4 + CD163 + macrophage population revealed two major subsets: the first population is characterized as LILRB5 + (leukocyte immunoglobulin like receptor B5) CD5L + MARCO + HMOX1 high and the second population is characterized as CD1C + FCER1A + (Fc epsilon receptor Ia) (Aizarani et al., 2019). Pathway enrichment analysis and differential gene expression analysis of these two macrophage subpopulations points to a metabolic/immunoregulatory signature in the LILRB5 + macrophage population and an enrichment for genes involved in MHC class II antigen presentation in the CD1C + macrophage population (Aizarani et al., 2019). Furthermore, in agreement with the above studies, VSIG4 has been identified as the best protein marker for human KCs, via cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis; additionally, FOLR2 (folate receptor β), CD163 and CD169 are also useful markers to identify KCs via flow cytometry and microscopy (Guilliams et al., 2022).
NASH liver -mouse. In mouse NASH livers, overlapping populations of macrophages named lipid associated macrophages (LAMs) or NASH-associated macrophages (NAMs) have been identified; both populations are characterized by high expression of triggering receptor expressed on myeloid cells 2 (TREM2), GPNMB and CD9 (Daemen, Gainullina et al., 2021;Xiong et al., 2019). These GPNMB + CD9 + macrophages are rarely present in healthy livers of mice, whereas they represent >60-70% of the macrophages in the NASH livers (Xiong et al., 2019). Of note, in patient samples, hepatic TREM2 mRNA levels are strongly associated with the NAFLD activity score and severity of fibrosis, steatosis, inflammation and hepatocyte ballooning (Xiong et al., 2019). Functionally, TREM2 high NAMs display a signature with strong activation of pathways of lysosomal degradation, endocytosis, MHC class II antigen presentation and extracellular matrix remodelling, as suggested by GO analysis of enriched genes (Xiong et al., 2019). Interestingly, reversal of NASH, either using a dual peroxisome proliferator-activated receptor (PPAR)α/PPARδ agonist or by switching the NASH-inducing diet back to a chow diet results in reduced expression of hepatic Trem2, Gpnmb and Cd9, suggesting that NAMs are highly responsive to dietary and pharmacological interventions that reverse NASH pathologies (Xiong et al., 2019). These GPNMB + CD9 + NAMs were initially described to originate from KCs (Xiong et al., 2019), but subsequent studies suggest that these cells are rather MoMfs. Specifically, another study described the presence of TREM2 + GPNMB + CD9 + macrophages in the liver mostly localized at crown-like structures (CLS) in a mouse model of high fat diet-induced liver injury (Daemen, Gainullina et al., 2021); these authors defined the cells as hepatic LAMs in analogy to the TREM2 + LAMs of the adipose tissue (Jaitin et al., 2019). However, the hepatic LAM-specific markers are not present in KCs (which in turn display high expression of Timd4, Clec4f, Marco and Cd163), suggesting that the hepatic LAMs are probably derived from newly recruited MoMfs and not from an alternative activation of KCs (Daemen, Gainullina et al., 2021). Indeed, lineage tracing experiments using CD115 CreER Rosa26 TdTomato and Flt3 Cre Rosa26 TdTomato mice were performed to directly demonstrate that these hepatic LAMs develop from MoMfs and not from KCs (Daemen, Gainullina et al., 2021). Using bone marrow transplantation experiments, another study demonstrated that the CD9 + TREM2 + hepatic LAMs found during MAFLD derive from the bone marrow and that these cells closely resemble the LAMs seen in the obese adipose tissue (Remmerie et al., 2020). Consistent with their monocyte origin, deficiency in the C-C chemokine receptor type 2 (CCR2) led to a reduced number of LAMs and decreased formation of CLS in NASH mice; interestingly the decrease in LAMs was associated with enhanced fibrosis, suggesting that LAMs may also have a protective role in the NASH liver (Daemen, Gainullina et al., 2021).
Furthermore, in a mouse model of Western diet-induced NASH, three clusters of MoMf were identified in the liver: cluster 1 expresses high levels of Mgst1 (microsomal glutathione S-transferase 1), Fn1 (fibronectin 1) and Msrb1 (methionine sulfoxide reductase B1); cluster 2 expresses high levels of Chil1 (chitinase-like 1); and cluster 3 expresses high levels of Il1b (Krenkel et al., 2020). All three clusters of MoMfs are also present in livers of normal diet fed healthy mice, but clusters 2 and 3 are increased in the NASH livers as compared to healthy livers, whereas cluster 1 remains the same in both groups (Krenkel et al., 2020). Additionally, S100a8 and S100a9, which encode the two subunits of the inflammatory protein calprotectin, are highly expressed in all three MoMf clusters under normal diet as compared to Western diet, whereas Plin2 (perilipin 2), which is associated with the formation of lipid droplets, is upregulated in the MoMf clusters 1 and 3 upon Western diet feeding (Krenkel et al., 2020). However, in contrast to other recent studies describing the liver macrophage populations in mice during MAFLD (Daemen, Gainullina et al., 2021;Remmerie et al., 2020;Xiong et al., 2019), the study by Krenkel et al did not identify the LAM/NAM macrophage subtypes.
NASH liver -human. Similar to the mouse studies, in human cirrhotic livers, a population of scar-associated macrophages (SAMs) was identified that are TREM2 + CD9 + (Ramachandran et al., 2019). SAMs expand in fibrotic livers and localize in the proximity of the collagen positive scar areas (Ramachandran et al., 2019). Moreover, SAMs express high levels of inflammatory and fibrosis-promoting genes such as SPP1, encoding for osteopontin, and IL1B. Conditioned medium derived from SAMs promotes collagen production in primary human HSCs in culture, highlighting the potential profibrogenic function of these macrophages (Ramachandran et al., 2019). In silico trajectory analysis of a combined dataset of liver resident mononuclear phagocytes and peripheral blood monocytes suggests that the SAMs derive from peripheral blood monocytes, with no differentiation from KCs (Ramachandran et al., 2019). Furthermore, in human NASH samples, the presence of SAMs positively correlates with increased histological NASH activity and the degree of fibrosis, suggesting that these cells expand early during the progression of liver disease (Ramachandran et al., 2019). Although TREM2 + profibrogenic macrophages accumulate in NASH livers and are associated with disease severity, macrophage TREM2 has been shown to suppress the initial progression of MAFLD by regulating hepatocyte mitochondrial function; this suggests that the function of macrophage TREM2 may vary at different stages of MAFLD (Daemen, Gainullina et al., 2021;Hou et al., 2021;Ramachandran et al., 2019;Xiong et al., 2019). Therefore, understanding the functional plasticity of TREM2-positive macrophages during MAFLD pathogenesis requires further investigation. In human NASH liver samples obtained from patients undergoing bariatric surgery, scRNA-seq analysis showed the presence of four macrophage clusters, consisting of KCs (expressing MARCO) and three MoMf clusters (expressing MNDA, myeloid cell nuclear differentiation antigen; LYZ and CSTA) (Fred et al., 2022). Furthermore, two of the three MoMf clusters are associated with fibrosis, with high expression of genes such as S100A8, S100A9 and VCAN (versican) (Fred et al., 2022). However, in contrast to the study by Ramachandran et al., this study found very low numbers of TREM2 + CD9 + macrophages (only 1% of total macrophages) in human NASH samples (Fred et al., 2022).
Altogether, although recent studies using single-cell analysis techniques have identified substantial phenotypic and functional heterogeneity in liver macrophages (Table 1), further investigation is required to determine the specific functions of the various liver macrophage subpopulations during the entire spectrum of MAFLD pathogenesis.

The complex function of macrophages in MAFLD pathogenesis
Owing to their central role in liver homeostasis as well as their high expression of pattern recognition receptors  (PRRs), such as toll like receptors (TLR), KCs are the first responders to liver injury (Wen et al., 2021). A multitude of signals may induce the activation of KCs during MAFLD initiation. These include pathogen associated molecular patterns, which increase in the circulation due to higher intestinal permeability and altered gut microbiome, ROS, damage associated molecular patterns, ATP and mitochondrial DNA, which may be released by damaged or dying hepatocytes (Nati et al., 2022;Wen et al., 2021). As the first responders, KCs secrete different chemokines and cytokines such as chemokine (C-C motif) ligand 2 (CCL2) and TNFα to recruit monocytes and other inflammatory cells that further promote liver inflammation and fibrosis during MAFLD (Huang et al., 2010;Miura et al., 2012;Tosello-Trampont et al., 2012). In addition, KCs promote hepatic steatosis by regulating triglyceride storage in hepatocytes, a mechanism mediated by interleukin (IL)-1β-dependent suppression of PPARα expression and activity (Stienstra et al., 2010). Indeed, depletion of KCs results in reduced infiltration of Ly6C + monocytes, inflammation, hepatic steatosis and fibrosis development in rodent models of NASH (Huang et al., 2010;Miura et al., 2012;Reid et al., 2016;Tosello-Trampont et al., 2012). Furthermore, in mice during NASH, increased apoptotic cell death of embryonic KCs enables repopulation by monocyte-derived macrophages (Reid et al., 2016;Seidman et al., 2020;Tran et al., 2020). Such monocyte-derived KCs exhibit a pro-inflammatory phenotype, compared to embryonic KCs, thereby leading to exacerbated liver damage and fibrosis; in contrast, embryonic KCs are more capable of promoting hepatic triglyceride storage during NASH (Tran et al., 2020).
Interestingly, these monocyte-derived KCs engraft the KC pool and remain long after disease regression, with potential long-term impact on liver function, thereby altering the liver response to lipids (Tran et al., 2020). In contrast to the study by Tran et al., another study showed that embryonic KCs and monocyte-derived KCs, that is, MoMfs that occupy the KC-depleted niche, exhibit similar functional biological roles (Seidman et al., 2020). In mice, when embryonic KCs are depleted and the KC niche is repopulated with monocyte-derived cells, followed by feeding with a NASH-inducing diet, no differences were found in gene expression in the liver, as shown by RNA sequencing (Seidman et al., 2020). Further studies are required to investigate in depth the phenotypic and functional similarities and differences between embryonic KCs and monocyte-derived KCs during NASH. Monocyte recruitment during liver injury is mainly mediated by the chemokine receptor CCR2; inhibiting monocyte recruitment in a mouse model of NASH using an inhibitor for CCR2/C-C chemokine receptor type 5 (CCR5) reduced hepatic steatosis, inflammation and fibrosis (Krenkel et al., 2018;Wen et al., 2021). On the contrary, CCR2 inactivation in high-fat, high-sucrose-diet-induced NASH resulted in increased fibrosis, associated with a decrease in LAMs (Daemen, Gainullina et al., 2021). Chemokine (C-C motif) receptor 8 (CCR8) is also involved in macrophage recruitment to the liver following injury, as shown in a mouse model of carbon tetrachloride (CCl 4 )-induced liver injury (Heymann et al., 2012). Additionally, the C-C chemokine receptor type 9 (CCR9)-chemokine (C-C motif) ligand 25 (CCL25) axis recruits MoMfs to the liver, as shown in a rodent model of CCL 4 -induced liver injury (Chu et al., 2013). Infiltrating MoMfs release pro-inflammatory/pro-fibrotic factors further propagating inflammation and activating HSCs (Nati et al., 2022;Wen et al., 2021).

Lipids and hepatic macrophage activation.
During NASH, lipids such as free cholesterol, cholesterol crystals, fatty acids and oxidized low-density lipoprotein (oxLDL) induce macrophage activation and enhance their pro-inflammatory phenotype (Subramanian et al., 2022). A critical lipotoxic substrate in NASH is free cholesterol (Ioannou, 2016). In a rodent model of MAFLD, dietary fat and cholesterol act in a synergistic manner to promote inflammation and fibrosis (McGettigan et al., 2019). KCs acquire cholesterol through uptake of circulating lipoproteins such as the cholesterol-rich LDL, via receptor-mediated endocytosis (Ioannou, 2016). The uptake of non-modified LDL via the LDL receptor (LDLR) is regulated by feedback inhibition, thereby preventing cholesterol overload in the cells; however, the uptake of modified LDL, for example oxLDL, via scavenger receptors is not subject to feedback regulation resulting in excess intracellular cholesterol levels and formation of 'foam cells' (Ioannou, 2016). Uptake of oxLDL by KCs results in lysosomal accumulation of these lipids, a phenomenon linked to increased hepatic inflammation, as shown in LDLR-knockout mice injected with modified or unmodified LDL (Bieghs et al., 2013). Of note, the scavenger receptors CD36 and macrophage scavenger receptor 1 (MSR1) mediate the uptake of modified lipids in KCs (Bieghs et al., 2012). Accordingly, MSR1 transcript levels are associated with the severity of steatosis, hepatocyte ballooning and NASH in human MAFLD (Govaere et al., 2022). In primary mouse macrophages, MSR1 facilitates palmitic acid-induced lipid accumulation and enhanced expression of Tnfa and Il6 mRNA (Govaere et al., 2022). Interestingly, fat-laden KCs produce higher levels of pro-inflammatory cytokines and chemokines in response to lipopolysaccharide (LPS) stimulation (Leroux et al., 2012). Lipid droplet accumulation in macrophages is essential for the production of inflammatory factors such as IL-1β, prostaglandin E2 and IL-6 (Castoldi et al., 2020).
Furthermore, during NASH, cholesterol crystals are present in lipid droplets of steatotic hepatocytes both in humans and in mice (Ioannou et al., 2013). KCs and MoMfs process the lipids of dead hepatocytes thereby forming the CLS; additionally, liver macrophages become activated by cholesterol crystals resulting in NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, and the production of pro-inflammatory/pro-fibrotic cytokines such as IL-1β, CCL2 and transforming growth factor-β (TGFβ) (Ioannou, 2016;Ioannou et al., 2013Ioannou et al., , 2017. Indeed, blockade of NLRP3 inflammasome using an inhibitor results in reduced hepatic inflammation, liver damage and fibrosis in a mouse model of NASH (Mridha et al., 2017). Furthermore, fatty acids derived from an unbalanced diet or from the obese adipose tissue can promote macrophage activation and hepatic inflammation during MAFLD (Nati et al., 2022). Palmitic acid-stimulated macrophages generate ROS; this process is mediated via the TLR4-MD2 complex, ultimately leading to increased pro-IL1β expression in macrophages (Kim et al., 2017). Of note, another study shows that palmitate does not directly activate TLR4; however, activation of TLR4 by other ligands such as LPS alters cellular lipid metabolism and gene expression, changes that are required for palmitate-induced inflammation (Lancaster et al., 2018). Additionally, palmitic acid impairs autophagy in macrophages via upregulation of hypoxia-inducible factor 1α (HIF-1α), and additionally HIF-1α mediates NF-κB activation and CCL2 production .

Efferocytosis and hepatic macrophage activation.
Hepatocyte death, a process fostering macrophage recruitment and activation, also drives HSC activation and fibrosis development during NASH (Subramanian et al., 2022). Following hepatocyte death, macrophages clear the apoptotic cells via efferocytosis, which initiates resolution of inflammation pathways (Kourtzelis et al., 2020). Efferocytic macrophages produce factors such IL-10 and TGFβ; the latter is a major driver of HSC activation and fibrogenesis (Kourtzelis et al., 2020;Subramanian et al., 2022). C-mer tyrosine kinase (MerTK) signalling in macrophages, which is linked to dead cell clearance, leads to extracellular signal-regulated kinase 1/2 pathway-dependent upregulation of TGFβ thereby promoting fibrosis development in a mouse model of NASH (Cai et al., 2020).

Figure 1. Role of macrophages in metabolic dysfunction associated fatty liver disease (MAFLD)
Under homeostatic conditions, the liver resident KCs exhibit self-renewal capacity, are located in close proximity to the LSECs, express the markers CLEC4F, TIM4 and CLEC2 and exert homeostatic actions. On the contrary, during MAFLD the KCs exhibit impaired self-renewal capacity. A multitude of signals such as PAMPs, ROS, DAMPs, ATP and mtDNA can activate the KCs, which in turn produce pro-inflammatory molecules, for example CCL2 and TNFα, thereby recruiting blood monocyte-derived macrophages (MoMfs) to the liver. Monocyte recruitment during liver injury is mediated by the chemokine receptors CCR2, CCR8 and CCR9. Hepatic macrophages may become lipid-laden macrophages (foamy Mf) owing to the uncontrolled uptake of lipids; the macrophage scavenger receptor 1 (MSR1) plays a critical role in mediating lipid uptake in these macrophages. Additionally, macrophage-derived IL-1β and TNFα promote lipid accumulation and inflammation in hepatocytes. Various processes and factors, such as efferocytosis, lipid accumulation, palmitic acid, cholesterol crystals, free cholesterol and oxLDL, can stimulate macrophages to promote profibrogenic HSC activation and their differentiation to myofibroblasts in a paracrine fashion by secreting a wide range of molecules, including TGFβ, TNFα, IL-1β, galectin-3, OPN and ROS. Monocyte-derived macrophages develop into hepatic LAMs, mainly localized in the crown like structures. Liver macrophages thus play complex roles during MAFLD progression. ATP, adenosine triphosphate; CCR2, C-C chemokine receptor type 2; CCR8, chemokine (C-C motif) receptor 8; CCR9, C-C chemokine receptor type 9; CCL2, chemokine (C-C motif) ligand 2; chol-crystals, cholesterol crystals; CLEC4F, C-type lectin domain family 4 member F; CLEC2, C-type lectin-like receptor 2; DAMPs, damage associated molecular patterns; IL-1β, interleukin 1β; KC, Kupffer cell; LAM, lipid associated macrophage; LSEC, liver sinusoidal endothelial cells; MFB, myofibroblasts; Mf, macrophage; MoMf, monocyte-derived macrophage; mtDNA, mitochondrial DNA; MSR1, macrophage scavenger receptor 1; OPN, osteopontin; oxLDL, oxidized low-density lipoprotein; PAMPs, pathogen associated molecular patterns; ROS, reactive oxygen species; TGFβ, transforming growth factor-β; TIM4, T-cell immunoglobulin and mucin domain containing 4; TREM2, triggering receptor expressed on myeloid cells 2; TNFα, tumour necrosis factor-α. fibrosis and cirrhosis model in rats, treatment with galectin inhibitors abrogated hepatic fibrosis and reversed cirrhosis (Subramanian et al., 2022;Traber et al., 2013). Together, multiple studies point to a central role of macrophages in MAFLD pathogenesis and progression to fibrosis, thereby suggesting that these cells represent therapeutic targets.

Conclusions
The prevalence of NAFLD/MAFLD and NASH is rapidly growing, with fibrosis severity being the major predictor of mortality. Macrophages play complex roles in the liver. Recent single-cell analysis approaches have demonstrated substantial phenotypic and functional heterogeneity in liver macrophages. While KCs promote homeostasis under healthy conditions (Krenkel & Tacke, 2017), recruited MoMfs rather contribute to propagation of NASH inflammation and fibrosis, as implicated by animal models and clinical studies (Wen et al., 2021) ( Fig. 1). However, recent studies have also suggested that specific macrophage subpopulations (e.g. LAMs) may also protect against fibrosis (Daemen, Gainullina et al., 2021b). Nevertheless, macrophage activation and their pro-inflammatory/pro-fibrotic products might represent potential therapeutic targets for NASH pathogenesis or progression. The chemokine receptor CCR2 is an important player in monocyte/macrophage recruitment during NASH; a dual CCR2-CCR5 inhibitor, cenicriviroc, demonstrated positive effects after the first year of treatment, with a significant number of patients showing improved fibrosis, and these findings were confirmed after 2 years (Friedman et al., 2018;Ratziu et al., 2020). However, the respective phase III trial (NCT03028740) was terminated due to lack of efficacy. This finding suggests that inhibiting recruitment of all monocyte-derived macrophages (instead of specifically targeting subpopulations thereof) is probably not the right therapeutic approach for NASH and fibrosis. Conversely, PPARs are nuclear transcription factors with various functions in MAFLD such as regulating inflammation and lipid metabolism (Wen et al., 2021). The pan-PPAR agonist lanifibranor has shown promising results, supporting further investigation (Barreby et al., 2022;Francque et al., 2021).
In conclusion, recent studies have expanded our knowledge on the diversity of macrophages, their turnover and their complex functions during MAFLD pathogenesis. However, further studies elucidating the specific functions of the various liver macrophage subpopulations during the complete spectrum of NASH pathogenesis are warranted, as they could likely yield novel therapeutic targets.