Humanitas Clinical and Research Center, Rozzano, Italy
BIOMETRA Department, University of Milan, Milan, Italy
Address reprint requests to: Alberto Mantovani, M.D., Humanitas Clinical and Research Center, via Manzoni 56, 20089 Rozzano, Milan, Italy. E-mail: firstname.lastname@example.org; fax: +39 02 82245101.
Potential conflict of interest: Dr. Mantovani and Dr. Sica received grants from Johnson & Johnson.
This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC), Italy, Fondazione Cariplo, Italy, Ministero Università Ricerca (MIUR) e Salute, and by the European Commission (ERC project no.: 233417 HIIS; to A.M.).
A full reference list is available as Supporting Information. We apologize to colleagues who, because of space limitations, are only cited there.
Resident and recruited macrophages are key players in the homeostatic function of the liver and in its response to tissue damage. In response to environmental signals, macrophages undergo polarized activation to M1 or M2 or M2-like activation states. These are extremes of a spectrum in a universe of activation states. Progress has been made in understanding the molecular mechanisms underlying the polarized activation of mononuclear phagocytes. Resident and recruited macrophages are a key component of diverse homeostatic and pathological responses of hepatic tissue. Polarized macrophages interact with hepatic progenitor cells, integrate metabolic adaptation, mediate responses to infectious agents, orchestrate fibrosis in a yin-yang interaction with hepatic stellate cells, and are a key component of tumor-promoting inflammation. Conclusion: A better understanding of macrophage diversity and plasticity in liver homeostasis and pathology may pave the way to innovative diagnostic and therapeutic approaches. (Hepatology 2014;59:2034–2042)
Though not considered a canonical lymphoid organ, the liver hosts a complex range of immunocompetent cells under physiological and pathological conditions.[1, 2] Cells of the monocyte-macrophage lineage are a major component of the host defense repertoire present in the liver.[1-3]
Tissue-resident and -recruited macrophages are a major component of mechanisms of innate resistance and a link between inflammation and cancer (Fig. 1). In the liver, resident macrophages (Kupffer cells; KCs), fulfill homeostatic functions, orchestrate tissue remodeling in ontogenesis, and regulate metabolic functions.[1-7] KCs are strategically positioned in liver sinusoids, where they trap, phagocytose, and clear microbes in the circulation, and act as a first line of resistance against blood-borne pathogens.[1, 2]
Resident and recruited cells of the monocyte-macrophage lineage exert a dual function in liver pathology.[8, 9] Here, we review selected aspects of the plasticity and polarized activation of cells of the monocyte-macrophage as well as its molecular mechanisms and relevance to liver immunopathology. Understanding and harnessing the yin-yang role of macrophages in liver inflammation and related diseases, including cancer, may pave the way to the development of innovative diagnostic and therapeutic strategies.
Macrophage Plasticity and Polarization
In addition to being the home of a major resident population of mononuclear phagocytes, the liver plays an important role in the ontogeny of the mononuclear phagocyte system. In the mouse, mononuclear phagocytes belong to different lineages, which originate from the yolk sac (YS), the YS and the liver, or the bone marrow (BM). The actual functional significance of distinct macrophage lineages and existence in humans remains to be elucidated. Interestingly murine KCs as well as other resident macrophages (e.g., microglia) originate from the YS in a colony-stimulating factor-1/receptor (CSF-1/R)-dependent and Myb-independent way. The KC population has been suggested to be maintained by local proliferation, which results in extensive mitosis after hepatectomy. Recent evidence suggests that macrophage accumulation can be sustained by local proliferation,[12, 13] in particular, during inflammation sustained by T-helper (Th)2 cells. It remains to be established what the relevance to the liver of these findings is. Evidence also suggests that BM-derived cells can differentiate into KCs, and two subsets of F4/80highCD11low KCs have been identified, respectivelys as radiosensitive, replaceable by BM precursors, and radioresistant or “sessile” ones, the former participating in immunoinflammatory reactions. Analysis of differentiation markers and homing receptor revealed only an increased expression of the costimulatory molecule CD80 (B7-1) in BM-derived KCs.
Interleukin (IL)-34 is an additional CSF-1R ligand sustaining brain microglia and skin macrophages[2, 3, 15] (Fig. 2). Genetic inactivation of the CSF-1R or CSF-1 gene has revealed that KCs are dependent on this pathway for development. Genetic ablation of the second CSF-1R ligand, IL-34, had little effect on liver macrophages. Progress has been made in defining the transcriptional and post-transcriptional networks involved in macrophage differentiation, but little specific information is available on liver macrophages.
KCs sense either exogenous or endogenous danger signals (e.g., bacterial products or necrotic cell debris) through pattern recognition receptors (PRRs). In response to Toll-like receptor (TLR) ligands and interferon-gamma (IFN-γ) or IL-4/IL-13, macrophages undergo M1 (classical) or M2 (alternative) activation, which mirror the TH1- TH2 polarization and represent extremes of a continuum in a universe of activation states.[2, 3, 7] The M1 phenotype is characterized by the expression of high levels of proinflammatory cytokines, high production of reactive nitrogen and oxygen intermediates, promotion of Th1 response, and strong microbicidal and tumoricidal activity. In contrast, M2 macrophages are involved in parasite containment, promote tissue remodeling and tumor progression, and have immunoregulatory functions. They are characterized by efficient phagocytic activity, high expression of scavenging, mannose, and galactose receptors, production of ornithine and polyamines through the arginase pathway, and an IL-12loIL-10hiIL-1decoyRhiIL-1RAhi phenotype. Other signals, including IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes, also polarize macrophages to an M2-like phenotype with some similarity to IL-4-activated macrophages and immunoregulatory and protumoral functions (Fig. 2).
Mechanisms of Macrophage Polarization
Macrophage polarization is controlled by different molecular mechanisms, including signaling pathways, transcription factors, epigenetic mechanisms, and post-transcriptional regulators. Imprinting of macrophage function by environmental signals, including polarizing molecules, has been variably referred to as “memory,” “short-term memory,” adaptive response, and training.[16, 17] Figure1B shows a simplified view of key regulators of macrophage polarization belonging to the interferon regulatory factor/signal transducer and activator of transcription/suppressor of cytokine signaling (IRF-STAT-SOCS) families. IFN and TLR-activated IRF-STAT-signaling pathways orient macrophage function toward the M1 phenotype (through STAT1), whereas IL-4 and IL-13 activate the M2 phenotype through STAT6. IL-10 activates STAT3-mediated expression of genes (Il10, Tgfb1, and Mrc1) associated with an M2-like phenotype,[18-20] and IL-3-mediated STAT5 activation was suggested to promote M2 polarization. IRF5 and IRF8 (through Notch) are part of the M1-associated transcriptional network.[3, 21, 22]
SOCS family members are key regulators of STAT activity in macrophages. IL-4 and IFN-γ, the latter in concert with TLR stimulation, up-regulate SOCS1 and SOCS3, which, in turn, inhibit the action of STAT1 and STAT3, respectively.[23, 24] Recently, RBP-J-mediated Notch signaling has been implicated in regulating macrophage polarization in a SOCS3-dependent manner. Furthermore, SOCS2 and SOCS3 were demonstrated to play as key opposite regulators of M1- and M2-like macrophage polarization and inflammatory response, respectively.
Downstream of, or in parallel with, the IRF-STAT-SOCS pathway, a panel of transcription factors contributes to polarized macrophage activation. These include the nuclear receptors Peroxisome proliferator-activated receptor (PPAR)-γ and PPAR-δ, Krüppel-like factor 4, and c-Myc. Induction of p50 nuclear factor kappa B (NF-κB) homodimers is essential for M2 polarization in vitro and in vivo. In addition, c-Jun N-terminal kinase (JNK) activation is required for M1 polarization of macrophages during obesity-induced inflammation and insulin resistance (IR).
A critical role in differentiation of tissue-resident M2-like macrophages was recently described for the adaptor protein, Trib1. Mice lacking Trib1 in hematopoietic cells showed a reduced number of M2-like macrophages and eosinophils, diminished adipose tissue mass, and increased lipolysis. In response to high-fat diet, these mice developed hypertriglyceridemia and IR.
Epigenetic changes and noncoding microRNAs (miRs) also participate in directing macrophage activation and polarization.[16, 17, 30, 31] For instance, IL-4 up-regulates the histone demethylase, Jumonji D3, in mouse macrophages, which alters chromatin modifications to promote expression of M2 genes and inhibit M1 genes. Moreover, overexpression of miRNA let-7c, that targets the transcription factor CCAAT/enhancer-binding protein, promotes M2 and inhibits M1 macrophage polarization, whereas expression of miR-19a-3p promotes M2 macrophage polarization and induced expression of the Fra-1 proto-oncogene.
Functions of Liver Macrophages
The origin of KCs has been thought to involve two mechanisms: replenishment by local self- proliferation and renewal and recruitment from circulating BM-derived monocytes. KCs represent a subset of CD11b+/F4/80+ cells expressing CD68 (macrosialin) and are the largest group of resident liver macrophages, lying within the periportal area of hepatic sinusoids. KCs have high phagocytic activity to uptake particulates, apoptotic cells present within the portal circulation, and microorganisms.
Regeneration is a key feature of hepatic tissue. Evidence suggests that macrophages play a key role in the orchestration of hepatic progenitor cell (HPC) fate and differentiation. This function of macrophages is mediated by expression of WNT ligands and inhibition of Notch signaling. It is tempting to speculate that orchestration of HPCs in liver regeneration is an aspect of the capacity of macrophages to interact with stem cells and contribute to organogenesis.[35-37]
In support of this, a delay in hepatic regeneration has been observed subsequent to selective depletion of KCs with liposome-encapsuled dichloromethylene diphosphonate (Cl2MDP). KC depletion also results in both decreased levels of tumor necrosis factor alpha (TNF-α) and IL-6 and delayed liver regeneration, which was attributed to impaired NF-κB activation.
In response to danger signals, KCs secrete various inflammatory mediators, including reactive oxygen and nitrogen species, proinflammatory cytokines (IL-1, IL-6, TNF-α, and granulocyte macrophage colony-stimulating factor), and chemokines (e.g., chemokine [C-C motif] ligand [CCL]3 and CCL5). These inflammatory mediators contribute to tissue damage in ischemia reperfusion, endotoxemia, and acetaminophen-induced liver hepatotoxicity and alcohol-induced liver steatosis, the latter being markedly attenuated in vivo by administration of recombinant IL-1 receptor antagonist.[40, 41] KCs may also express immunoregulatory molecules, such as IL-10 and transforming growth factor beta (TGF-β), and the regulatory check-point molecule, B7-H1 (PD-L1).[42, 43] Unbalanced production of pro- and anti-inflammatory mediators by KCs can lead to liver injury.
KCs undergo polarized inflammatory programs. They play a central role in the pathogenesis of alcoholic liver disease, by expressing high levels of proinflammatory cytokines (e.g., TNF-α). Cannabinoid CB2 receptor agonists and adiponectin were recently shown to shift KC polarization to the M2/anti-inflammatory phenotype.[45, 46] Adiponectin prevented the progression of nonalcoholic steatohepatitis in mice, whereas lack of adiponectin enhanced the progression of hepatic steatosis, fibrosis, and hepatic tumor formation.
Along with recruited macrophages, KCs play a major role in the metabolic adaptation of hepatocytes during increased caloric intake. By regulating the oxidation of fatty acids, KCs promote increased lipid storage in hepatocytes during obesity, which results in hepatic IR. This event is triggered by the secretion of inflammatory cytokines (e.g., TNF, IL-6, and IL-1β), thus suggesting a beneficial role for alternatively M2-activated KCs in metabolic syndrome and type 2 diabetes.[50, 51] A key function of the liver is to orchestrate metabolic adaptation (glucose, lipid) primarily in response to diverse signals of dietary, hormonal, and immunologic origins. KCs and their M2 activation are essential players in the regulation of fatty acid oxidation. Indeed, IL-4/IL-13-driven M2 activation involves PPAR-δ and its selective inactivation resulted in defective M2 activation of KCs, steatosis, and IR.[50, 52] KC depletion resulted in a similar phenotype, though the limitations intrinsic to macrophage depletion strategies caution against overinterpretation. Thus, through, as yet, not fully defined pathways, M2-skewed KCs instruct lipid metabolism by hepatocytes.
Macrophages and Liver Fibrosis
Fibrosis is a key feature of chronic liver inflammation,[4, 54] and activated macrophages exert a dual function in the orchestration of matrix deposition and remodeling. This function is reminiscent of homeostatic tissue remodeling during oncofetal life and in selected tissues in adulthood.[6, 55]
Hepatic stellate cells (HSCs) in response to damage differentiate into myofibroblast-like cells, which produce most extracellular matrix (ECM) components in pathology. HSCs and their progeny engage in a bidirectional interaction with resident and recruited macrophages. Depending on context and activation signals, macrophages can exert dual functions on HSC and ECM deposition (Fig. 3). p53 drives a senescence program in HSCs and it was recently demonstrated that, during chronic liver injury, ablation of p53 in HSCs increases liver fibrosis and cirrhosis, associated with hepatocellular carcinoma (HCC) development. By releasing factors that stimulate M2 macrophage polarization, p53-deficient stellate cells create a tumor-promoting environment.
Phagocytosis of dying necrotic cells and debris triggers production of TGF-β by KCs and recruited macrophages.[57-60] On the other hand, phagocytosis of apoptotic hepatocytes and cholangiocytes has been shown to dampen the development of fibrosis.[61, 62] In addition, macrophages can directly clear excess collagen, and, recently, the CD11BhighF4/80intLy-6Clow macrophage subset was shown to orchestrate regression of murine liver fibrosis. Thus, macrophage-mediated phagocytosis can exert dual influence on fibrotic responses.
Although various cell types can be a source of TGF-β1, there is strong evidence that macrophages are a prime source of this profibrotic cytokine. The prototypic M2 polarization signal, IL-13, induces production of TGF-β as well as its activation by matrix metalloproteinase (MMP)-9.
Activated resident and recruited macrophages are a source of growth factors (e.g., insulin-like growth factor 1 and platelet-derived growth factor), osteopontin, cytokines and chemokines (e.g. CCL2) which recruit circulating monocytes and affect the function of HSC and fibroblasts. In particular there is genetic evidence for a role in liver fibroblastic responses of the chemokine receptors, C-C chemokine receptor (CCR)2, CCR1, and CCR5.[67-69] Of note, CCR2highLy6Clow monocyte-derived macrophages have been found to exhibit an M2 polarization state in different pathological situations associated with fibrosis infiltration. These cells are thought to interact with HSCs through TGF-β to promote fibrosis.[54, 65, 70]
Il-4 and IL-13, produced by Th2 cells, as well as other cell types, such as innate lymphoid cells and, possibly, KC themselves, have, in general, profibrotic activity. They do so by eliciting alternative M2 activation of macrophages. In general, polarized myeloid cells play a major role in orchestrating liver fibrosis in response to parasites. In addition, IL-13 promotes the activation of TGF-β1 (see above) and directly stimulates collagen synthesis.[71-73] For instance, in Schistosoma infection and in CCL4-induced liver fibrosis, Th2 and Th1 responses are associated with pro- and antifibrotic activity, respectively.[4, 5, 54]
Macrophages can also exert antifibrotic activity and promote resolution of fibrosis. Interstitial collagenases produced by macrophages (e.g., MMP-13) can facilitate fibrous tissue degradation. Thus, recruited and liver-resident mononuclear phagocytes can exert dual influences on the fibrotic evolution of liver inflammation, depending on their polarization state, underlying pathology as well as largely undefined environmental cues. Dissecting signals and cell populations involved in the pro- and antifibrotic action of liver macrophages may pave the way to innovative approaches.
The pathogenesis of HCC has been associated with hepatocyte death, infiltration of inflammatory cells, and compensatory liver regeneration, which is dependent on the production of hepatic mitogenic cytokines produced by KCs, such as IL-6. Therefore, it is impellent to clarify the molecular events driving these processes. In this regard, NF-κB and related regulatory pathways (e.g., NF-κB essential modulator and inhibitor of nuclear factor kappa-B kinase subunit gamma) are central orchestrators of liver carcinogenesis.[75, 76] The function of NF-κB is affected by the model utilized, but its genetic or pharmacologic inhibition in myeloid cells invariably resulted in tumor inhibition.[75, 76] Additional inflammation-related molecular pathways involved in liver carcinogenesis include TGF-β-activated kinase, β-catenin, lymphotoxin receptors, and STAT3.[77, 78]
Acquisition of protumoral M2 functions by tumor-associated macrophages (TAMs) is driven by various cytokines and signals expressed within the tumor microenvironment, which can be provided by either cancer or stromal cells. In particular, B lymphocytes and Th2 polarized lymphocytes concur to release cytokines and/or antibodies to promote the alternative M2 polarization of TAM.[3, 79] Of interest, in patients with HCC, galectin-9 expressing KCs were suggested to promote immune escape by negatively regulating Th1-mediated immune responses through interaction with T-cell immunoglobulin- and mucin-domain-containing molecule. Moreover, CD69(+) T cells present in HCC tissues instruct tumor macrophages to produce greater amounts of the immunosuppressive enzyme, indoleamine 2 3-dioxygenase.
As in most human tumors, TAM infiltration was generally associated with poor prognosis in HCC.[82, 83] Similarly, in cholangiocarcinoma, high counts of CD163(+) (a marker of M2 macrophages) macrophages were associated with poor disease-free survival. Consistently with these findings, a refined 17-gene signature, associated with overexpression of the macrophage growth factor, CSF-1, and representative of a Th1- to Th2-like profile switch, was validated as a predictor of HCC venous metastases.
Recently, the proinflammatory myeloid cell surface receptor, triggering receptor expressed on myeloid cells 1, was shown to be a pivotal determinant of KC activation in diethylnitrosamine-induced HCC, and its ablation resulted in diminished activation of JNK and NF-κB. IL-22, a cytokine secreted by Th17 cells, was recently reported to be a novel inflammation driver through STAT3-signaling activation, leading to HCC growth and progression.
TAMs have been shown to affect both angio- and lymphangiogenesis, by producing various angiogenic (e.g., vascular endothelial growth factor [VEGF], TNF-α, basic fibroblast growth factor) as well as lymphangiopoietic (e.g., VEGF-C and -D) factors. High peritumoral coexpression of VEGF-C, VEGF receptor (VEGFR)-1, and VEGFR-3 was recently associated with higher peritumoral distribution of macrophages, as well as with a poorer outcome after hepatectomy.
These findings, pointing to a protumor function of TAM in liver cancer, raise the issue of therapeutic targeting. Sorafenib is a multikinase inhibitor reported to improve survival rates in patients with advanced HCC and to inhibit the suppressive immune cell populations, T-regulatory cells and myeloid-derived suppressor cells. A combination of sorafenib with zoledronic-acid-driven macrophage depletion better inhibited tumor progression, tumor angiogenesis, and lung metastasis, compared with mice treated with sorafenib alone. Thus, liver cancer may represent a prime context for the development of macrophage-targeting antitumor strategies.
Cells of the monocyte-macrophage lineage are a key component of the homeostatic functions of the liver and of its response to damage. In response to damaged tissue components, microbial moieties, and cytokines, macrophages undergo polarization into M1 (classical) or M2 (alternative) activation states. As discussed above, activated macrophages mirroring in vitro polarized mononuclear phagocytes are indeed present in the liver, and polarized M2 or M2-like activated macrophages orchestrate metabolic activity, tissue responses to parasites (e.g., Schistosoma) and toxic xenobiotics (CCl4), and fibrosis. However, in many pathological conditions, the plasticity of macrophages defies a simple M1 versus M2 classification. Moreover, macrophages can exert dual influence on the fibrotic evolution of liver inflammation and its resolution. Considerable efforts are currently devoted to the development of macrophage-targeting therapeutic strategies.[3, 92] The complexity and yin-yang function of macrophages in hepatic immunopathology raises the issue of macrophage reorientation, rather than inhibition, as a perspective in the development of therapeutic strategies.