Accepted Manuscript Frontiers in Liver Transplantation Macrophage heterogeneity in liver injury and fibrosis Frank Tacke, Henning W. Zimmermann PII: DOI: Reference:

S0168-8278(14)00005-1 http://dx.doi.org/10.1016/j.jhep.2013.12.025 JHEPAT 4988

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

18 November 2013 19 December 2013 22 December 2013

Please cite this article as: Tacke, F., Zimmermann, H.W., Macrophage heterogeneity in liver injury and fibrosis, Journal of Hepatology (2013), doi: http://dx.doi.org/10.1016/j.jhep.2013.12.025

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Clinical Application of Basic Science - JHEPAT-D-13-01872 REVISED MANUSCRIPT; CHANGES ARE UNDERLINED

Macrophage heterogeneity in liver injury and fibrosis

Frank Tacke, Henning W. Zimmermann University Hospital Aachen, Dept of Medicine III, Pauwelsstr. 30, 52074 Aachen, Germany

Electronic word count (main text): 2488 words Number of figures: 2 Number of tables: 1 List of abbreviations: CCL, C-C motif chemokine ligand; CCl4, carbon tetrachloride; CCR, CC motif chemokine receptor; CSF, colony stimulating factor; CX3CL1, fractalkine; CX3CR1, fractalkine receptor; DAMPs, damage-associated molecular pattern molecules; FACS, fluorescence activated cell sorter; HSC, hepatic stellate cell; IFN, interferon; IL, interleukin; iNOS,

inducible

nitric

oxide

synthase;

LPS,

lipopolysaccharide;

Ly-6C,

monocyte/macrophage differentiation antigen; M1/M2, macrophage polarization status 1/2; MMP, matrix metalloproteinases; NF, nuclear factor; PAMPs, pathogen-associated molecular patterns; PDGF, platelet derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor Financial support: This work was supported by the German Research Foundation (DFG Ta434/2-1 & SFB/TRR57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. Conflict of interest: The authors disclose no competing interests. Corresponding author: Frank Tacke, M.D., PhD Department of Medicine III RWTH-University Hospital Aachen Pauwelsstrasse 30, 52074 Aachen, Germany Phone: -49-241-80-35848 Fax: -49-241-80-82455 Email: [email protected]

Summary Hepatic macrophages are central in the pathogenesis of chronic liver injury and have been proposed as potential targets in combatting fibrosis. Recent experimental studies in animal models revealed that hepatic macrophages are a remarkably heterogeneous population of immune cells that fulfill diverse functions in homeostasis, disease progression and regression from injury. These range from clearance of pathogens or cellular debris and maintenance of immunological tolerance in steady state conditions; central roles in initiating and perpetuating inflammation in response to injury; promoting liver fibrosis via activating hepatic stellate cells in chronic liver damage; and, finally, resolution of inflammation and fibrosis by degradation of extracellular matrix and release of anti-inflammatory cytokines. Cellular heterogeneity in the liver is partly explained by the origin of macrophages. Hepatic macrophages can either arise from circulating monocytes, which are recruited to the injured liver via chemokine signals, or from self-renewing embryo-derived local macrophages, termed Kupffer cells. Kupffer cells appear essential for sensing tissue injury and initiating inflammatory responses, while infiltrating Ly-6C+ monocyte-derived macrophages are linked to chronic inflammation and fibrogenesis. In addition, proliferation of local or recruited macrophages may possibly further contribute to their accumulation in injured liver. During fibrosis regression, monocyte-derived cells differentiate into Ly-6C (Ly6C, Gr1) low expressing ‘restorative’ macrophages and promote resolution from injury. Understanding the mechanisms that regulate hepatic macrophage heterogeneity, either by monocyte subset recruitment, by promoting restorative macrophage polarization or by impacting distinctive macrophage effector functions, may help to develop novel macrophage subset-targeted therapies for liver injury and fibrosis.

Summary word count: 248 words Keywords: macrophage, monocyte, liver fibrosis, liver inflammation, chemokines

Introduction Hepatic macrophages hold a central position in the pathogenesis of chronic liver injury and have been proposed as potential targets in combatting fibrosis [1]. The ambivalence of macrophage activity in experimental liver damage and the identification of functionally opposing macrophage subsets, though, have impeded the development of macrophagebased interventional strategies so far. In congruence with the fact that liver fibrosis is not an unidirectional irreversible process, hepatic macrophages can actually exert dual functions in the context of experimental liver fibrosis by either promoting or abrogating the excessive deposition of extracellular matrix [2]. Intriguing questions have arisen from this finding, and current research focuses on unscrambling mechanisms of functional diversity underlying the opposing tasks of hepatic macrophages throughout the evolution of liver scarring. Important aspects include the origin of the macrophage subsets (derived from circulating monocyte precursors vs. resident Kupffer cells), their differentiation (oftentimes classified as M1 vs. M2 polarization) as well as their effector functions in the context of liver diseases.

Macrophage heterogeneity Macrophage heterogeneity is expressed by a high diversity in cytokines released, cell surface markers and transcriptional profiles. In order to accommodate for the broad spectrum of macrophage function and phenotypes, these cells have been classified either into ‘proinflammatory’ M1 or ‘immunoregulatory’ M2 macrophages, though this simple dichotomous nomenclature does not fully reflect the complex biology of macrophage subsets [3]. Consequently, M2 macrophages are now further categorized into various subtypes that pursue wound healing or anti-inflammation but may also promote inflammation in some circumstances. M1 macrophages are intimately linked to Th1 primed CD4 T-cells, whereas M2 macrophages reciprocally engage with Th2 CD4 T-cells. M1 macrophages are typically induced by IL-12, IFN-γ and LPS in response to acute deleterious incidents, whereas M2 macrophages are controlled via IL-4 and IL-13 [4]. Key effector functions of ‘classical macrophages’ (M1) are bacterial clearance, antiviral activity and release of pro-inflammatory cytokines (such as TNF, IL-1β, IL-12, reactive oxygen species), while ‘alternatively activated macrophages’ (M2) promote defense against parasitic infections, are involved in tissue remodeling and secrete immune-modulatory mediators (such as IL-10, TGF-β, IL-4, IL-13) [3]. However, in disease conditions that are not exclusively skewed towards one end of the spectrum such as acute bacterial peritonitis (M1) or chronic helminth (M2) infection, it is very difficult to assign tissue macrophages to classical or alternative activation. In fact, liver

macrophages appear to simultaneously express markers of M1 and M2 differentiation [5], indicating that this dichotomous concept cannot be entirely applied to hepatic diseases. Rodent models of injury rather indicate that the function of hepatic macrophage subsets in the context of liver diseases largely depends on their origin [6]. Therefore, we propose to distinguish resident hepatic macrophages, termed Kupffer cells, and infiltrating bone marrowderived macrophages, originating from circulating monocytes, to characterize macrophage heterogeneity in the liver.

Resident hepatic macrophages in health and disease Owing to its unique vascular supply the liver is constantly exposed to high concentrations of blood-borne food antigens and bacterial constituents derived from the commensal intestinal flora (Figure 1). Therefore, highly orchestrated innate immune mechanisms in the liver are required to prevent the instigation of inflammatory responses towards those harmless substrates. Due to their potent phagocytic capacity, high density of surface scavenger and pattern recognition receptors as well as the ability to release numerous mediators that govern the local immunological milieu, resident hepatic macrophages meet the prerequisite to balance this incessant immunogenic stimulus and promote tolerance (e.g., dampening of T cell activation) [6]. Actually, under steady state conditions the liver harbors the most abundant pool of macrophages in the whole body. It has long been debated whether circulating monocytes contribute to the Kupffer cell pool. Data from bone marrow and liver transplanted mice demonstrated that monocytes can in principle give rise to functional hepatic macrophages without overt inflammatory stimuli to the liver [7]. However, already in 1997, Naito and coworkers provided experimental data indicating that Kupffer cells almost exclusively originate from fetal yolk sac precursors and self-renew throughout adult life in homeostasis, depending on proliferative signals via M-CSF [8]. These findings could be recently recapitulated using sophisticated cell tracking techniques. These experiments revealed that Kupffer cells delineate from local precursors and constantly renew themselves dependent on the growth factors GM-CSF and M-CSF [9]. During inflammation the hepatic macrophage pool is even expanded, and a startling scientific debate is ongoing regarding the origin and underlying mechanisms of macrophage enrichment. In the early phase after a hazardous incident, sessile hepatic macrophages rapidly secrete pro-inflammatory cytokines and chemokines such as IL-1β, TNF, CCL2 and CCL5, resulting in the paracrine activation of protective or apoptotic signaling pathways of hepatocytes and the recruitment of additional immune cells that booster hepatic injury [10] (Figure 1). In addition, not only inflammatory stimuli, but also metabolic signals may

modulate the activation of hepatic macrophages, as evidenced for the overload of lipids and distinct cholesterol derivatives in Kupffer cells in models of fatty liver disease and steatohepatitis [11, 12]. The central location of Kupffer cells in the sinusoids also allows intimate interactions with other non-parenchymal hepatic cell populations (Figure 1). On the one hand, hepatic macrophages interact with other immune cells; for instance, they secrete the chemokine CXCL16 that attracts NKT cells, which in turn can activate pro-inflammatory signals in macrophages [13]. On the other hand, there is clear evidence from in vitro and in vivo studies that Kupffer cells can activate hepatic stellate cells (HSC) to transdifferentiate into myofibroblasts, the major collagen-producing cell type in hepatic fibrosis [14, 15]. Kupffer cells activate HSC via paracrine mechanisms, likely involving the potent profibrotic and mitogenic cytokines TGF-β and PDGF (Figure 1) [15]. These profibrotic functions of Kupffer cells during chronic hepatic injury remain functionally relevant, even if the infiltration of additional inflammatory monocytes is blocked via pharmacological inhibition of the chemokine CCL2 [16]. Moreover, hepatic macrophages can express several matrix metalloproteinases (MMP), including MMP-9, MMP-12 and MMP-13, that are involved in matrix degradation and therefore favor resolution of liver injury and fibrosis [17, 18]. Although it appears plausible that Kupffer cells, which have tolerogenic and immune-suppressive functions in homeostasis, may undergo a phenotypic switch and promote tissue remodeling, experimental evidence assigning such antifibrotic functions to resident macrophages are currently lacking (Figure 2). The opposing effects of macrophage activation in homeostasis and inflammation indicate the versatile nature of Kupffer cells that could possibly rest on heterogeneous subsets constituting total hepatic macrophages or on the plasticity of the cells that may adopt various phenotypes according to the hepatic microenvironment. Due to their high phagocytic and endo(pino)cytic capacity, local Kupffer cells can be relatively easy targeted by biofunctionalized nanoparticles intended to influence macrophage polarization as well as by carrier tools designed to deliver drugs directly to Kupffer cells (Table) [19, 20]. In order to translate such concepts into clinical applications, however, the precise contribution of local macrophages to liver injury, fibrosis and resolution in relation to invading monocyte-derived macrophages needs to be fully dissected.

Monocytes as precursors of hepatic macrophages While circulating monocytes are likely dispensable for replenishing the hepatic macrophage pool in homeostasis, hepatic metabolic or toxic damage results in the massive infiltration of monocyte-derived macrophages into the liver (Figure 1). Murine models revealed that ‘inflammatory’ Ly-6Chi expressing monocytes accumulate in injured liver, dependent on the chemokine – receptor interactions CCL2 / CCR2 or CCL1 / CCR8 [21-24]. One of the major sources of CCL2 are HSCs, which are activated through TLR4 ligands and thereby guide monocyte recruitment [25]. Freshly infiltrating (monocyte-derived) macrophages are characterized as CD11b+ F4/80+ cells by FACS in mice, whereas matured monocyte-derived and resident Kupffer cells are CD11blo F4/80hi [20, 23]. Targeted deletion of macrophages in CD11b-diphteria toxin receptor (DTR) transgenic mice ameliorates liver fibrosis similar to the abrogation of chemokine pathways that control monocyte influx [2, 23, 26, 27], suggesting that infiltrating monocytes exert major profibrotic functions in fibrosis progression (Figure 1). Apart from directly stimulating matrix-secreting HSC, hepatic macrophages may aggravate scarring by promoting HSC survival via IL-1 and TNF induced NF-κB activation [15]. Interestingly, the profibrogenic effect of infiltrating monocytes in mouse models depends on the underlying genetic background. Balb/c mice, in which Th2 immune responses inherently prevail, are more protected from liver damage and ensuing fibrosis by the disruption of monocyte infiltration than Th1-dominated C57Bl/6 mice, indicating that M1 and M2 polarization might directly influence the outcome in chronic hepatic injury [21]. Another important factor determining the fate of invading monocytes as to the extent of liver damage is the fractalkine receptor CX3CR1, predominantly present on non-classical Ly-6Clo monocytes, in the absence of which monocyte-derived macrophages differentiate into detrimental iNOS and TNF-α producing effector cells [28]. As outlined above, macrophages can also enhance fibrosis resolution in a phase-dependent fashion through the secretion of matrix metalloproteinases (Figure 2) [17, 29]. Hence, not only the interference with the recruitment of ‘profibrogenic’ monocytes was tested in experimental settings, but also the cell transfer of ‘fibrolytic’ monocytes / macrophages. As such, the delivery of syngeneic mature macrophages, but not immature (precursor) monocytes into CCl4-treated mice reduced liver scarring by attracting other immune cells resulting into enhanced levels of antifibrotic IL-10 and increased MMP-9 and -13 activation, accompanied by elevated local growth factor levels IGF-1, VEGF and CSF-1 [30]. The fact that only terminally differentiated macrophages bear the potential to facilitate fibrosis regression in contrast to their progenitors suggests that hepatic macrophage function varies according to disease kinetics and environmental cues.

Following this concept, early infiltrating monocytes elicit organ impairment, whereas after local differentiation into resident macrophages, these cells can gain the ability to restore liver integrity (Figure 2). Recently, ‘restorative macrophages’, characterized by Ly-6Clo expression in mice, have been identified that are competent in resolving fibrosis and accumulated in the restorative phase after tissue damage [5]. These Ly-6Clo macrophages directly delineate from infiltrating Ly-6Chi monocytes/macrophages [5], thus undergo a functional switch in the course of liver injury due to mechanisms that warrant further elucidation. Of note, transcriptome analysis revealed that these Ly-6Clo hepatic macrophages display a profile that cannot be classified according to the M1/M2 nomenclature. A striking feature of those restorative macrophages is that they are postphagocytic, because injection of liposomes boosted the degradation of extracellular matrix [5]. Interestingly, even after cessation of liver injury, a sustained influx of Ly-6Chi macrophages can be observed, that dampens spontaneous fibrosis regression by producing pro-inflammatory cytokines such as TNF. Blocking the CCL2-dependent influx in the phase of fibrosis resolution therefore even enhances clearance of scar fibers [31]. These findings substantiate the theory that freshly infiltrating macrophages worsen liver injury, whereas restoration is elicited by locally matured monocyte-derived macrophages.

Proliferation of hepatic macrophages in liver injury? Fate mapping studies revealed that Kupffer cells originate prenatally from either the yolk sac or local precursors and maintain themselves by self-renewal through proliferation [8, 9]. In line, the absence of the transcription factor Myb1, critical for hematopoietic stems cells, does not affect the presence of Kupffer cells, indicating that myeloid precursor cells are dispensable for the population of hepatic macrophages in homeostasis [32]. However, there is increasing evidence that even during certain types of inflammation, accumulation of macrophages derives from the division of resident cells. In Th2-dominated helminth infection alternatively activated pleural macrophages proliferate in response to IL-4 beyond physiological borders set by the availability of CSF-1 [33, 34]. Importantly, IL-4 also drives replication of Kupffer cells, denoting that this phenomenon may hold true for the liver as well [33]. Of note, alternatively activated arginase-expressing macrophages are capable of constraining liver fibrosis in an archetypical Th2 inflammation model and it is feasible that they also derive from local precursors [35]. However, more detailed studies are necessary to assess the contribution of in situ proliferating macrophages for expanding the macrophage pool during infections and sterile liver inflammation. Moreover, it is currently unclear whether only resident Kupffer cells or also monocyte-derived macrophages are capable of proliferating upon inflammation (Figure 1). In conditions of atherosclerosis or peritonitis,

infiltrated monocyte-derived as well as tissue resident macrophages locally proliferated in response to inflammatory stimuli [36, 37]. In case of progressing liver injury, the contribution of local macrophage proliferation within the liver remains to be determined.

Heterogeneity

of

human

hepatic

macrophages

and

translation

into

clinical

applications A major obstacle for the development of novel therapies for liver fibrosis targeting macrophages is the significant paucity of functional data in man. Distinct macrophage populations can also be found in human liver, including ‘classical’ CD14++CD16- and ‘nonclassical’ CD14+CD16+ monocytes/macrophages as well as CD16++ cells that include atypical macrophages and dendritic cells [38]. The progression of chronic liver diseases from hepatitis to fibrosis to cirrhosis is closely associated with an enrichment of ‘non-classical’ CD14+CD16+ monocyte-derived macrophages in the liver of patients with various disease etiologies [39]. The accumulation of these cells is likely based on two mechanisms: facilitated recruitment across inflamed sinusoidal endothelium and local transdifferentiation from CD14++CD16- precursor cells [38], reminiscent of the maturation of murine Ly-6C+ to Ly-6Clo macrophages in experimental rodent liver injury [5, 23]. Circulating human CD14+CD16+ monocytes share phenotypic features with murine Ly-6Clo monocytes [40], but intrahepatic human CD14+CD16+ macrophages are strikingly similar to mouse inflammatory monocyte-derived macrophages with respect to functional aspects, as these cells release proinflammatory as well as fibrogenic mediators [38]. On the other hand, human intrahepatic CD14+CD16+ macrophages possess a high phagocytic activity, which has been identified as a feature of ‘restorative’ (Ly-6Clo) hepatic macrophages in mice [5]. These partly overlapping and partly opposing features of human and mouse macrophage subsets require further translational research. Very likely, our current definitions of the distinct hepatic macrophages still comprise mixed populations of functionally distinct cell subsets, whose differentiating marker and gene expression profiles remain to be deciphered. Another impediment in translating findings from mouse models to human disease is the wide spectrum of chronic liver disorders in humans. It is highly conceivable that the functionality of hepatic macrophage subsets is influenced by the nature of the underlying liver disease (e.g., lipid overload in fatty liver disease; antigen-specific responses in autoimmune hepatitis; impact of bile acids in cholangiopathies). Current studies have focused on the stage of disease progression (fibrosis, cirrhosis) rather than the nature of the underlying injury [38, 39]. Hence, further translational research is warranted to address disease-specific characteristics of hepatic monocyte and macrophage heterogeneity.

Nevertheless, many of the pathways characterized in mice for monocyte recruitment and macrophage effects are also strongly regulated in patients with liver diseases, suggesting well conserved mechanisms of macrophage activation across species [41]. Such pathways may possibly serve as novel targets for therapeutic approaches (for a summary of selected potential targets, see Table). For instance, activation of the CCL2-CCR2 axis is associated with monocyte infiltration in patients with chronic liver diseases and parallels fibrogenesis [39, 42]. Also, CD14+CD16+ monocyte-derived macrophages are capable of activating human stellate cells in vitro, partially dependent on TGF-β release [39]. The way human hepatic macrophage subsets shape the outcome of chronic liver disease is still unresolved and needs to be clarified before new therapeutic agents aiming at macrophage infiltration or polarization can enter clinical trials.

Acknowledgments This work was supported by the German Research Foundation (DFG Ta434/2-1 & SFB/TRR57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen.

Key points •

Macrophages exert critical functions in liver homeostasis, in the initiation of inflammation in response to hepatic injury and induction of fibrogenesis, but also in resolution of inflammation and fibrosis



Animal models from experimental liver injury revealed a remarkable heterogeneity of hepatic macrophages with diverse functions



Important aspects of hepatic macrophage heterogeneity include the origin of the macrophage subsets (derived from circulating monocyte precursors vs. resident Kupffer cells), their differentiation (oftentimes termed M1 vs. M2 polarization) as well as their effector functions in the context of liver diseases



Macrophage subpopulations with distinct functional properties have been also identified in human livers from patients with chronic liver diseases and fibrosis



Understanding the mechanisms that regulate hepatic macrophage heterogeneity, either by monocyte subset recruitment, by promoting restorative macrophage polarization or by impacting distinctive macrophage effector functions, may help to develop novel macrophage subset-targeted therapies for liver injury and fibrosis

References [1] Schuppan D, Kim YO. Evolving therapies for liver fibrosis. The Journal of clinical investigation 2013;123(5): 1887-1901. [2] Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, et al. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. The Journal of clinical investigation 2005;115(1): 56-65. [3] Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nature reviews Immunology 2011;11(11): 723-737. [4] Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nature reviews Immunology 2008;8(12): 958-969. [5] Ramachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, et al. Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis. Proceedings of the National Academy of Sciences of the United States of America 2012;109(46): E3186-3195. [6] Zimmermann HW, Trautwein C, Tacke F. Functional role of monocytes and macrophages for the inflammatory response in acute liver injury. Frontiers in physiology 2012;3: 56. [7] Klein I, Cornejo JC, Polakos NK, John B, Wuensch SA, Topham DJ, et al. Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages. Blood 2007;110(12): 4077-4085. [8] Naito M, Hasegawa G, Takahashi K. Development, differentiation, and maturation of Kupffer cells. Microscopy research and technique 1997;39(4): 350-364. [9] Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 2013;38(1): 79-91. [10] Luedde T, Schwabe RF. NF-kappaB in the liver--linking injury, fibrosis and hepatocellular carcinoma. Nature reviews Gastroenterology & hepatology 2011;8(2): 108118. [11] Leroux A, Ferrere G, Godie V, Cailleux F, Renoud ML, Gaudin F, et al. Toxic lipids stored by Kupffer cells correlates with their pro-inflammatory phenotype at an early stage of steatohepatitis. Journal of hepatology 2012;57(1): 141-149. [12] Bieghs V, Hendrikx T, van Gorp PJ, Verheyen F, Guichot YD, Walenbergh SM, et al. The cholesterol derivative 27-hydroxycholesterol reduces steatohepatitis in mice. Gastroenterology 2013;144(1): 167-178 e161. [13] Wehr A, Baeck C, Heymann F, Niemietz PM, Hammerich L, Martin C, et al. Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis. Journal of immunology 2013;190(10): 5226-5236. [14] De Minicis S, Seki E, Uchinami H, Kluwe J, Zhang Y, Brenner DA, et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology 2007;132(5): 1937-1946. [15] Pradere JP, Kluwe J, De Minicis S, Jiao JJ, Gwak GY, Dapito DH, et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 2013;58(4): 1461-1473. [16] Baeck C, Wehr A, Karlmark KR, Heymann F, Vucur M, Gassler N, et al. Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury. Gut 2012;61(3): 416-426. [17] Fallowfield JA, Mizuno M, Kendall TJ, Constandinou CM, Benyon RC, Duffield JS, et al. Scar-associated macrophages are a major source of hepatic matrix metalloproteinase-13 and facilitate the resolution of murine hepatic fibrosis. Journal of immunology 2007;178(8): 5288-5295. [18] Pellicoro A, Aucott RL, Ramachandran P, Robson AJ, Fallowfield JA, Snowdon VK, et al. Elastin accumulation is regulated at the level of degradation by macrophage

metalloelastase (MMP-12) during experimental liver fibrosis. Hepatology 2012;55(6): 19651975. [19] Melgert BN, Olinga P, Van Der Laan JM, Weert B, Cho J, Schuppan D, et al. Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats. Hepatology 2001;34(4 Pt 1): 719-728. [20] Bartneck M, Ritz T, Keul HA, Wambach M, Bornemann J, Gbureck U, et al. Peptidefunctionalized gold nanorods increase liver injury in hepatitis. ACS nano 2012;6(10): 87678777. [21] Galastri S, Zamara E, Milani S, Novo E, Provenzano A, Delogu W, et al. Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis. Clinical science 2012;123(7): 459-471. [22] Heymann F, Hammerich L, Storch D, Bartneck M, Huss S, Russeler V, et al. Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice. Hepatology 2012;55(3): 898909. [23] Karlmark KR, Weiskirchen R, Zimmermann HW, Gassler N, Ginhoux F, Weber C, et al. Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis. Hepatology 2009;50(1): 261-274. [24] Miura K, Yang L, van Rooijen N, Ohnishi H, Seki E. Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2. American journal of physiology Gastrointestinal and liver physiology 2012;302(11): G1310-1321. [25] Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, et al. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nature medicine 2007;13(11): 1324-1332. [26] Seki E, de Minicis S, Inokuchi S, Taura K, Miyai K, van Rooijen N, et al. CCR2 promotes hepatic fibrosis in mice. Hepatology 2009;50(1): 185-197. [27] Mitchell C, Couton D, Couty JP, Anson M, Crain AM, Bizet V, et al. Dual role of CCR2 in the constitution and the resolution of liver fibrosis in mice. The American journal of pathology 2009;174(5): 1766-1775. [28] Karlmark KR, Zimmermann HW, Roderburg C, Gassler N, Wasmuth HE, Luedde T, et al. The fractalkine receptor CX(3)CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes. Hepatology 2010;52(5): 17691782. [29] Imamura M, Ogawa T, Sasaguri Y, Chayama K, Ueno H. Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats. Gastroenterology 2005;128(1): 138-146. [30] Thomas JA, Pope C, Wojtacha D, Robson AJ, Gordon-Walker TT, Hartland S, et al. Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function. Hepatology 2011;53(6): 2003-2015. [31] Baeck C, Wei X, Bartneck M, Fech V, Heymann F, Gassler N, et al. Pharmacological inhibition of the chemokine CCL2 (MCP-1) accelerates liver fibrosis regression by suppressing Ly-6C+ macrophage infiltration. Hepatology 2013;in press. [32] Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard N, Kierdorf K, et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 2012;336(6077): 86-90. [33] Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, van Rooijen N, et al. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 2011;332(6035): 1284-1288. [34] Jenkins SJ, Ruckerl D, Thomas GD, Hewitson JP, Duncan S, Brombacher F, et al. IL4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. The Journal of experimental medicine 2013;210(11): 2477-2491. [35] Pesce JT, Ramalingam TR, Mentink-Kane MM, Wilson MS, El Kasmi KC, Smith AM, et al. Arginase-1-expressing macrophages suppress Th2 cytokine-driven inflammation and fibrosis. PLoS pathogens 2009;5(4): e1000371. [36] Robbins CS, Hilgendorf I, Weber GF, Theurl I, Iwamoto Y, Figueiredo JL, et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nature medicine 2013;19(9): 1166-1172.

[37] Davies LC, Rosas M, Jenkins SJ, Liao CT, Scurr MJ, Brombacher F, et al. Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation. Nature communications 2013;4: 1886. [38] Liaskou E, Zimmermann HW, Li KK, Oo YH, Suresh S, Stamataki Z, et al. Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics. Hepatology 2013;57(1): 385-398. [39] Zimmermann HW, Seidler S, Nattermann J, Gassler N, Hellerbrand C, Zernecke A, et al. Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis. PloS one 2010;5(6): e11049. [40] Ingersoll MA, Spanbroek R, Lottaz C, Gautier EL, Frankenberger M, Hoffmann R, et al. Comparison of gene expression profiles between human and mouse monocyte subsets. Blood 2010;115(3): e10-19. [41] Zimmermann HW, Tacke F. Modification of chemokine pathways and immune cell infiltration as a novel therapeutic approach in liver inflammation and fibrosis. Inflammation & allergy drug targets 2011;10(6): 509-536. [42] Marra F, DeFranco R, Grappone C, Milani S, Pastacaldi S, Pinzani M, et al. Increased expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis: correlation with monocyte infiltration. The American journal of pathology 1998;152(2): 423-430.

Table Conceivable therapeutic applications targeting hepatic macrophage heterogeneity (derived from experimental models of chronic liver injury) Target

Possible approach (selected from experimental models) +

Inhibition of (Ly-6C ) inflammatory monocyte influx

• Pharmacological antagonism of CCL2 (MCP-1) small using inhibitory RNA molecules (so-called Spiegelmers)[16] • Pharmaceutical inhibition of CCR2 [24] • Intestinal decontamination by antibiotics, leading to lower hepatic levels of LPS and reduced monocyte recruitment [25]

Counteraction of proinflammatory cytokines released by hepatic macrophages

• Neutralization of TNF and IL-1 to promote apoptosis of activated hepatic myofibroblasts [15]

Switching / modulating hepatic macrophage polarization (M1-M2)

• Biofunctionalized nanoparticles influencing hepatic macrophage polarization [20]

Augmentation of restorative hepatic macrophages

• Delivery of “polarizing” drugs (e.g., dexamethasone) to hepatic macrophages [19]

• Administration of CX3CL1 to induce a protective hepatic macrophage phenotype [28] • Application of IL-4 to force local proliferation of tissue-remodeling macrophages [33] • Autologous cell transfer of in vitro matured and polarized macrophages [30] • Inhibition of inflammatory monocyte influx during the regression of fibrosis [31]

Figure Legends

Figure 1. Hepatic macrophage heterogeneity during initiation and progression of chronic liver injury and fibrosis (mouse models). In the initial phase of hepatic injury, epithelial cell damage affecting hepatocytes or cholangiocytes perish and release DangerAssociated-Molecular-Pattern molecules (DAMPs) such as RNA, DNA, or alarmins (like HMBG-1) that activate resident Kupffer cells (KC), located at the luminal side of the liver sinusoidal endothelium (LSEC). In turn, Kupffer cells secrete pro-inflammatory cytokines like IL-1β and TNF that perpetuate parenchymal damage by inducing apoptosis. Furthermore, increased sinusoidal levels of Pathogen-Associated-Molecular-Pattern molecules (PAMPs) including lipopolysaccharide (LPS) stimulate Kupffer cells but also hepatic stellate cells (HSC) through Toll-like-Receptor 4 (TLR4) activation that leads to TGF-β sensitization and secretion of CCL2 by HSC as well as potent activation of KCs. Increased levels of CCL2 promote chemotaxis of bone marrow-derived ‘classical’ Ly-6Chi monocytes that enter the liver, where they develop into infiltrating Ly-6C+ macrophages exhibiting a pro-inflammatory phenotype. Freshly infiltrating hepatic macrophages booster the progression of chronic liver injury and fibrosis through TGF-β/PDGF-mediated HSC transdifferentiation and proliferation. The contribution of locally proliferating hepatic macrophages to the initiation and progression of chronic liver injury remains elusive. Overall, these mechanisms lead to the accumulation of collagen forming scar tissue.

Figure 2. Hepatic macrophage heterogeneity during regression of chronic liver injury and fibrosis (mouse models). In the course of chronic liver injury, Ly-6Chi macrophages can adopt a restorative phenotype characterized by Ly-6Clo expression and capacity to degrade excessive extracellular matrix proteins via metalloproteinases (MMP-9,-12,-13) and to induce HSC apoptosis. CX3CL1 (fractalkine) promotes survival of intrahepatic macrophages and may induce maturation of Ly-6C+ cells. Engulfment of cell fragments from apoptotic cells can further promote restorative features of hepatic macrophages. The contribution of freshly recruited Ly-6Clo monocytes into regenerating liver remains to be established. Overall, these mechanisms lead to the degradation of extracellular matrix during resolution of liver fibrosis.

Figure 1

cholangiocytes

hepatocytes

DAMPs

CCL2

chemotaxis

INITIATION

stellate cells

LSEC

Kupffer cell PAMPs (LPS)

CCL2

Ly-6Chi monocytes

proliferation

Ly-6C+ MΦ

PROGRESSION

TNF, IL1β

activation

recruitment

apoptotic HSC

activated myofibroblast

proliferation

stellate cells

ECM degradation transdifferentiation

ECM deposition

proven mechanism potential mechanism

scar formation

Figure 2

Kupffer cell Ly-6Clo monocytes

IL10

Ly-6Clo MΦ

recruitment

recruitment

switch

Ly-6Chi monocytes

Ly-6C+ MΦ restorative KC

matrix resolution

CX3CL1 (fractalkine) MMP-9,-12,-13

phagocytosis

REGRESSION

differentiation / maturation

ECM deposition

apoptotic parenchymal cells apoptotic HSC ECM degradation

scar regression proven mechanism potential mechanism

Macrophage heterogeneity in liver injury and fibrosis.

Hepatic macrophages are central in the pathogenesis of chronic liver injury and have been proposed as potential targets in combatting fibrosis. Recent...
2MB Sizes 1 Downloads 0 Views