Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice
Potential conflict of interest: Nothing to report.
This study was supported by NIH grants R01DK076920 and U54CA163111 (both to Robert F. Schwabe). Jean-Philippe Pradere was supported by a postdoctoral fellowship from the American Liver Foundation. Johannes Kluwe was supported by the German Research Foundation (grant KL2140/2-1) and a Sheila Sherlock fellowship from the European Association for the Study of the Liver. Ingmar Mederacke was supported by the German Research Foundation (grant ME3723/1-1). Dianne Dapito was supported by NIH grant 1F31DK091980. Costica Aloman was supported by NIH grant 1K08DK088954.
Address reprint requests to: Robert F. Schwabe, Department of Medicine, Columbia University, College of Physicians & Surgeons, Russ Berrie Pavilion, Room 415, 1150 St. Nicholas Ave, New York, NY 10032; E-mail: firstname.lastname@example.org; fax: 212-851-5461
Although it is well established that hepatic macrophages play a crucial role in the development of liver fibrosis, the underlying mechanisms remain largely elusive. Moreover, it is not known whether other mononuclear phagocytes such as dendritic cells (DCs) contribute to hepatic stellate cell (HSC) activation and liver fibrosis. We show for the first time that hepatic macrophages enhance myofibroblast survival in a nuclear factor kappa B (NF-κB)–dependent manner and thereby promote liver fibrosis. Microarray and pathway analysis revealed no induction of HSC activation pathways by hepatic macrophages but a profound activation of the NF-κB pathway in HSCs. Conversely, depletion of mononuclear phagocytes during fibrogenesis in vivo resulted in suppressed NF-κB activation in HSCs. Macrophage-induced activation of NF-κB in HSCs in vitro and in vivo was mediated by interleukin (IL)−1 and tumor necrosis factor (TNF). Notably, IL-1 and TNF did not promote HSC activation but promoted survival of activated HSCs in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in coculture experiments and genetic ablation of IL-1 and TNF receptor in vivo. Coculture and in vivo ablation experiments revealed only a minor contribution to NF-κB activation in HSCs by DCs, and no contribution of DCs to liver fibrosis development, respectively. Conclusion: Promotion of NF-κB–dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis. (Hepatology 2013;58:1461–1473)
The development of liver fibrosis constitutes one of the major complications of chronic liver disease, with many clinical consequences such as the development of esophageal varices and ascites being directly related to the presence of liver fibrosis. The hepatic wound healing response is a concerted action of multiple resident and nonresident cell types that not only provides a scaffold for structural stability but also involves the removal of cellular debris by infiltrating hepatic macrophages (HM) and the regeneration of functional parenchyma.[2, 3] Hepatic stellate cells (HSCs) are considered the main fibrogenic cell type in the liver and are responsible for the production of various types of extracellular matrix.[2, 3] HSCs undergo a well-characterized activation process, during which they lose their characteristic vitamin A and lipid stores and obtain a myofibroblastic phenotype.[2, 3] The activation of HSCs is controlled by multiple soluble mediators, including transforming growth factor β and platelet-derived growth factor, and is part of a complex cellular network that controls the hepatic wound healing response. Previous studies have demonstrated that multiple cell populations—including HMs, myeloid-derived suppressor cell, B cells, T cells, and natural killer cells—influence the development of liver fibrosis.[4-12] Among those, HMs exert a profound effect on HSCs and hepatic fibrosis as shown by genetic or pharmacologic models of macrophage depletion.[6, 7, 13] At the same time, HMs also contribute to fibrosis resolution through MMP13 and matrix remodeling.[6, 14, 15] However, the mechanisms by which HMs promote liver fibrosis remain largely elusive. Dendritic cells (DCs) are developmentally closely related to macrophages and exert a profound effect on liver fibrosis regression and the cytokine microenvironment during fibrogenesis, but their contribution to liver fibrosis development remains unknown.
In the present study, we uncovered the promotion of HSC/myofibroblast survival as a novel mechanism through which macrophages promote fibrosis. Moreover, we demonstrate for the first time that neither classical DCs (cDCs) nor plasmacytoid DCs (pDCs) contribute to fibrogenesis.
Hepatic fibrogenesis involves multiple resident and recruited cell populations. HSCs represent the center component of this wound healing response, but other populations, including macrophages, are known positive modulators of fibrogenesis. Here, we uncover a novel function of macrophages, the promotion of HSC/myofibroblast survival. A second novel finding of our study lies in the discovery that DCs do not contribute to liver fibrosis.
Employing microarray and pathway analysis, we discovered that NF-κB, the best-characterized antiapoptotic signaling pathway[29, 30] and an important regulator of liver injury and fibrosis, was a key pathway activated in HSCs by HMs. The relevance and physiologic nature of the employed in vitro coculture system is validated by the finding that this system achieves HSC gene expression patterns highly similar to those found in in vivo–activated HSCs, and that all gene expression changes and functional consequences of NF-κB activation were confirmed in vivo. Activation of the NF-κB pathway was further established by reporter assays, western blots, immunofluorescence, and qRT-PCR. Most importantly, NF-κB was activated in HSCs from fibrotic livers, and macrophage depletion reduced NF-κB activation in HSCs. The activation of NF-κB in HSCs in liver fibrosis is consistent with a previous study, but points toward macrophages instead of angiotensin II as the main trigger of NF-κB activation in HSCs. Surprisingly, coculture with macrophages and macrophage-secreted cytokines such as IL-1β and TNFα did not promote HSC activation, and is consistent with the reported minor or insignificant inductions of α-SMA and Col1a1 mRNA, and absence of increased α-SMA protein expression in most studies that cocultured human and murine HSCs with macrophages.[33, 34] Only one previous study found a profound and significant activation of rat HSCs by HMs. In our study, macrophage-induced NF-κB activation rendered activated HSCs more resistant to cell death in vitro and in vivo, thereby promoting the persistence of activated HSCs and fibrosis. Although the rate of 1% HSC apoptosis in fibrotic livers appeared low, it reflects the rapid removal of apoptotic cells in vivo (as opposed to their accumulation in vitro), and is virtually identical to peak apoptosis rates reported by Iredale et al. Thus, the observed increase to 5% HSC apoptosis is biologically highly significant, reducing the number activated myofibroblasts and limiting fibrogenic responses as reported.[11, 22, 32, 36] It is likely that increased NF-κB activation protects activated HSCs from both intrinsic and extrinsic inducers of cell death. Accordingly, our study also found that HMs induce the expression of Trail decoy receptors in HSCs in an NF-κB–dependent manner. This finding is of interest because natural killer cells, which are particularly enriched in the liver and activated during liver injury, contribute significantly to the killing of activated HSCs during liver fibrosis in a Trail-dependent manner.[11, 37, 38]
Our study identified IL-1 and TNF as main factors of HM-mediated NF-κB activation and cytoprotection in HSCs. Notably, we observed no effect of IL-1β or TNFα on HSC activation. The key role of HM-derived IL-1 and TNF in NF-κB activation and protection from HSC death was found not only in vitro but also in vivo, as demonstrated by the profound decrease in NF-κB–responsive genes in unplated, ultrapure HSC isolates from TNFR1/IL1R1 dko mice, and increased apoptosis of desmin-positive cells in TNFR1/IL1R1 dko livers after BDL. Previous studies have demonstrated reduced fibrogenesis in mice deficient in TNFR1 or IL1-R.[39, 40] In contrast to these studies, we could not observe reduced liver fibrosis in IL-1R knockout mice in three different models of liver fibrosis. This is consistent with the notion that both TNFα and IL-1β are powerful NF-κB activators, that they can likely functionally substitute each other.
Our study employed F4/80-positive HM from bile duct-ligated livers for co-culture experiments and therefore exposed HSCs to a mixture of resident and recruited macrophages typically found during fibrogenesis. Accordingly, our data show that these HM have a mixed M1/M2 phenotype as previously reported. Based on our observations that converting HM into M1 phenotype increased, and into M2 phenotype reduced their ability to induce NF-κB-dependent gene expression in HSCs, we conclude that the inflammatory/M1 HM subpopulation contributes to NF-κB activation and HSC survival. It should be emphasized that the M1/M2 classification does not fully account for diverse and often overlapping biological functions of macrophage populations, particularly in the liver. It is conceivable that different HM populations collaborate for the induction fibrosis in vivo, with inflammatory M1-type HM promoting HSC survival and M2-type HM affecting HSCs through other pathways. We did not find a significant impact of Gr1 status in HM on NF-κB activation in HSCs (data not shown), suggesting that both recruited and resident macrophages are capable of promoting NF-κB activation in HSCs. Clodronate did not affect HSC activation directly, nor did it alter NF-κB activation in HSCs. Moreover, our results employing DC depletion additionally excluded DC as potential contributors to clodronate effects, as we did not see a contribution of this cell type to liver fibrosis.
DCs are key regulators of inflammation and the cytokine milieu in the fibrotic liver. Moreover, DCs contribute to the regression of liver fibrosis through an MMP9-dependent mechanism. However, the contribution of DCs to fibrogenesis is unknown. Although we found that CD11c-positive DCs induce a moderate degree of NF-κB activation in HSCs via TNF and IL-1 production, we did not observe a role for pDC or cDC in promoting liver fibrosis in BDL- and CCl4-induced liver fibrosis. Most likely, the much lower number of DCs in the liver in comparison to HMs and the lower potency of NF-κB activation by DCs renders the contribution of DC-derived TNF and IL-1 to the overall pool and NF-κB–mediated HSC survival insignificant. In this regard, the ratio of DCs to HSCs in our coculture experiments is at least one or two magnitudes higher than the ratio that can be achieved in a fibrotic liver. Another possible explanation may be the critical role of DCs in NK cell activation, cells with well-established antifibrogenic potential.[11, 41] None of the available CD11c-DTR based ablation strategies can achieve a completely selective depletion of cDCs without affecting the composition of other immune cells.[26, 27] Even recent transgenic mouse models that avoid early neutrophilia after DC depletion still lead to neutrophilia after 2 days. Although neutrophilia represents a confounding factor, we consider it unlikely that neutrophilia affects fibrogenesis based on previous studies that did not show effects on liver fibrosis. These data suggest that DCs interact with and regulate other immune cells, or that increased granulopoiesis after ablation causes these secondary effects. Nonetheless, the ablation strategies employed in our study (1) avoid common side effects described for DC ablation due to our use of CD11c-DTR chimeric mice and 120G8 antibody, (2) address the role of both cDC and pDC, and (3) investigate the role of cDCs in two common models of liver fibrosis.
Our study contains several limitations. First, because we observed more than 1,400 HM-regulated genes, it is likely that genes besides NF-κB–regulated genes affect HSC responses. Further studies are required to unravel the relevance of NF-κB–independent genes and pathways regulated by HM. These may include additional mediators secreted from HMs such as IL-6 and transforming growth factor β.[35, 42] Accordingly, our IPA analysis revealed Stat1/3/5 as an HM-activated pathway. Second, our studies were performed in mouse models, and further studies are required to determine whether HM-induced NF-κB activation plays a role in human fibrogenesis. As patients develop fibrosis slowly over decades, pathways that promote long-term myofibroblast survival may be particularly relevant. IL-1 and TNF inhibitors may be considered for antifibrotic therapies but may cause severe side effects. In conjunction with previous studies,[32, 43] our data support the concept that targeting the NF-κB pathway in HSCs and subsequent induction of HSC apoptosis may be a more suitable antifibrogenic strategy.
In conclusion, our study shows that HMs provide a novel link between inflammation, HSC survival, and liver fibrosis and suggests that inflammatory signaling pathways may provide additional targets for antifibrotic therapies in the liver. Future studies are needed to determine whether macrophage-mediated promotion of myofibroblast survival also promotes fibrosis in other organs.