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Connolly MK, Bedrosian AS, Mallen-St Clair J, Mitchell AP, Ibrahim J, Stroud A, et al. In liver fibrosis, dendritic cells govern hepatic inflammation in mice via TNF-alpha. J Clin Invest 2009;119:3213-3225. (Reprinted with permission of the American Society for Clinical Investigation; permission conveyed through Copyright Clearance Center, Inc.)

Abstract

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Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8- fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-α. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease. © 2010 American Society for Clinical Investigation.

Comment

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Inflammatory responses after liver injury are a prerequisite for organ fibrosis. Several recent experimental approaches have provided evidence that intrahepatic inflammation is a highly regulated process involving the targeted recruitment and differentiation of distinct immune cell subsets into the hepatic microenvironment.1, 2 In a recent article, Connolly et al.3 add to this evolving picture by describing a role for liver dendritic cells (DCs) during fibrogenesis. Upon induction of experimental fibrosis in mice, they report a large increase of intrahepatic DCs, which were found to secrete several proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) and to activate natural killer (NK) cells, cytotoxic T cells, and hepatic stellate cells.3 The findings point toward a potentially interesting new aspect of the profibrogenic immune cell activation.

Intrahepatic leukocyte composition is very complex and includes numerous immune cell subtypes that enable the liver to function as a main site of innate immunity.4 Liver DCs represent, in the steady-state, only a minor population from the intrahepatic leukocytes; their major function after encountering an antigen is to initiate the adaptive immune responses or, in the absence of inflammation, immune tolerance.5 Moreover, other cell populations in the liver, including sinusoidal endothelial cells, macrophages/Kupffer cells, and even hepatic stellate cells, can also serve as antigen-presenting cells under certain conditions.1 Analysis of DCs primarily by flow cytometry (fluorescence-activated cell sorting [FACS]) requires meticulous efforts to exclude related cells that may express DC markers. The most widely used marker to identify the DC population is CD11c, which is a β2-integrin with unclear function, and which is expressed at different levels on a wide variety of cells including NK cells, NKT cells, monocytes, and even T or B cells (Fig. 1).6, 7 In the presence of inflammation, the analysis of DCs by FACS requires exclusion of autofluorescence, which is normally present in normal liver and is augmented in the setting of inflammation.8 More than that, digestion of the fibrotic tissue results in cell suspension with variable cell doublets and significant numbers of nonhematopoietic cells that may also express CD11c (e.g., stellate cells).9 For these reasons, the analysis of the intrahepatic DC population by FACS needs to be carefully validated by a sorting and cytospin approach to confirm that the cells analyzed are corresponding morphologically to DC populations.7, 10

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Figure 1. CD11c-positive cell populations in murine liver fibrosis. Several immune cell populations of hematopoietic origin (CD45+) can express the surface marker CD11c in mice, including NK cells (NK1.1+DX5+, MHC-II, CD11b), monocytes/macrophages (CD11b+F4/80+, Ly6G, NK1.1) or dendritic cell (DC) subsets, i.e., myeloid DC (CD11b+MHC-II+, CD8a), lymphoid DC (CD8a+MHCII+, CD11b), or plasmacytoid DC (B220+Gr1+PDCA+MHC-II+, CD11b, CD8a). Experimental results from murine models indicate that NK cells inhibit and (inflammatory) Gr1+ monocytes promote hepatic fibrosis progression. The role of DCs for initiation, progression, or regression of fibrosis is currently unclear.

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In the article by Connolly et al.,3 the authors investigate in a mouse model of liver fibrosis the composition of hepatic nonparenchymal CD11c+ cells and assess the impact of CD11c+ cells and “DC depletion” on the inflammatory environment. They showed, primarily by using the tool of flow cytometry, that 20%-27% of the nonparenchymal cells during fibrosis progression are CD11c+ “DCs”. These cells express variable levels of costimulatory molecules (CD40 and major histocompatibility complex II [MHC-II]), suggesting their involvement in antigen presentation. Further in the article, the CD11c+ cell population from fibrotic livers was isolated by CD11c immunomagnetic beads and was assessed in terms of the level of cytokine production; with or without toll-like receptor stimulation, this cell population has a high capacity to produce TNF-α and interleukin-6 (IL-6). Ex vivo depletion of CD11c+ cells isolated from fibrotic liver results in attenuated cytokine production. When a transgenic mouse model of conditional depletion of CD11c+ cells was used, cytokine production in the liver was diminished during the inflammatory process upon transitory “DC depletion”. Additionally, the authors showed that CD11c+ cells (labeled as “DCs”) isolated from the fibrotic livers are able to stimulate NK cells in vivo and in vitro, can be loaded by specific peptides, and induced a significant cytotoxic T lymphocyte response and T cell proliferative response. All these antigen-presentation properties of CD11c+ cells were confirmed in a model of tumor growth challenge; immunization of mice with CD11c+ cells loaded with ovalbumin peptide resulted in protection from tumor development by a cell line that expressed the peptide.

Although the main focus of the experiments is the modulation of the inflammatory process by the CD11c+ cell population during fibrosis progression, a possible link between this population and hepatic stellate cell function during fibrosis is provided by direct coculture experiments showing the augmentation of cytokine production and increased proliferative responses of hepatic stellate cells. In summary, the current study provides data suggestive of major dynamic changes of CD11c+ cells during fibrosis progression that impact the inflammatory environment of the liver. As the authors also stated in their discussion section, clear evidence of the role of CD11c-positive cells or DCs on liver fibrosis progression was not assessed.

There are a few aspects of the study that require a careful interpretation of the findings. First of all, the high fraction of cells identified as “DCs” among the inflammatory infiltrate is very surprising, because no other peripheral organs during an inflammatory state reportedly have such high numbers of DCs. The gating strategy used by Connolly and coworkers to identify the “DC population” included only the gates for the forward-scatter/side-scatter and side-scatter/CD11c plots. Based on the reasons stated above, this fraction may include NK, NKT, T, and B cells, and probably a high population of monocytes/macrophages that are massively recruited during the inflammatory process (Fig. 1).11 No mention of gating for viable cells, exclusion of doublets, and exclusion of nonhematopoietic cells was reported. Without clear evidence by cytospin analysis of specific DC morphology, labeling the whole CD11c+ population as DCs is far from complete, and the existence of a high (>30%) proportion of MHC-II–negative “DCs” by this group further underscores the shortcoming of the applied FACS protocol. In a similar fashion, the isolation of DCs using magnetic beads for the in vitro experiments described in the article used CD11c-positivity as a marker of DCs and was not associated in a combination protocol of depletion of the cells that may express CD11c but are not DCs.6

The second aspect that needs to be considered is the role of liver NK cell activation by DCs during fibrosis progression. The process of NK activation by DCs is a well-defined process12, 13; however, the impact of NK cell activation by DCs on liver fibrosis is unclear at this point because there is clear evidence that NK cell activity is protective during fibrosis progression.14, 15 Furthermore, the process of NK activation by liver DCs seems to be TNFα-dependent rather than IL-15–dependent.13 This latter result should raise major concerns regarding the contamination with monocytes/macrophages during isolation from fibrotic livers. In support of this conclusion, some of these “DC” features resemble “TNFα/inducible nitric oxide synthase (iNOS)-producing DCs” (Tip-DCs) that are Ly6C+/Gr1+ monocyte-derived macrophages commonly found in inflamed tissue.16

Moreover, the functional characterization of DCs in liver fibrosis remains an open question. The authors neither used DC depletion, which could have been performed in wild-type mice that underwent transplantation with CD11c-DTR (diphtheria toxin receptor) bone marrow and repetitive diphtheria toxin injections, nor expansion of the DC pool, which could have been achieved by the administration of growth factors such as fms-related tyrosine kinase 3 ligand during fibrogenesis, or interference with DC migration, which could have been addressed by using chemokine (C-C motif) receptor 7–deficient or plt-transgenic mice. Therefore, we believe that the article published by Connolly and coworkers addresses an important aspect of the liver microenvironment regarding the role of DCs in fibrosis progression. Additional studies are warranted to clarify the functional and potentially clinically relevant contribution of DCs for the progression or regression of liver fibrosis.

References

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  2. Abstract
  3. Comment
  4. References