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Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion
Research Article
Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion Bastian Höchst1,⇑, , Frank A. Schildberg1, , Pia Sauerborn1, Yvonne A. Gäbel1, Heidrun Gevensleben2, Diane Goltz2, Lukas C. Heukamp3, Andreas Türler4, Matthias Ballmaier5, Friederike Gieseke6, Ingo Müller7, Jörg Kalff8, Christian Kurts1, Percy A. Knolle1, Linda Diehl1,⇑ 1 Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Germany; 2Institute for Pathology, Universitätsklinikum Bonn, Germany; 3Institute for Pathology, University of Cologne, Germany; 4Department of General and Abdominal Surgery, Johanniter-Krankenhaus Bonn, Germany; 5Department of Pediatric Hematology and Oncology, Medizinische Hochschule Hannover, Germany; 6Research Institute, Children’s Cancer Center, Hamburg, Germany; 7Clinic for Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 8Department of Surgery, University Hospital Bonn, Germany
Background & Aims: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. Methods: We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14+HLA-DR/low monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14+HLA-DR/low cells by using blocking antibodies or knockdown technology. Results: Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14+HLA-DR/low cells. Such CD14+HLA-DR/low monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSCinduced development of CD14+HLA-DR/low monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44.
Keywords: Hepatic stellate cell; Myeloid derived suppressor cell; CD44; Arginase. Received 15 January 2013; received in revised form 2 April 2013; accepted 20 April 2013; available online 9 May 2013 ⇑ Corresponding authors. Address: Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Tel.: +49 228 287 11038; fax: +49 228 287 11052. E-mail addresses: [email protected] (B. Höchst), [email protected] (L. Diehl). These authors contributed equally to this work. Abbreviations: HSC, hepatic stellate cell; IFN, interferon; MDSC, myeloid derived suppressor cell; PGE2, prostaglandin E2; SCF, stem cell factor; siRNA, small interfering RNA; shRNA, small hairpin RNA; VEGF, vascular endothelial growth factor.
Conclusions: Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction The immune system is precisely balanced regarding inflammation and tolerance. This balance is upheld by both effector and regulatory/suppressive cells. Myeloid derived suppressor cells (MDSCs) can exert immunosuppressive functions [1] and comprise a group of immature myeloid cells or progenitors of mature macrophages, granulocytes or dendritic cells. MDSCs accumulate in the blood, bone-marrow, spleen, and tumor of patients and mice under pathological conditions and can establish a local immunosuppressive environment. In humans, MDSCs are associated with the suppression of T-cell responses [1], the induction of regulatory T cells [2] as well as poor prognosis in cancer patients [3]. MDSCs generally express CD33, however, different types of tumors harbor distinct subsets of MDSCs. They can be divided into three phenotypic subpopulations: Lin-HLA-DR/low [4], CD14+HLA-DR/low [5], and CD15+HLA-DR/low [6] MDSCs. How MDSCs are induced and from which cells they arise is still a matter of debate. Many soluble factors are reported to be involved in the induction of MDSCs, such as CSF-1, and -2 [7,8]. However, they are also important for physiological development or function of myeloid cells.
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JOURNAL OF HEPATOLOGY Under steady state conditions, hepatic stellate cells (HSCs) are vitamin A storing pericytes situated in the space of Dissé. Chronic inflammation leads to HSC activation, proliferation, and transdifferentiation into myofibroblasts resulting in fibrotic remodeling of the liver. In a murine model of islet-transplantation in vivo, transfer of HSCs increased MDSC numbers [9], however, the exact mechanism by which this occurs could not be identified. As the frequency of HSCs increases during persistent inflammation and MDSCs are especially increased in liver diseases [10], we investigated whether human HSCs are involved in the induction of MDSCs. Here, we show that hepatic stellate cells can induce CD14+HLA-DR/low myeloid derived suppressor cells from mature peripheral blood monocytes. Furthermore, we provide evidence that this MDSC induction requires cell-cell contact and is mediated via CD44.
Materials and methods Suppression assay CD14+ monocytes were cultured with HSCs or control cell-lines for 3 days or were left untreated. CD14+HLA-DR/low MDSCs were isolated ex vivo as described before [2]. T cells were labeled with 0.1 lM carboxyfluorescein-succinimidylester (CFSE) and were stimulated with T-cell activation and expansion kit (Miltenyi, Bergisch Gladbach, Germany). CD14+ cells were titrated as indicated. Proliferation of T cells was measured after 4 days based on the dilution of CFSE. Where indicated, L-N-monomethyl-arginine citrate (L-NMMA), N-hydroxylL-arginine (L-NOHA) (10 lM each) (all Sigma-Aldrich, Seelze, Germany) or antiTGF-b blocking antibody (Clone 1D11, 40 lg/ml) (R&D Systems, Minneapolis, MN) were added.
on monocytes (Fig. 1A). This effect was HSC-specific as we found no such effect upon co-culture with hepatocyte cell-lines (HepG2, HuH-7), a liver sinusoidal endothelial cell-line (SK-Hep1), primary B-cells or the colon carcinoma cell-line CCL253 (Fig. 1A and B). Moreover, activated pHSCs were more potent inducers of CD14+HLA-DR/low monocytes compared to freshly isolated pHSCs (Fig. 1A) and 7 days after isolation, primary HSCs were as efficient in HLA-DR downregulation as the HSC cell-line LX2 (Fig. 1A), indicating that only activated HSCs could functionally modulate circulating monocytes. The number of HLA-DR/low cells among CD14+ monocytes increased over time in co-culture with HSCs (Fig. 1C). These CD14+ cells did not proliferate (Supplementary Fig. 1A) or undergo increased apoptosis (data not shown), indicating that these cells trans-differentiated from mature monocytes and did not arise from immature progenitors that may have been present in the culture. Furthermore, we excluded that HSCs in general downregulate HLA-DR expression in immune cells, as co-culture with LX2 did not downregulate HLA-DR on B-cells or myeloid dendritic cells (mDC) (Fig. 1D). In addition, we did not find HSCs to be able to induce other phenotypical MDSC populations, such as Lin-CD11bCD33+ cells, from PBMC (Supplementary Fig. 1B). MHC-class-II downregulation by HSCs required cell-cell contact, because LX2 culture supernatant alone or culturing LX2 separately from monocytes in a transwellsystem did not affect HLA-DR (Fig. 1E). Together, these data indicate that human HSCs induce CD14+HLA-DR/low monocytes from circulating HLA-DRhi monocytes in a cell-cell-contact dependent fashion. CD14+HLA-DR/low monocytes induced by hepatic stellate cells phenotypically resemble MDSCs
Gene silencing using siRNA LX2 cells were trypsinized and 106 cells were resuspended in 200 ll Nucleofector Solution (Lonza, Verviers, Belgium) containing 300 nmol/L siRNA. Cells were transfected using Nucleofector II according to the manufacturers’ protocol using the protocol for human monocytes. Statistical analysis Statistical analysis was done using Student’s t test. Data are depicted, as mean ± SEM. p values 60.05 were considered significant. ⁄p 60.05, ⁄⁄p 60.01, p 60.001.
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Results Primary human hepatic stellate cells downregulate HLA-DR expression on peripheral blood monocytes Given the complex regulatory functions of HSCs described in the literature [9,11,12], we investigated whether human HSCs were capable of generating MDSCs. It is known that hematopoietic stem cells or early myeloid progenitors have the capacity to develop into MDSCs [13], but some MDSC subsets in humans express the monocyte marker CD14 [5]. Therefore, we investigated whether circulating CD14+ monocytes, which pass through hepatic sinusoids within the blood stream, would develop into MDSCs after contact with HSCs. Therefore, we cultured freshly isolated peripheral blood CD14+HLA-DR+ monocytes on primary human HSCs (pHSCs) or the HSC cell-line LX2. Co-culture with HSCs resulted in marked downregulation of HLA-DR expression
To further characterize HSC-induced CD14+HLA-DR/low monocytes, we analyzed their surface marker expression profile and compared it to monocytes cultured with other cell lines or freshly isolated CD14+HLA-DR+ monocytes. CD14+HLA-DR/low MDSCs isolated from healthy individuals served as control. There were no differences with respect to CD15, CD40, and CD80 expression levels on freshly isolated monocytes and HSC-co-cultured monocytes, also ex vivo MDSCs did not express these molecules (Fig. 2). While co-culture with hepatocyte cell lines led to upregulation of co-stimulatory molecules like CD40 and CD80 on peripheral blood monocytes, such regulation was not observed when monocytes were co-cultured with HSCs (Fig. 2). Upon contact with HSCs, however, monocytes showed increased levels of IL-4Ra (Fig. 2), a phenotype also observed in MDSCs isolated from patients suffering from melanoma and colorectal carcinoma [14]. Interestingly, monocytes cultured on HSCs downregulated CD11b (Fig. 2). Thus, phenotypic characterization of surface molecules did not allow us to identify a profile typical for MDSCs in monocytes co-cultured with HSCs. This led us to perform quantitative PCR in order to determine the expression levels of genes specifically associated with MDSCs [15]. S100A12, that is overexpressed in MDSCs from hepatocellular and colorectal carcinoma patients [16], was selectively expressed in CD14+HLA-DR/low monocytes cultured on LX2 and CD14+HLA-DR/low MDSCs ex vivo (Supplementary Fig. 2). Also arginase-I, which is involved in the MDSC-mediated suppression of T-cell function, [5] was expressed selectively in HSC-induced CD14+HLA-DR/low monocytes and ex vivo CD14+HLA-DR/low MDSCs. In contrast, the expression of iNOS,
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Fig. 1. Hepatic stellate cells induce an MDSC-like phenotype in peripheral blood monocytes. (A–C) Monocytes were cultured alone, on primary human HSCs, the HSC cell-lines LX2, HepG2, HuH7, SK-Hep-1 or CCL253 for 3 days, and the expression of HLA-DR was measured by flow cytometry. Representative contour plots (A), % CD14+HLA-DR+ expression from 5 independent experiments (B) or absolute number (C) are shown. (D) Isolated monocytes, B-cells or myeloid dendritic cells were added to a confluent layer of LX2 in a 24-well plate. The expression of HLA-DR was analyzed on CD14+, CD19+ or CD1c+ cells by flow cytometry. Cumulative data from 3 independent experiments are shown. (E) Monocytes were cultured alone, on LX2, with LX2 conditioned media or with LX2 cells separated by a transwell-insert for 3 days. The frequency of CD14+HLA-DR/low cells is plotted cumulative for 3 independent experiments. ⁄p 60.05, ⁄⁄p 60.01, ⁄⁄⁄p 60.001.
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Fig. 2. Monocytes cultured on hepatic stellate cells upregulate IL-4Ra. CD14+ cells were cultured as described before, alone, co-cultured with different cell lines for 3 days or analyzed directly ex vivo. The expression of surface markers was analyzed by flow cytometry gating on CD14+, or CD14+HLA-DR+ or CD14+HLA-DR/low cells. The corresponding MFI is shown. Representative histograms from 5 experiments are shown.
which has also been described to mediate T-cell suppression [17], was not restricted to HSC-cultured monocytes or ex vivo MDSCs 530
(Supplementary Fig. 2). Taken together, CD14+ monocytes cultured on HSCs downregulate HLA-DR, express IL-4Ra and
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JOURNAL OF HEPATOLOGY S100A12, and upregulate Arg-I mRNA, which indicates that these cells also may exert suppressive function.
MDSCs mediated suppression of T cells (Fig. 3D). Moreover, neither the addition of blocking antibodies against IL-10 or IL6R, nor co-culture with an IDO inhibitor (1-MT) or the ROS inhibitors (YCG063 or MnTBAP) prevented suppression of T-cell proliferation (Supplementary Fig. 3). Together, both phenotypically and functionally HSC-induced CD14+HLA-DR/low monocytes represent a myeloid derived suppressor cell population that exert the suppressive function through arginase-I.
HSC-induced CD14+HLA-DR/low monocytes suppress T-cell proliferation in an arginase-I-dependent manner We next investigated whether HSC-induced CD14+HLA-DR/low monocytes could suppress T-cell proliferation. To this end, human CD8+ T cells were stimulated using anti-CD2/CD3/CD28coated beads, and CD14+HLA-DR/low monocytes previously cultured with LX2 were added. Addition of increasing numbers of HSC-induced CD14+HLA-DR/low monocytes to a fixed number of CD8 T cells progressively reduced T-cell proliferation (Fig. 3A). This effect was only observed with CD14+ cells cultured on LX2, pHSCs and freshly isolated MDSCs (Fig. 3B), but not with those monocytes cultured together with LSECs (SK-Hep1) or the hepatocyte (HepG2, HuH-7) cell lines. The same inhibitory effect on proliferation was also observed for CD4 T cells (data not shown). In addition to T-cell proliferation, IFN-c production was also inhibited by LX2-induced CD14+HLA-DR/low monocytes and correlated inversely with the addition of increasing numbers of LX2-induced CD14+HLA-DR/low monocytes into the culture (Fig. 3C). MDSCs can mediate the suppressive function via several mechanisms [18]. By adding specific inhibitors or blocking antibodies into the co-culture of T cells with HSC-induced CD14+HLA-DR/low monocytes, we found that only L-NOHA, a specific arginase-I inhibitor, prevented the suppression of T-cell proliferation. Neither the pan-inhibitor of NO-synthase (LNMMA) nor blocking antibodies against TGF-b was able to block
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The induction of CD14+HLA-DR/low monocyte-derived MDSCs by HSCs depends on the expression of CD44 CD44 expression has been shown to be increased on hepatic stellate cells from injured rat livers and is associated with their increased motility [19]. Moreover, in toxic liver injury, CD44 was shown to prevent exacerbation of inflammation via control of macrophage function infiltrating the liver [20]. In humans, hepatic CD44 expression is increased in patients with liver disease [21] and is involved in progression of various cancer types including hepatocellular carcinoma [22,23], in which MDSCs are increased and involved in immune-suppression [5,16]. To test if CD44 is involved in the induction of MDSCs, we added increasing concentrations of anti-CD44 blocking antibodies into the co-culture of LX2 and monocytes. Because we have previously shown, that CD54 on HSCs is important for the veto-function towards CD8 T cells [12], we also included blocking antibodies for CD54. In contrast to HSC-mediated CD8 T-cell inhibition, CD54 was not involved in induction of HLA-DR/low monocytes by HSCs, but blocking CD44 reduced the induction of MDSCs from monocytes dose dependently (Fig. 4A). Moreover, blocking CD44 inter-
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Fig. 3. Hepatic stellate cell-modified monocytes suppress T cells in an arginase-I-dependent mechanism. Monocytes were cultured for 3 days as indicated. Isolated CSFE labeled CD8 T cells were stimulated using CD2/CD3/CD28-coated beads and CD14+ cells were added at different ratios. Proliferation was analyzed (A, B, and D) after four days. The percentage proliferation compared to maximal proliferation (T cells cultured alone) is depicted (B). IFN-c production was quantified by ELISA (ratio T cells to CD14+HLA-DR/low cells 1:0.1/0.3/1/3) (C). Blocking antibodies or inhibitors were added during the co-culture of monocytes with T cells as indicated (D). Representative histograms (A) or cumulative results from 6 (B and C) or 3 (D) independent experiments are shown. ⁄p 60.05, ⁄⁄p 60.01, ⁄⁄⁄p 60.001, n.s., not significant.
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Fig. 4. Induction of CD14 HLA-DR cells by hepatic stellate cells is mediated by CD44. CD14 cells were cultured on LX2 as described before. Blocking antibody against CD54, CD44 or appropriate isotype controls were added as indicated (A, B, F, and G). The frequency of CD14+HLA-DR/low cells is plotted from 4 independent experiments (A) or 2 experiments pHSCs (F and G). CD14+ cells were isolated after culture with LX2 in the absence or presence of blocking CD44 antibody and were cocultured with CD8 T cells. Proliferation of T cells was measured on day 4. Cumulative results of 4 independent experiments are shown (B). CD44 was silenced in LX2 using siRNA as described. As control, scrambled siRNA or untreated cells were used as controls. After 24 h, expression of CD44 on LX2 cells was analyzed by flow cytometry. Histograms represent mean fluorescence intensity of CD44 (C). Monocytes were co-cultured with different siRNA-transfected LX2 cells and the expression of HLA-DR was analyzed by flow cytometry. Dot plots show percentages of HLA-DR/low cells of 3 independent experiments (D). Primary isolated human hepatic stellate cells were isolated and cultured for 6 days. Cells were analyzed every day for the mean fluorescence intensity of CD44 and GeoMean of autofluorescence by flow cytometry. Representative results from 2 independent experiments are shown (E). ⁄p 60.05, ⁄⁄p 60.01, ⁄⁄⁄p 60.001.
actions between monocytes and HSCs prevented acquisition of the suppressive capacity by those monocytes (Fig. 4B). To investigate whether HSC-expressed CD44 was important for induction of MDSCs, we efficiently knocked down the expression of CD44 on LX2 cells using two different siRNAs (Fig. 4C). In absence of CD44 expression, LX2 cells were not able to downregulate HLADR on monocytes anymore (Fig. 4D), indicating that HSC-dependent MDSCs induction is CD44-mediated. As we found primary human HSCs to become more efficient in inducing MDSCs from monocytes after activation by culture on plastic, we investigated expression kinetics of CD44 on primary HSCs. As HSCs become activated, they lose vitamin A containing lipid droplets and thereby autofluorescence (Fig. 4E). Primary HSCs did not express CD44, but markedly upregulated CD44 during activation in vitro (Fig. 4E), achieving levels comparable to LX2. More importantly, the induction of HLA-DR/low monocytes by primary HSCs 532
depended on CD44 expression, as it was abrogated in the presence of anti-CD44 blocking antibodies (Fig. 4F and G). Together, these data suggest that activated hepatic stellate cells can efficiently induce MDSCs in a CD44-dependent manner from mature monocytes.
Discussion The liver has an extraordinary immunological function determined by the unique micro-environment and its organ-resident cells that function as antigen-presenting cells to regulate antigen-specific immune responses [24]. Antigen-specific immunity can eradicate infections with hepatotropic pathogens from the liver [25]. Nevertheless, persistence of pathogens in hepatocytes or development of hepatocellular carcinoma in the context of
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JOURNAL OF HEPATOLOGY chronic inflammation demonstrates that the tolerogenic function of hepatic cell populations may impede efficient immune surveillance under certain circumstances. Here, we report that activated HSCs, which arise as a consequence of hepatic inflammation, induce myeloid derived suppressor cells (MDSCs) in a cell-contact and CD44-dependent manner from peripheral blood monocytes. HSCs can exert regulatory immune function via inhibition of DC-mediated CD8 T-cell activation [12,26], induction of T regulatory cells [11,26] or PDL1-mediated induced apoptosis of T cells [27]. Furthermore, adoptive transfer of murine HSCs leads to the induction of MDSCs [9], which are prominent regulatory cells known to locally regulate adaptive immunity [28]. Data from in vitro studies showed that MDSCs can be generated from bone-marrow cells in the presence of various cytokine cocktails [29]. Therefore, it is thought that MDSCs in vivo originate from precursors in the bone-marrow and represent a population of immature myeloid cells that are recruited to sites of inflammation. Our data provide evidence that CD14+HLA-DR/low MDSCs can also be generated from circulating CD14+ monocytes in a peripheral organ such as the liver. Interestingly, MDSCs induced by HSCs are similar to those found in patients suffering from hepatocellular carcinoma [5] or HCV infection [30], indicating that also in humans HSCs may induce MDSCs in vivo. Thus, the local generation of MDSCs by HSCs may provide a novel mechanism as to how systemic adaptive immunity is locally downregulated in the liver. We discovered that activated, but not freshly isolated HSCs, had the capacity to generate CD14+HLA-DR/low monocytederived MDSCs. During chronic inflammatory conditions, HSCs are activated in vivo and trans-differentiate into pro-fibrogenic myofibroblasts [31]. We, therefore, investigated the role of molecules known to be upregulated upon activation of HSCs. A number of soluble factors, like GM-CSF, M-CSF, and VEGF, have been implicated to play a role in the induction of MDSCs [32]. Recently, murine HSC were found to mediate MDSC induction via the complement factor C3 upon adoptive transfer [33]. However, we found that soluble factors do not play a role in the induction of CD14+HLA-DR/low MDSCs from monocytes by HSCs. This could be due to species differences or the origin of the MDSC, as murine HSC were used to generate MDSC from bone-marrow precursors and not mature monocytes [29]. Subsequently, we focused on cell surface molecules expressed by HSCs. CD54 expression on HSCs is critically important for direct inhibitory effects on naïve T-cell activation and proliferation [12]. In the generation of CD14+HLA-DR/low monocyte-derived MDSCs by HSCs, CD54 was not involved. HSC activation also leads to increased expression levels of CD44 [34,35]. We found evidence for the involvement of CD44 in generation of CD14+HLA-DR/low monocyte-derived MDSCs by using blocking antibodies to CD44 or siRNA-mediated CD44 knockdown in HSCs. It remains unclear how CD44 induced generation of CD14+HLA-DR/low monocyte-derived MDSCs from circulating monocytes because CD44 is not selectively expressed by activated HSCs. CD44 is involved in cell recruitment to tissues such as liver or bone marrow [36,37] and locomotion of T cells within tissues or tumors [38]. CD44 has several interaction partners including extracellular matrix proteins such as hyaluronic acid, mediators such as osteopontin, and the VLA-4 molecule to establish cell-cell interaction [39]. Although VLA-4 has mainly been found to mediate cell adhesion, a signaling func-
tion for this molecule has been reported for T cells [40], indicating that potentially VLA-4 could be involved in signaling processes in CD14+ monocytes leading to MDSC differentiation. Moreover, specificity of CD44 in the induction of MDSCs could also be mediated via its differential splicing that can lead to altered functionality [41]. Indeed, rat hepatic stellate cells express a distinct set of CD44 splice variants necessary for migration during liver injury [19]. Thus, it is possible that a particular splice variant of CD44 upregulated on HSCs is involved in induction of MDSCs. Our findings raise the question whether monocytes or monocyte-derived cells present in the liver all function as MDSCs. In the hepatic sinusoid, fenestrated LSECs without a basement membrane, do not provide a physical barrier and allow direct physical contact between HSCs in the space of Dissé and Kupffer cells or circulating monocytes [42]. However, only activated HSCs have the capacity to generate monocyte-derived MDSCs. Therefore, it is unlikely that, under steady state non-inflammatory conditions, MDSCs are generated from monocytes or Kupffer cells. Consistently, Kupffer cells isolated from the normal liver function as antigen-presenting cells, but not as suppressive MDSCs [43]. However, under inflammatory conditions, Kupffer cells may also be modulated, as we found LX2 cells to be able to downregulate HLA-DR on LPS-stimulated Kupffer cells (data not shown). The induction of MDSCs by activated HSCs in the chronically inflamed liver may thus represent a negative feedback loop in the attempt to dampen or prevent immunopathology in the liver. As the activation of HSCs is accompanied by increased expression of chemokines and adhesion molecules such as CD54 and CD44 that can lead to attraction of circulating monocytes [35], chronic inflammation and the consequent HSC activation may thus trigger both increased recruitment of monocytes and differentiation into MDSCs. It is unclear whether activated HSCs also bear the capacity to modulate Kupffer-cell function and cause their differentiation into MDSCs. The induction of CD14+HLA-DR/low monocyte-derived MDSCs by HSCs did not result from proliferation of precursor cells, but represents a differentiation process that was so far unknown to be involved in MDSC generation [44]. It is important to note that HSC-induced CD14+HLA-DR/low monocyte-derived MDSCs did not maintain their surface and functional phenotype for more than 2 days after losing HSC contact (data not shown), indicating that the increase in numbers of MDSCs in the liver will only be transient and strictly linked to persistent hepatic inflammation. It also suggests that MDSCs generated from monocytes require continuous contact with activated HSCs to maintain their function as suppressor cells. The dynamic induction of MDSCs from monocytes by activated mesenchymal cells, such as alphasmooth muscle antigen (aSMA) positive hepatic HSCs, indicates that aSMA+ tumor stroma cells, whose appearance in tumor tissue is often directly correlated with patient survival [45], may also locally induce MDSCs thereby creating a regulatory tumor micro-environment that impairs immune clearance of cancer cells. It also provides an interesting link to the observation that tumors mostly arise in organs during chronic inflammatory conditions [46]. Taken together, these results demonstrate that activated human HSCs can induce differentiation of monocytes into MDSCs, which provides evidence for a so far unrecognized mechanism in the generation of MDSCs via a re-differentiation step of already differentiated monocytes. The HSC-mediated
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Research Article induction of CD14+HLA-DR/low monocyte-derived MDSCs also attributes further immune competence to peripheral organs like the liver because attenuation of local inflammation may not only occur as a consequence of increased chemokine-mediated recruitment of circulating MDSCs, but also rely on a local and dynamic differentiation process initiated by organ-resident HSCs. Financial support This work was supported by a BONFOR grant of the University of Bonn medical faculty to BH.
Conflict of interest The authors who have taken part in this study do not have a relationship with the manufacturers of the drugs involved either in the past or present and did not receive funding from the manufacturers to carry out their research. BH was supported by a grant from the University of Bonn Medical Faculty (BONFOR). LD was supported by a grant from the german research council (SFB704).
Authors’ contributions LD and BH were responsible for the study concept, interpretation of data and drafting of the manuscript. BH, FAS, LCH, YAG, and PS performed experiments and BH and FAS analyzed the data. MB, HG, DG, LCH, FG, AT, JCK and IM provided the material. LD, CK, and PK provided important intellectual content as well as critical revision of the manuscript.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.04. 033.
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Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion Bastian Höchst1,⇑, , Frank A. Schildberg1, , Pia Sauerborn1, Yvonne A. Gäbel1, Heidrun Gevensleben2, Diane Goltz2, Lukas C. Heukamp3, Andreas Türler4, Matthias Ballmaier5, Friederike Gieseke6, Ingo Müller7, Jörg Kalff8, Christian Kurts1, Percy A. Knolle1, Linda Diehl1,⇑ 1 Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Germany; 2Institute for Pathology, Universitätsklinikum Bonn, Germany; 3Institute for Pathology, University of Cologne, Germany; 4Department of General and Abdominal Surgery, Johanniter-Krankenhaus Bonn, Germany; 5Department of Pediatric Hematology and Oncology, Medizinische Hochschule Hannover, Germany; 6Research Institute, Children’s Cancer Center, Hamburg, Germany; 7Clinic for Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 8Department of Surgery, University Hospital Bonn, Germany
Background & Aims: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. Methods: We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14+HLA-DR/low monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14+HLA-DR/low cells by using blocking antibodies or knockdown technology. Results: Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14+HLA-DR/low cells. Such CD14+HLA-DR/low monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSCinduced development of CD14+HLA-DR/low monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44.
Keywords: Hepatic stellate cell; Myeloid derived suppressor cell; CD44; Arginase. Received 15 January 2013; received in revised form 2 April 2013; accepted 20 April 2013; available online 9 May 2013 ⇑ Corresponding authors. Address: Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Tel.: +49 228 287 11038; fax: +49 228 287 11052. E-mail addresses: [email protected] (B. Höchst), [email protected] (L. Diehl). These authors contributed equally to this work. Abbreviations: HSC, hepatic stellate cell; IFN, interferon; MDSC, myeloid derived suppressor cell; PGE2, prostaglandin E2; SCF, stem cell factor; siRNA, small interfering RNA; shRNA, small hairpin RNA; VEGF, vascular endothelial growth factor.
Conclusions: Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction The immune system is precisely balanced regarding inflammation and tolerance. This balance is upheld by both effector and regulatory/suppressive cells. Myeloid derived suppressor cells (MDSCs) can exert immunosuppressive functions [1] and comprise a group of immature myeloid cells or progenitors of mature macrophages, granulocytes or dendritic cells. MDSCs accumulate in the blood, bone-marrow, spleen, and tumor of patients and mice under pathological conditions and can establish a local immunosuppressive environment. In humans, MDSCs are associated with the suppression of T-cell responses [1], the induction of regulatory T cells [2] as well as poor prognosis in cancer patients [3]. MDSCs generally express CD33, however, different types of tumors harbor distinct subsets of MDSCs. They can be divided into three phenotypic subpopulations: Lin-HLA-DR/low [4], CD14+HLA-DR/low [5], and CD15+HLA-DR/low [6] MDSCs. How MDSCs are induced and from which cells they arise is still a matter of debate. Many soluble factors are reported to be involved in the induction of MDSCs, such as CSF-1, and -2 [7,8]. However, they are also important for physiological development or function of myeloid cells.
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JOURNAL OF HEPATOLOGY Under steady state conditions, hepatic stellate cells (HSCs) are vitamin A storing pericytes situated in the space of Dissé. Chronic inflammation leads to HSC activation, proliferation, and transdifferentiation into myofibroblasts resulting in fibrotic remodeling of the liver. In a murine model of islet-transplantation in vivo, transfer of HSCs increased MDSC numbers [9], however, the exact mechanism by which this occurs could not be identified. As the frequency of HSCs increases during persistent inflammation and MDSCs are especially increased in liver diseases [10], we investigated whether human HSCs are involved in the induction of MDSCs. Here, we show that hepatic stellate cells can induce CD14+HLA-DR/low myeloid derived suppressor cells from mature peripheral blood monocytes. Furthermore, we provide evidence that this MDSC induction requires cell-cell contact and is mediated via CD44.
Materials and methods Suppression assay CD14+ monocytes were cultured with HSCs or control cell-lines for 3 days or were left untreated. CD14+HLA-DR/low MDSCs were isolated ex vivo as described before [2]. T cells were labeled with 0.1 lM carboxyfluorescein-succinimidylester (CFSE) and were stimulated with T-cell activation and expansion kit (Miltenyi, Bergisch Gladbach, Germany). CD14+ cells were titrated as indicated. Proliferation of T cells was measured after 4 days based on the dilution of CFSE. Where indicated, L-N-monomethyl-arginine citrate (L-NMMA), N-hydroxylL-arginine (L-NOHA) (10 lM each) (all Sigma-Aldrich, Seelze, Germany) or antiTGF-b blocking antibody (Clone 1D11, 40 lg/ml) (R&D Systems, Minneapolis, MN) were added.
on monocytes (Fig. 1A). This effect was HSC-specific as we found no such effect upon co-culture with hepatocyte cell-lines (HepG2, HuH-7), a liver sinusoidal endothelial cell-line (SK-Hep1), primary B-cells or the colon carcinoma cell-line CCL253 (Fig. 1A and B). Moreover, activated pHSCs were more potent inducers of CD14+HLA-DR/low monocytes compared to freshly isolated pHSCs (Fig. 1A) and 7 days after isolation, primary HSCs were as efficient in HLA-DR downregulation as the HSC cell-line LX2 (Fig. 1A), indicating that only activated HSCs could functionally modulate circulating monocytes. The number of HLA-DR/low cells among CD14+ monocytes increased over time in co-culture with HSCs (Fig. 1C). These CD14+ cells did not proliferate (Supplementary Fig. 1A) or undergo increased apoptosis (data not shown), indicating that these cells trans-differentiated from mature monocytes and did not arise from immature progenitors that may have been present in the culture. Furthermore, we excluded that HSCs in general downregulate HLA-DR expression in immune cells, as co-culture with LX2 did not downregulate HLA-DR on B-cells or myeloid dendritic cells (mDC) (Fig. 1D). In addition, we did not find HSCs to be able to induce other phenotypical MDSC populations, such as Lin-CD11bCD33+ cells, from PBMC (Supplementary Fig. 1B). MHC-class-II downregulation by HSCs required cell-cell contact, because LX2 culture supernatant alone or culturing LX2 separately from monocytes in a transwellsystem did not affect HLA-DR (Fig. 1E). Together, these data indicate that human HSCs induce CD14+HLA-DR/low monocytes from circulating HLA-DRhi monocytes in a cell-cell-contact dependent fashion. CD14+HLA-DR/low monocytes induced by hepatic stellate cells phenotypically resemble MDSCs
Gene silencing using siRNA LX2 cells were trypsinized and 106 cells were resuspended in 200 ll Nucleofector Solution (Lonza, Verviers, Belgium) containing 300 nmol/L siRNA. Cells were transfected using Nucleofector II according to the manufacturers’ protocol using the protocol for human monocytes. Statistical analysis Statistical analysis was done using Student’s t test. Data are depicted, as mean ± SEM. p values 60.05 were considered significant. ⁄p 60.05, ⁄⁄p 60.01, p 60.001.
⁄⁄⁄
Results Primary human hepatic stellate cells downregulate HLA-DR expression on peripheral blood monocytes Given the complex regulatory functions of HSCs described in the literature [9,11,12], we investigated whether human HSCs were capable of generating MDSCs. It is known that hematopoietic stem cells or early myeloid progenitors have the capacity to develop into MDSCs [13], but some MDSC subsets in humans express the monocyte marker CD14 [5]. Therefore, we investigated whether circulating CD14+ monocytes, which pass through hepatic sinusoids within the blood stream, would develop into MDSCs after contact with HSCs. Therefore, we cultured freshly isolated peripheral blood CD14+HLA-DR+ monocytes on primary human HSCs (pHSCs) or the HSC cell-line LX2. Co-culture with HSCs resulted in marked downregulation of HLA-DR expression
To further characterize HSC-induced CD14+HLA-DR/low monocytes, we analyzed their surface marker expression profile and compared it to monocytes cultured with other cell lines or freshly isolated CD14+HLA-DR+ monocytes. CD14+HLA-DR/low MDSCs isolated from healthy individuals served as control. There were no differences with respect to CD15, CD40, and CD80 expression levels on freshly isolated monocytes and HSC-co-cultured monocytes, also ex vivo MDSCs did not express these molecules (Fig. 2). While co-culture with hepatocyte cell lines led to upregulation of co-stimulatory molecules like CD40 and CD80 on peripheral blood monocytes, such regulation was not observed when monocytes were co-cultured with HSCs (Fig. 2). Upon contact with HSCs, however, monocytes showed increased levels of IL-4Ra (Fig. 2), a phenotype also observed in MDSCs isolated from patients suffering from melanoma and colorectal carcinoma [14]. Interestingly, monocytes cultured on HSCs downregulated CD11b (Fig. 2). Thus, phenotypic characterization of surface molecules did not allow us to identify a profile typical for MDSCs in monocytes co-cultured with HSCs. This led us to perform quantitative PCR in order to determine the expression levels of genes specifically associated with MDSCs [15]. S100A12, that is overexpressed in MDSCs from hepatocellular and colorectal carcinoma patients [16], was selectively expressed in CD14+HLA-DR/low monocytes cultured on LX2 and CD14+HLA-DR/low MDSCs ex vivo (Supplementary Fig. 2). Also arginase-I, which is involved in the MDSC-mediated suppression of T-cell function, [5] was expressed selectively in HSC-induced CD14+HLA-DR/low monocytes and ex vivo CD14+HLA-DR/low MDSCs. In contrast, the expression of iNOS,
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Fig. 1. Hepatic stellate cells induce an MDSC-like phenotype in peripheral blood monocytes. (A–C) Monocytes were cultured alone, on primary human HSCs, the HSC cell-lines LX2, HepG2, HuH7, SK-Hep-1 or CCL253 for 3 days, and the expression of HLA-DR was measured by flow cytometry. Representative contour plots (A), % CD14+HLA-DR+ expression from 5 independent experiments (B) or absolute number (C) are shown. (D) Isolated monocytes, B-cells or myeloid dendritic cells were added to a confluent layer of LX2 in a 24-well plate. The expression of HLA-DR was analyzed on CD14+, CD19+ or CD1c+ cells by flow cytometry. Cumulative data from 3 independent experiments are shown. (E) Monocytes were cultured alone, on LX2, with LX2 conditioned media or with LX2 cells separated by a transwell-insert for 3 days. The frequency of CD14+HLA-DR/low cells is plotted cumulative for 3 independent experiments. ⁄p 60.05, ⁄⁄p 60.01, ⁄⁄⁄p 60.001.
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Fig. 2. Monocytes cultured on hepatic stellate cells upregulate IL-4Ra. CD14+ cells were cultured as described before, alone, co-cultured with different cell lines for 3 days or analyzed directly ex vivo. The expression of surface markers was analyzed by flow cytometry gating on CD14+, or CD14+HLA-DR+ or CD14+HLA-DR/low cells. The corresponding MFI is shown. Representative histograms from 5 experiments are shown.
which has also been described to mediate T-cell suppression [17], was not restricted to HSC-cultured monocytes or ex vivo MDSCs 530
(Supplementary Fig. 2). Taken together, CD14+ monocytes cultured on HSCs downregulate HLA-DR, express IL-4Ra and
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JOURNAL OF HEPATOLOGY S100A12, and upregulate Arg-I mRNA, which indicates that these cells also may exert suppressive function.
MDSCs mediated suppression of T cells (Fig. 3D). Moreover, neither the addition of blocking antibodies against IL-10 or IL6R, nor co-culture with an IDO inhibitor (1-MT) or the ROS inhibitors (YCG063 or MnTBAP) prevented suppression of T-cell proliferation (Supplementary Fig. 3). Together, both phenotypically and functionally HSC-induced CD14+HLA-DR/low monocytes represent a myeloid derived suppressor cell population that exert the suppressive function through arginase-I.
HSC-induced CD14+HLA-DR/low monocytes suppress T-cell proliferation in an arginase-I-dependent manner We next investigated whether HSC-induced CD14+HLA-DR/low monocytes could suppress T-cell proliferation. To this end, human CD8+ T cells were stimulated using anti-CD2/CD3/CD28coated beads, and CD14+HLA-DR/low monocytes previously cultured with LX2 were added. Addition of increasing numbers of HSC-induced CD14+HLA-DR/low monocytes to a fixed number of CD8 T cells progressively reduced T-cell proliferation (Fig. 3A). This effect was only observed with CD14+ cells cultured on LX2, pHSCs and freshly isolated MDSCs (Fig. 3B), but not with those monocytes cultured together with LSECs (SK-Hep1) or the hepatocyte (HepG2, HuH-7) cell lines. The same inhibitory effect on proliferation was also observed for CD4 T cells (data not shown). In addition to T-cell proliferation, IFN-c production was also inhibited by LX2-induced CD14+HLA-DR/low monocytes and correlated inversely with the addition of increasing numbers of LX2-induced CD14+HLA-DR/low monocytes into the culture (Fig. 3C). MDSCs can mediate the suppressive function via several mechanisms [18]. By adding specific inhibitors or blocking antibodies into the co-culture of T cells with HSC-induced CD14+HLA-DR/low monocytes, we found that only L-NOHA, a specific arginase-I inhibitor, prevented the suppression of T-cell proliferation. Neither the pan-inhibitor of NO-synthase (LNMMA) nor blocking antibodies against TGF-b was able to block
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The induction of CD14+HLA-DR/low monocyte-derived MDSCs by HSCs depends on the expression of CD44 CD44 expression has been shown to be increased on hepatic stellate cells from injured rat livers and is associated with their increased motility [19]. Moreover, in toxic liver injury, CD44 was shown to prevent exacerbation of inflammation via control of macrophage function infiltrating the liver [20]. In humans, hepatic CD44 expression is increased in patients with liver disease [21] and is involved in progression of various cancer types including hepatocellular carcinoma [22,23], in which MDSCs are increased and involved in immune-suppression [5,16]. To test if CD44 is involved in the induction of MDSCs, we added increasing concentrations of anti-CD44 blocking antibodies into the co-culture of LX2 and monocytes. Because we have previously shown, that CD54 on HSCs is important for the veto-function towards CD8 T cells [12], we also included blocking antibodies for CD54. In contrast to HSC-mediated CD8 T-cell inhibition, CD54 was not involved in induction of HLA-DR/low monocytes by HSCs, but blocking CD44 reduced the induction of MDSCs from monocytes dose dependently (Fig. 4A). Moreover, blocking CD44 inter-
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Fig. 3. Hepatic stellate cell-modified monocytes suppress T cells in an arginase-I-dependent mechanism. Monocytes were cultured for 3 days as indicated. Isolated CSFE labeled CD8 T cells were stimulated using CD2/CD3/CD28-coated beads and CD14+ cells were added at different ratios. Proliferation was analyzed (A, B, and D) after four days. The percentage proliferation compared to maximal proliferation (T cells cultured alone) is depicted (B). IFN-c production was quantified by ELISA (ratio T cells to CD14+HLA-DR/low cells 1:0.1/0.3/1/3) (C). Blocking antibodies or inhibitors were added during the co-culture of monocytes with T cells as indicated (D). Representative histograms (A) or cumulative results from 6 (B and C) or 3 (D) independent experiments are shown. ⁄p 60.05, ⁄⁄p 60.01, ⁄⁄⁄p 60.001, n.s., not significant.
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Fig. 4. Induction of CD14 HLA-DR cells by hepatic stellate cells is mediated by CD44. CD14 cells were cultured on LX2 as described before. Blocking antibody against CD54, CD44 or appropriate isotype controls were added as indicated (A, B, F, and G). The frequency of CD14+HLA-DR/low cells is plotted from 4 independent experiments (A) or 2 experiments pHSCs (F and G). CD14+ cells were isolated after culture with LX2 in the absence or presence of blocking CD44 antibody and were cocultured with CD8 T cells. Proliferation of T cells was measured on day 4. Cumulative results of 4 independent experiments are shown (B). CD44 was silenced in LX2 using siRNA as described. As control, scrambled siRNA or untreated cells were used as controls. After 24 h, expression of CD44 on LX2 cells was analyzed by flow cytometry. Histograms represent mean fluorescence intensity of CD44 (C). Monocytes were co-cultured with different siRNA-transfected LX2 cells and the expression of HLA-DR was analyzed by flow cytometry. Dot plots show percentages of HLA-DR/low cells of 3 independent experiments (D). Primary isolated human hepatic stellate cells were isolated and cultured for 6 days. Cells were analyzed every day for the mean fluorescence intensity of CD44 and GeoMean of autofluorescence by flow cytometry. Representative results from 2 independent experiments are shown (E). ⁄p 60.05, ⁄⁄p 60.01, ⁄⁄⁄p 60.001.
actions between monocytes and HSCs prevented acquisition of the suppressive capacity by those monocytes (Fig. 4B). To investigate whether HSC-expressed CD44 was important for induction of MDSCs, we efficiently knocked down the expression of CD44 on LX2 cells using two different siRNAs (Fig. 4C). In absence of CD44 expression, LX2 cells were not able to downregulate HLADR on monocytes anymore (Fig. 4D), indicating that HSC-dependent MDSCs induction is CD44-mediated. As we found primary human HSCs to become more efficient in inducing MDSCs from monocytes after activation by culture on plastic, we investigated expression kinetics of CD44 on primary HSCs. As HSCs become activated, they lose vitamin A containing lipid droplets and thereby autofluorescence (Fig. 4E). Primary HSCs did not express CD44, but markedly upregulated CD44 during activation in vitro (Fig. 4E), achieving levels comparable to LX2. More importantly, the induction of HLA-DR/low monocytes by primary HSCs 532
depended on CD44 expression, as it was abrogated in the presence of anti-CD44 blocking antibodies (Fig. 4F and G). Together, these data suggest that activated hepatic stellate cells can efficiently induce MDSCs in a CD44-dependent manner from mature monocytes.
Discussion The liver has an extraordinary immunological function determined by the unique micro-environment and its organ-resident cells that function as antigen-presenting cells to regulate antigen-specific immune responses [24]. Antigen-specific immunity can eradicate infections with hepatotropic pathogens from the liver [25]. Nevertheless, persistence of pathogens in hepatocytes or development of hepatocellular carcinoma in the context of
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JOURNAL OF HEPATOLOGY chronic inflammation demonstrates that the tolerogenic function of hepatic cell populations may impede efficient immune surveillance under certain circumstances. Here, we report that activated HSCs, which arise as a consequence of hepatic inflammation, induce myeloid derived suppressor cells (MDSCs) in a cell-contact and CD44-dependent manner from peripheral blood monocytes. HSCs can exert regulatory immune function via inhibition of DC-mediated CD8 T-cell activation [12,26], induction of T regulatory cells [11,26] or PDL1-mediated induced apoptosis of T cells [27]. Furthermore, adoptive transfer of murine HSCs leads to the induction of MDSCs [9], which are prominent regulatory cells known to locally regulate adaptive immunity [28]. Data from in vitro studies showed that MDSCs can be generated from bone-marrow cells in the presence of various cytokine cocktails [29]. Therefore, it is thought that MDSCs in vivo originate from precursors in the bone-marrow and represent a population of immature myeloid cells that are recruited to sites of inflammation. Our data provide evidence that CD14+HLA-DR/low MDSCs can also be generated from circulating CD14+ monocytes in a peripheral organ such as the liver. Interestingly, MDSCs induced by HSCs are similar to those found in patients suffering from hepatocellular carcinoma [5] or HCV infection [30], indicating that also in humans HSCs may induce MDSCs in vivo. Thus, the local generation of MDSCs by HSCs may provide a novel mechanism as to how systemic adaptive immunity is locally downregulated in the liver. We discovered that activated, but not freshly isolated HSCs, had the capacity to generate CD14+HLA-DR/low monocytederived MDSCs. During chronic inflammatory conditions, HSCs are activated in vivo and trans-differentiate into pro-fibrogenic myofibroblasts [31]. We, therefore, investigated the role of molecules known to be upregulated upon activation of HSCs. A number of soluble factors, like GM-CSF, M-CSF, and VEGF, have been implicated to play a role in the induction of MDSCs [32]. Recently, murine HSC were found to mediate MDSC induction via the complement factor C3 upon adoptive transfer [33]. However, we found that soluble factors do not play a role in the induction of CD14+HLA-DR/low MDSCs from monocytes by HSCs. This could be due to species differences or the origin of the MDSC, as murine HSC were used to generate MDSC from bone-marrow precursors and not mature monocytes [29]. Subsequently, we focused on cell surface molecules expressed by HSCs. CD54 expression on HSCs is critically important for direct inhibitory effects on naïve T-cell activation and proliferation [12]. In the generation of CD14+HLA-DR/low monocyte-derived MDSCs by HSCs, CD54 was not involved. HSC activation also leads to increased expression levels of CD44 [34,35]. We found evidence for the involvement of CD44 in generation of CD14+HLA-DR/low monocyte-derived MDSCs by using blocking antibodies to CD44 or siRNA-mediated CD44 knockdown in HSCs. It remains unclear how CD44 induced generation of CD14+HLA-DR/low monocyte-derived MDSCs from circulating monocytes because CD44 is not selectively expressed by activated HSCs. CD44 is involved in cell recruitment to tissues such as liver or bone marrow [36,37] and locomotion of T cells within tissues or tumors [38]. CD44 has several interaction partners including extracellular matrix proteins such as hyaluronic acid, mediators such as osteopontin, and the VLA-4 molecule to establish cell-cell interaction [39]. Although VLA-4 has mainly been found to mediate cell adhesion, a signaling func-
tion for this molecule has been reported for T cells [40], indicating that potentially VLA-4 could be involved in signaling processes in CD14+ monocytes leading to MDSC differentiation. Moreover, specificity of CD44 in the induction of MDSCs could also be mediated via its differential splicing that can lead to altered functionality [41]. Indeed, rat hepatic stellate cells express a distinct set of CD44 splice variants necessary for migration during liver injury [19]. Thus, it is possible that a particular splice variant of CD44 upregulated on HSCs is involved in induction of MDSCs. Our findings raise the question whether monocytes or monocyte-derived cells present in the liver all function as MDSCs. In the hepatic sinusoid, fenestrated LSECs without a basement membrane, do not provide a physical barrier and allow direct physical contact between HSCs in the space of Dissé and Kupffer cells or circulating monocytes [42]. However, only activated HSCs have the capacity to generate monocyte-derived MDSCs. Therefore, it is unlikely that, under steady state non-inflammatory conditions, MDSCs are generated from monocytes or Kupffer cells. Consistently, Kupffer cells isolated from the normal liver function as antigen-presenting cells, but not as suppressive MDSCs [43]. However, under inflammatory conditions, Kupffer cells may also be modulated, as we found LX2 cells to be able to downregulate HLA-DR on LPS-stimulated Kupffer cells (data not shown). The induction of MDSCs by activated HSCs in the chronically inflamed liver may thus represent a negative feedback loop in the attempt to dampen or prevent immunopathology in the liver. As the activation of HSCs is accompanied by increased expression of chemokines and adhesion molecules such as CD54 and CD44 that can lead to attraction of circulating monocytes [35], chronic inflammation and the consequent HSC activation may thus trigger both increased recruitment of monocytes and differentiation into MDSCs. It is unclear whether activated HSCs also bear the capacity to modulate Kupffer-cell function and cause their differentiation into MDSCs. The induction of CD14+HLA-DR/low monocyte-derived MDSCs by HSCs did not result from proliferation of precursor cells, but represents a differentiation process that was so far unknown to be involved in MDSC generation [44]. It is important to note that HSC-induced CD14+HLA-DR/low monocyte-derived MDSCs did not maintain their surface and functional phenotype for more than 2 days after losing HSC contact (data not shown), indicating that the increase in numbers of MDSCs in the liver will only be transient and strictly linked to persistent hepatic inflammation. It also suggests that MDSCs generated from monocytes require continuous contact with activated HSCs to maintain their function as suppressor cells. The dynamic induction of MDSCs from monocytes by activated mesenchymal cells, such as alphasmooth muscle antigen (aSMA) positive hepatic HSCs, indicates that aSMA+ tumor stroma cells, whose appearance in tumor tissue is often directly correlated with patient survival [45], may also locally induce MDSCs thereby creating a regulatory tumor micro-environment that impairs immune clearance of cancer cells. It also provides an interesting link to the observation that tumors mostly arise in organs during chronic inflammatory conditions [46]. Taken together, these results demonstrate that activated human HSCs can induce differentiation of monocytes into MDSCs, which provides evidence for a so far unrecognized mechanism in the generation of MDSCs via a re-differentiation step of already differentiated monocytes. The HSC-mediated
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Research Article induction of CD14+HLA-DR/low monocyte-derived MDSCs also attributes further immune competence to peripheral organs like the liver because attenuation of local inflammation may not only occur as a consequence of increased chemokine-mediated recruitment of circulating MDSCs, but also rely on a local and dynamic differentiation process initiated by organ-resident HSCs. Financial support This work was supported by a BONFOR grant of the University of Bonn medical faculty to BH.
Conflict of interest The authors who have taken part in this study do not have a relationship with the manufacturers of the drugs involved either in the past or present and did not receive funding from the manufacturers to carry out their research. BH was supported by a grant from the University of Bonn Medical Faculty (BONFOR). LD was supported by a grant from the german research council (SFB704).
Authors’ contributions LD and BH were responsible for the study concept, interpretation of data and drafting of the manuscript. BH, FAS, LCH, YAG, and PS performed experiments and BH and FAS analyzed the data. MB, HG, DG, LCH, FG, AT, JCK and IM provided the material. LD, CK, and PK provided important intellectual content as well as critical revision of the manuscript.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.04. 033.
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