Identifizierung und Charakterisierung von myeloiden Suppressorzellen bei Patienten mit hepatozellulärem Karzinom

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Differential Induction of Ly6G and Ly6C Positive Myeloid Derived Suppressor Cells in Chronic Kidney and Liver Inflammation and Fibrosis

<div><p>CD11b⁺Gr1⁺ myeloid derived suppressor cells (MDSC) are known to be very potent suppressors of T cell immunity and can be further stratified into granulocytic MDSC and monocytic MDSC in mice based on expression of Ly6G or Ly6C, respectively. Here, using these markers and functional assays, we aimed to identify whether MDSC are induced during chronic inflammation leading to fibrosis in both kidney and liver and whether additional markers could more specifically identify these MDSC subsets. In an adenine-induced model of kidney inflammation/fibrosis suppressive Ly6G^pos MDSC were induced. The suppressive function within the Ly6G⁺ MDSC population was exclusively present in IFNγRβ expressing cells. In contrast, in chronic inflammation in the liver induced by bile duct ligation, suppressive capacity was exclusively present in the Ly6C^pos MDSC subset. Gene expression analyses confirmed the differential origins and regulation of those MDSC subsets. Additionally, depletion of MDSC in either kidney or liver fibrosis enhanced fibrosis markers, indicating a protective role for MDSC in organ fibrosis. Thus, our data demonstrate that during liver inflammation and kidney fibrosis MDSC with similar function arise bearing a distinct marker profile and arising from different cell populations.</p></div

Pancreatic Premalignant Lesions Secrete Tissue Inhibitor of Metalloproteinases-1, Which Activates Hepatic Stellate Cells Via CD63 Signaling to Create a Premetastatic Niche in the Liver

Background &amp; Aims Pancreatic ductal adenocarcinoma (PDAC) metastasizes to liver at early stages, making this disease highly lethal. Tissue inhibitor of metalloproteinases-1 (TIMP1) creates a metastasis-susceptible environment in the liver. We investigated the role of TIMP1 and its receptor CD63 in metastasis of early-stage pancreatic tumors using mice and human cell lines and tissue samples. Methods We obtained liver and plasma samples from patients in Germany with chronic pancreatitis, pancreatic intra-epithelial neoplasia, or PDAC, as well as hepatic stellate cells (HSCs). We performed studies with Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP (CPK) mice, Pdx-1+/Cre;Kras+/LSL-G12D;Trp53+/LSL-R172H (KPC) mice, and their respective healthy littermates as control, and Cd63−/− mice with their wild-type littermates. KPC mice were bred with Timp1−/− mice to produce KPCxTimp1−/− mice. TIMP1 was overexpressed and CD63 was knocked down in mice using adenoviral vectors AdTIMP1 or AdshCD63, respectively. Hepatic susceptibility to metastases was determined after intravenous inoculation of syngeneic 9801L pancreas carcinoma cells. Pancreata and liver tissues were collected and analyzed by histology, immunohistochemical, immunoblot, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction analyses. We analyzed the effects of TIMP1 overexpression or knockdown and CD63 knockdown in transduced human primary HSCs and HSC cell lines. Results Chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients expressed higher levels of TIMP1 protein than normal pancreas. The premalignant pancreatic lesions that developed in KPC and CPK mice expressed TIMP1 and secreted it into the circulation. In vitro and in vivo, TIMP1 activated human or mouse HSCs, which required interaction between TIMP1 and CD63 and signaling via phosphatidylinositol 3-kinase, but not TIMP1 protease inhibitor activity. This signaling pathway induced expression of endogenous TIMP1. TIMP1 knockdown in HSCs reduced their activation. Cultured TIMP1-activated human and mouse HSCs began to express stromal-derived factor-1, which induced neutrophil migration, a marker of the premetastatic niche. Mice with pancreatic intra-epithelial neoplasia–derived systemic increases in TIMP1 developed more liver metastases after injections of pancreatic cancer cells than mice without increased levels of TIMP1. This increase in formation of liver metastases from injected pancreatic cancer cells was not observed in TIMP1 or CD63 knockout mice. Conclusions Expression of TIMP1 is increased in chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients. TIMP1 signaling via CD63 leads to activation of HSCs, which create an environment in the liver that increases its susceptibility to pancreatic tumor cells. Strategies to block TIMP1 signaling via CD63 might be developed to prevent PDAC metastasis to the liver.</p

Differential distribution of monocytic and granulocytic myeloid derived suppressor cells within Gr-1 positive cells in liver and kidney inflammation and fibrosis.

<p>C57BL/6 mice underwent bile-duct ligation, were fed an adenine-enriched diet, or were injected i.v. with LPS/IFNγ. Furthermore, BM-MDSC were generated <i>in vitro</i> by culture of bone marrow cells with CSF2 (GM-CSF) (A, B). After the indicated times liver, kidney, spleen or bone marrow cells were isolated and analysed by flow cytometry. Histograms depict viable (Hoechst negative), non-parenchymal cells stained with CD11b and Gr-1 (A) or viable, CD11b^pos cells stained for Ly6G and Ly6C (B). Representative (A, B) and cumulative (C) data of 4 (liver, spleen) or 3 (kidney, bone marrow) independent experiments are shown (n>9). Absolute numbers of CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ meyloid cells in the liver (D) and kidney (E) at the indicated time-points after BDL or adenine-feeding, respectively. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001.</p

Fibrosis markers in the liver and kidney after all-trans-retinoic acid treatment.

<p>C57BL/6 mice underwent bile-duct ligation, were fed an adenine-enriched diet or as a control were left untreated. After 7 days mice were treated with 1g/L all-trans-retinoic acid (ATRA) in their drinking water (BDL: n = 7, Adenine: n = 4) or not (BDL: n = 8, adenine: n = 4)) for the remaining time until analysis. At day 14 (BDL and adenine feeding), total liver and kidney RNA was isolated for real-time PCR of fibrosis markers. Shown are mRNA expression levels for α-SMA, collagen IV, TGF-β and vimentin relative to the levels in non-treated mice (n = 3), which was set to 1. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001.</p

Surface marker expression on suppressive and non-suppressive myeloid subsets.

<p>C57BL/6 mice were treated and cells were isolated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119662#pone.0119662.g001" target="_blank">Fig. 1</a>. Myeloid cells from non-treated mice served as controls (steady state). Flow cytometric analysis of surface markers associated with MDSC induction/function on CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ myeloid cells are depicted in the histograms. Specific staining: black lines. Isotype controls: filled grey. Representative data of 3 independent experiments is shown.</p

Suppressive capacity of monocytic and granulocytic MDSC subsets.

<p>C57BL/6 mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119662#pone.0119662.g001" target="_blank">Fig. 1</a>. Gating strategy for sorting (A). At the indicated times myeloid subsets from liver (B), kidney (C), spleen (D) or in vitro bone marrow culture (E) were isolated and CD11b⁺ cells were sorted on the basis of their Ly6C or Ly6G expression and additional expression of IFNγRβ (A), yielding 4 separate subsets of CD11b⁺ myeloid cells. Naïve CFSE-labelled CD8 T cells were stimulated using αCD3/αCD28 coated beads and the different subsets of sorted myeloid cells were added at a 3:1 ratio (B-E). After 72h T cell proliferation was analysed by flow cytometry and the percentage of proliferated T cells is depicted (B-E). Cumulative data from 2 independent experiments are shown. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001. ND = not detectable.</p

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8+ T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1+ memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire

Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions

<div><p>Background</p><p>Activation of the immune system in terms of subseptic conditions during liver regeneration is of paramount clinical importance. However, little is known about molecular mechanisms and their mediators that control hepatocyte proliferation. We sought to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy (PH), and to uncover the impact of the integrin lymphocyte function-associated antigen-1 (LFA-1) on liver regeneration in a subseptic setting.</p><p>Methods</p><p>Wild-type (WT) and LFA-1^-/- mice underwent a 2/3 PH and low-dose lipopolysaccharid (LPS) application. Hepatocyte proliferation, immune cell infiltration, and cytokine profile in the liver parenchyma were determined.</p><p>Results</p><p>Low-dose LPS application after PH results in a significant delay of liver regeneration between 48h and 72h, which is associated with a reduced number of CD3⁺ cells within the regenerating liver. In absence of LFA-1, an impaired regenerative capacity was observed under low-dose LPS application. Analysis of different leukocyte subpopulations showed less CD3⁺NK1.1⁺ NKT cells in the liver parenchyma of LFA-1^-/- mice after PH and LPS application compared to WT controls, while CD3^-NK1.1⁺ NK cells markedly increased. Concordantly with this observation, lower levels of NKT cell related cytokines IL-12 and IL-23 were expressed in the regenerating liver of LFA-1^-/- mice, while the expression of NK cell-associated CCL5 and IL-10 was increased compared to WT mice.</p><p>Conclusion</p><p>A subseptic situation negatively alters hepatocyte proliferation. Within this scenario, we suggest an important impact of NKT cells and postulate a critical function for LFA-1 during processes of liver regeneration.</p></div

Characterization of immunocompetent cells in the regenerating liver.

<p>Number of Gr1⁺ (A) or CD3⁺ (C) immune cells per mm² liver parenchyma were quantified in the regenerating liver at distinct time points after PH, PH plus LPS application or sham-procedure plus LPS application in WT mice. The 0h timepoint represents liver tissue of WT mice (n = 2) without any treatment and before the operation (preoperative situation). Representative images for Gr1⁺ (B, green) or CD3⁺ (D, red) cells within the liver parenchyma 6h after sham procedure plus LPS, PH plus LPS or PH only in WT mice are shown. At least 5 sections per time point and treatment were analyzed. Each value represents the mean ± SD from 3 independent mice per time point. Statistical significances were analyzed by two-way ANOVA-test and refer to the comparison between PH and PH and LPS application. Scale bars indicate 50μm. Gr1⁺ cells in green, CD3⁺ cells in red, nuclei in blue (DAPI).</p