: As the predominant lymphocyte subset in the liver, natural killer (NK) cells have been shown to be highly associated with the outcomes of patients with chronic hepatitis B virus infection (CHB) and hepatocellular carcinoma (HCC). Previously, we reported that NKG2A, a checkpoint candidate, mediates human and murine NK cell dysfunction in CHB. However, NK cell exhaustion and, particularly, the level of NKG2A expression within liver tumors have not been reported. : In this study, we analyzed NKG2A expression and the related dysfunction of NK cells located in intra- or peritumor regions of liver tissue samples from 207 HCC patients, in addition to analyzing disease outcomes.: The expression of NKG2A in NK cells and the NKG2A ligand, HLA-E, in intratumor HCC tissues was observed to be increased. These NK cells, and particularly CD56 NK cells, with higher NKG2A expression showed features of functional exhaustion and were associated with a poor prognosis. The increase in NKG2A expression might be induced by IL-10, which was present at a high level in the plasma of HCC patients. Blocking IL-10 could specifically inhibit NKG2A expression in NK cells. : These findings indicate that NKG2A expression is influenced by factors from cancer nests and contributes to NK cell exhaustion, suggesting that NKG2A blockade has the potential to restore immunity against liver tumors by reversing NK cell exhaustion.

The local immune mechanisms responsible for either self-healing or sustained chronic infection are not clear, in the development of E. multilocularis larvae. Here, we developed a suitable experimental model that mimics naturally infected livers, according to the parasite load. We demonstrated that local cellular immunity and fibrogenesis are actually protective and fully able to limit metacestode growth in the liver of low or medium dose-infected mice (LDG or MDG), or even to clear it, while impairment of cellular immunity is followed by a more rapid and severe course of the disease in high dose-infected mice (HDG). And recruitment and/ or proliferation of memory T cells (including CD4 Tem, CD8 Tcm and CD8 Tem) and imbalance of T1/T2/T17/Treg-type T cells in liver were not only associated with clearance of the parasite infection in LDG, but also with increased hepatic injury in HDG; in particular the dual role of CD8 T cells depending on the parasite load and the various stages of metacestode growth. Besides, we first demonstrate the association between LAG3- or 2B4-expressing T cells exhaustion and HD inocula in late stages. Our quantitative experimental model appears fully appropriate to study immunomodulation as a therapeutic strategy for patients with Alveolar Echinococcosis.

Background and Aims The cestode Echinococcus multilocularis infection, a serious health problem worldwide, causes alveolar echinococcosis (AE), a tumor‐like disease predominantly located in the liver and able to spread to any organs. Until now, there have been few studies that explore how T‐cell exhaustion contributes to the parasite’s escape from immune attack and how it might be reversed. Approach and Results In this study, we found that liver T‐cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine‐based inhibitory motif domain (TIGIT) expression was significantly enhanced and positively correlated with lesion activity in AE patients. High TIGIT expression in both liver‐infiltrating and blood T cells was associated with their functional exhaustion, and its ligand CD155 was highly expressed by hepatocytes surrounding the infiltrating lymphocytes. In co‐culture experiments using human blood T cells and hepatic cell line HL‐7702, CD155 induced functional impairment of TIGIT+ T cells, and in vitro blockade with TIGIT antibody restored the function of AE patients’ T cells. Similar TIGIT‐related functional exhaustion of hepatic T cells and an abundant CD155 expression on hepatocytes were observed in E. multilocularis–infected mice. Importantly, in vivo blocking TIGIT prevented T‐cell exhaustion and inhibited disease progression in E. multilocularis–infected mice. Mechanistically, CD4+ T cells were totally and CD8+ T cells partially required for anti‐TIGIT–induced regression of parasite growth in mice. Conclusions This study demonstrates that E. multilocularis can induce T‐cell exhaustion through inhibitory receptor TIGIT, and that blocking this checkpoint may reverse the functional impairment of T cells and represent a possible approach to immunotherapy against AE.

Alveolar echinococcosis (AE) is characterized by the development of irreversible fibrosis and of immune tolerance towards Echinococcus multilocularis (E. multilocularis). Very little is known on the presence of transforming growth factor-β (TGF-β) and other components of TGF-β/Smad pathway in the liver, and on their possible influence on fibrosis, over the various stages of infection. Using Western Blot, qRT-PCR and immunohistochemistry, we measured the levels of TGF-β1, TGF-β receptors, and down-stream Smads activation, as well as fibrosis marker expression in both a murine AE model from day 2 to 360 post-infection (p.i.) and in AE patients. TGF-β1, its receptors, and down-stream Smads were markedly expressed in the periparasitic infiltrate and also in the hepatocytes, close to and distant from AE lesions. Fibrosis was significant at 180 days p.i. in the periparasitic infiltrate and was also present in the liver parenchyma, even distant from the lesions. Over the time course after infection TGF-β1 expression was correlated with CD4/CD8 T-cell ratio long described as a hallmark of AE severity. The time course of the various actors of the TGF-β/Smad system in the in vivo mouse model as well as down-regulation of Smad7 in liver areas close to the lesions in human cases highly suggest that TGF-β plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.

BackgroundAlveolar echinococcosis (AE) is characterized by the tumor-like growth of Echinococcus (E.) multilocularis. Very little is known on the influence of helminth parasites which develop in the liver on the proliferation/growth arrest metabolic pathways in the hepatocytes of the infected liver over the various stages of infection.Methodology/Principal FindingsUsing Western blot analysis, qPCR and immunohistochemistry, we measured the levels of MAPKs activation, Cyclins, PCNA, Gadd45β, Gadd45γ, p53 and p21 expression in the murine AE model, from day 2 to 360 post-infection. Within the early (day 2–60) and middle (day60–180) stages, CyclinB1 and CyclinD1 gene expression increased up to day30 and then returned to control level after day60; Gadd45β, CyclinA and PCNA increased all over the period; ERK1/2 was permanently activated. Meanwhile, p53, p21 and Gadd45γ gene expression, and caspase 3 activation, gradually increased in a time-dependent manner. In the late stage (day180–360), p53, p21 and Gadd45γ gene expression were significantly higher in infected mice; JNK and caspase 3 were activated. TUNEL analysis showed apoptosis of hepatocytes. No significant change in CyclinE, p53 mRNA and p-p38 expression were observed at any time.ConclusionsOur data support the concept of a sequential activation of metabolic pathways which 1) would first favor parasitic, liver and immune cell proliferation and survival, and thus promote metacestode fertility and tolerance by the host, and 2) would then favor liver damage/apoptosis, impairment in protein synthesis and xenobiotic metabolism as well as promote immune deficiency, and thus contribute to the dissemination of the protoscoleces after metacestode fertility has been acquired. These findings give a rational explanation to the clinical observations of hepatomegaly and of unexpected survival of AE patients after major hepatic resections, and of chronic liver injury, necrosis and of hepatic failure at an advanced stage and in experimental animals.

BackgroundAdhesion molecules distributed on the cell-surface depends upon their dynamic trafficking that plays an important role during cancer progression. ADP-ribosylation factor 6 (Arf6) is a master regulator of membrane trafficking. CD147, a tumor-related adhesive protein, can promote the invasion of liver cancer. However, the role of Arf6 in CD147 trafficking and its contribution to liver cancer progression remain unclear.MethodsStable liver cancer cell lines with Arf6 silencing and over-expression were established. Confocal imaging, flow cytometry, biotinylation and endomembrane isolation were used to detect CD147 uptake and recycling. GST-pull down, gelatin zymography, immunofluorescence, cell adhesion, aggregation and tight junction formation, Transwell migration, and invasion assays were used to examine the cellular phenotypes. GEPIA bioinformatics, patient’s specimens and electronic records collection, and immunohistochemistry were performed to obtain the clinical relevance for Arf6-CD147 signaling.ResultsWe found that the endocytic recycling of CD147 in liver cancer cells was controlled by Arf6 through concurrent Rab5 and Rab22 activation. Disruption of Arf6-mediated CD147 trafficking reduced the cell-matrix and cell-cell adhesion, weakened cell aggregation and junction stability, attenuated MMPs secretion and cytoskeleton reorganization, impaired HGF-stimulated Rac1 activation, and markedly decreased the migration and invasion of liver cancer cells. Moreover, high-expression of the Arf6-CD147 signaling components in HCC (hepatocellular carcinoma) was closely correlated with poor clinical outcome of patients.ConclusionsOur results revealed that Arf6-mediated CD147 endocytic recycling is required for the malignant phenotypes of liver cancer. The Arf6-driven signaling machinery provides excellent biomarkers or therapeutic targets for the prevention of liver cancer.

BackgroundAlveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.Methodology/Principal FindingsGene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.Conclusions E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.

BackgroundEchinococcus granulosus infection causes cystic echinococcosis (CE); the generation of liver fibrosis around the parasitic larval cyst (metacestode) may play a major role in the spontaneous limitation of the parasitic growth; however, fibrogenesis has received little attention in CE. It has been reported that miR-19b plays a role in various diseases, including infectious diseases, by regulating fibrogenesis. However, its function in the development of liver fibrosis in E. granulosus infection is unknown.MethodsThe expression of miR-19b and genes that are involved in liver fibrosis were analysed in E. granulosus-infected human livers using qRT-PCR. The role of miR-19b on hepatic stellate cells (LX-2 cells in vitro) treated with hydatid cyst fluid (HCF) was then analysed by 3-(4, 5-dimet-hylthiazol-2-yl)-2, 4-diphenyl-tetrazolium bromide (MTT) assay, qRT-PCR, Western blot and flow cytometry.ResultsThe results showed that the expression of miR-19 was significantly reduced in the pericystic collagen-rich liver tissue of CE patients, compared to normal liver. Incubation of LX-2 cells (in vitro) with HCF induced a decreased proliferation of these cells and a reduced expression of miR-19, inversely correlated with the expression of collagen 1A1 and TGF-β receptor II (TβRII). Conversely, overexpression of miR-19 by LX-2 cells inhibited the proliferation of these cells and led to decreased TβRII expression.ConclusionsOur study provides new evidence for the intervention of miRNAs in the regulation of fibrosis in infectious diseases; it suggests that E. granulosus can inhibit miR-19 liver expression and promote fibrosis through the increase in TβRII, the activation of hepatic stellate cells and extracellular matrix production.