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The spatial organization of immune cells within the tumor microenvironment (TME) largely determines the anti-tumor immunity and also highly predicts tumor progression and therapeutic response. Tim-3 is a well-accepted immune checkpoint and plays multifaceted immunoregulatory roles via interaction with distinct Tim-3 ligands (Tim-3L), showing great potential as an immunotherapy target. However, the cell sociology mediated by Tim-3/Tim-3L and their contribution to tumor development remains elusive. Here, we analyzed the spatial distribution of Tim-3/Tim-3L in TME using multiplex fluorescence staining and revealed that despite the increased Tim-3 expression in various tumor-infiltrated lymphocytes, Tim-3(+)CD4(+) cells were more accumulated in parenchymal/tumor region compared with stromal region and exhibited more close association with patient survival. Strikingly, CD4 T cells surrounding Tim-3L(+) cells expressed higher Tim-3 than other cells in cancerous tissues. In vivo studies confirmed that depletion of CD4 T cells completely abrogated the inhibition of tumor growth and metastasis, as well as the functional improvement of CD8 T and NK, mediated by Tim-3 blockade, which was further validated in peripheral lymphocytes from patients with hepatocellular carcinoma. In conclusion, our findings unravel the importance of CD4 T cells in Tim-3/Tim-3L-mediated immunosuppression and provide new thoughts for Tim-3 targeted cancer immunotherapy.

The strong regenerative ability of the liver safeguards the crucial hepatic functions. The balance between hepatocyte proliferation and death is critical for restoring liver size and physiology. Tumour necrosis factor (TNF) alpha-induced protein 8-like 1 (TIPE1) is highly expressed in liver and has been identified as a candidate regulator for cell proliferation and death, being involved in a variety of biological processes and diseases. However, the role of TIPE1 in liver regeneration remains unexplored. In the present study, we found that TIPE1 expression was elevated in the regenerating liver induced by either partial hepatectomy or 10% carbon tetrachloride administration. Mice with hepatocyte conditional Tipe1 knockout presented significantly impaired liver regeneration. Mechanistically, hepatic Tipe1 deficiency decreased the level of reactive oxygen species in hepatocytes, which in turn led to the inhibition of Forkhead box O1 acetylation and microtubule-associated protein 1 light chain 3 I to microtubule-associated protein 1 light chain 3 II conversion, and the accumulation of sequestosome 1. By contrast, forced expression of TIPE1 in hepatocyte significantly promoted liver regeneration following 70% partial hepatectomy and enhanced hepatocyte reactive oxygen species/acetylated-Forkhead box O1 level and autophagy. These findings indicate that TIPE1 plays a crucial role in liver regeneration by finely regulating the oxidative stress and autophagy and is a potential target for medical intervention of liver regeneration.

Natural killer (NK) cells are key players in tumor immunosurveillance, and metabolic adaptation manipulates their fate and functional state. The nicotinamide adenine dinucleotide (NADNADThese findings reveal NAD

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. Here, we identified zinc fingers and homeoboxes 2 (ZHX2), an HCC-associated tumor suppressor, as an important repressor of de novo lipogenesis. Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC1, and SCD1. In accordance with this, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Results from silencing and overexpression demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with the SREBP1c inhibitor fatostatin dampened the spontaneous formation of tumors in liver-specific Zhx2 knockout mice. Mechanistically, ZHX2 increased expression of miR-24-3p transcriptionally, which targeted SREBP1c and led to its degradation. In conclusion, our data suggest a novel mechanism through which ZHX2 suppresses HCC progression, which may provide a new strategy for the treatment of HCC. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Radiofrequency ablation (RFA) is a widely used and effective treatment for primary or metastatic liver cancer with small-size lesions. However, the therapeutic effectiveness of RFA in controlling metastatic lesion or recurrence is still limited. As the major cell population in tumor microenvironment (TME), macrophages have been reported to be recruited to RFA-treated lesion, but their roles are still unclear. Herein, we successfully established the mouse model mimicking RFA-induced abscopal effect, in which RFA eliminated the local orthotopic liver tumor but failed to control growth of distant tumor. Correspondently, RFA suppressed protumoral activation of local tumor-associated macrophages (TAMs), but failed to reprogram TAMs in distance. Importantly, although RFA led to reduced proportion of hepatic CD169(+) macrophages in local and decreased expression of immune inhibitory molecules Tim-3 and PD-L1, these alterations were not observed for CD169(+) macrophages in distant TME. Further RNA-seq and flow cytometry analysis showed that hepatic CD169(+) macrophages contributed to reprograming TME through recruiting CD8(+) T/NK cells and suppressing accumulation of MDSCs/Tregs. Consistently, depletion of CD169(+) macrophages in CD169-DTR mouse greatly promoted liver tumor progression and largely dampened RFA-induced tumor suppression. Notably, transfer of CD169(+) macrophages synergistically enhanced RFA-induced inhibition of distant tumor. To our knowledge, this is the first study which demonstrates hepatic CD169(+) macrophages as a key factor responsible for RFA-induced abscopal effect. Our data suggest RFA with transfer of CD169(+) macrophages as a promising combination therapy to lessen metastasis or recurrence of liver cancer in patients.

Objective Macrophage-mediated inflammation plays a significant role in the development and progression of diabetic nephropathy (DN). However, the underlying mechanisms remain unclear. Studies suggest that T cell immunoglobulin domain and mucin domain-3 (Tim-3) has complicated roles in regulating macrophage activation, but its roles in the progression of DN are still completely unknown. Methods We downregulated Tim-3 expression in kidney (intrarenal injection of Tim-3 shRNA expressing lentivirus or global Tim-3 knockout mice) and induced DN by streptozotocin (STZ). We analyzed the degree of renal injury, especially the podocyte injury induced by activated macrophages in vitro and in vivo. Then, we transferred different bone marrow derived macrophages (BMs) into STZ-induced Tim-3 knockdown mice to examine the effects of Tim-3 on macrophages in DN. Results First, we found that Tim-3 expression on renal macrophages was increased in patients with DN and in two diabetic mouse models, i.e. STZ-induced diabetic mice and db/db mice, and positively correlated with renal dysfunction of DN patients. Tim-3 deficiency ameliorated renal damage in STZ-induced diabetes with concurrent increase in protein levels of Nephrin and WT-1. Similar effects were observed in mice with Tim-3 knockdown diabetic mice. Second, adoptive transfer of Tim-3-expressing macrophages, but not Tim-3 knockout macrophages, accelerated diabetic renal injury in DN mice, suggesting a key role for Tim-3 on macrophages in the development of DN. Furthermore, we found NF-κB activation and TNF-α excretion were upregulated by Tim-3 in diabetic kidneys, and podocyte injury was associated with the Tim-3-mediated activation of the NF-κB/TNF-α signaling pathway in DN macrophages both in vivo and in vitro. Conclusions These results suggest that Tim-3 functions as a key regulator in renal inflammatory processes and serves as a potential therapeutic target for renal injury in DN., Highlights • Tim-3 aggravates the progression of diabetic nephropathy (DN) by triggering the NF-κB/TNF-α pathway in renal macrophages. • Tim-3 highly expresses on renal macrophages has positive correlation with bad renal function of DN patients. • Tim-3 expressed on macrophages accelerates podocyte injury in vitro and in vivo. • Tim-3 functions as a key regulator in renal inflammatory processes and serves as a potential therapeutic target in DN.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. METHODS: ZHX2 overexpression and knockdown were introduced into HCC cell lines. BODIPY staining and D-Glucose- 13C6 labeled lipid mass spectrometry were involved to estimate lipogenesis in HCC cell lines. Colony formation and cell proliferation assays were used to determine HCC development. Hepatocytes specific Zhx2 knockout mice (Zhx2-KOhep) were included to induce spontaneous liver tumor. qRT-PCR, western blotting and dual luciferase assays were used to detect gene regulation. FINDINGS: Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC and SCD1. In accordance, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Further studies demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with SREBP1c inhibitor, fatostatin, dampened the spontaneous tumor formation in Zhx2-KOhep mice. Mechanically, ZHX2 transcriptionally increased expression of miR-24-3p, which targets on SREBP1c. INTERPRETATION: ZHX2 inhibits de novo lipogenesis in HCC cells by down-regulating the expression of SREBP1c via miR-24-3p, and further inhibits the development of HCC. These new findings may provide a new strategy for the treatment of HCC. FUNDING STATEMENT: This work was supported by grants from the National Science Foundation of China (Key project 81830017 and 81902443,81702647,81902051,81972819), Taishan Scholarship (No.tspd20181201), National Key Research and Development Program (2018YFE0126500), National Science Foundation for Distinguished Young Scholars of China (81425012), Shandong Provincial Key Innovation project (No.2018FYJH0503), Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Key Research and Development Program of Shandong (2019GSF108238) . DECLARATION OF INTERESTS: All authors declare no potential conflicts of interest. ETHICS APPROVAL STATEMENT: The Medical Ethics Committee of Shandong University approved the use of human specimens in accordance with the Helsinki Declaration. All mice were housed under conditions free of specific pathogens and euthanized according to regulations established by the Animal Care Committee of Shandong University.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC-associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV-positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV-encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR-155, a well-known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR-155 levels were found in HBV-positive tumors, livers of HBV transgenic mice and HBx-overexpressing hepatoma cell lines. MiR-155 overexpression reduced ZHX2 levels via miR-155 seed sites in the ZHX2 3'UTR, whereas blocking miR-155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC-promoting properties of HBV may include ZHX2 silencing via a miR-155 dependent pathway and suggests a novel therapy for HBV-related HCC.

Increased expression of T cell immunoglobulin and mucin domain‐3 (Tim‐3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim‐3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim‐3 was up‐regulated on hepatic iNKT cells from HBV‐transgenic (Tg) mice or iNKT cells stimulated with α‐galactosylceramide (α‐Galcer). Compared with Tim‐3− iNKT cells, Tim‐3+ iNKT cells expressed more IFN‐γ, IL‐4 and CD107a, indicating a strong relationship between Tim‐3 and iNKT cell activation. Constantly, treatment of Tim‐3 blocking antibodies significantly enhanced the production of IFN‐γ, TNF‐α, IL‐4 and CD107a in iNKT cells both in vivo and in vitro. This Tim‐3− mediated suppression of iNKT cells was further confirmed in Tim‐3 knockout (KO) mice. Moreover, Tim‐3 blockade promoted α‐Galcer‐triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down‐regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim‐3 blockade in promoting iNKT cell‐mediated HBV inhibition. Therefore, combination of α‐Galcer with Tim‐3 blockade might be a promising approach in chronic hepatitis B therapy.

Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Metabolism orchestrates homeostasis of macrophages. However, the precise mechanism of macrophage metabolism during sepsis remains poorly elucidated. In this study, we identified the key role of zinc fingers and homeoboxes (Zhx2), a ubiquitous transcription factor, in macrophage glycolysis and sepsis by enhancing 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) expression. Mice with myeloid Zhx2-specific deletion (abbreviated as MKO) showed more resistance to cecal ligation and puncture and LPS-induced sepsis, exhibiting as prolonged survival, attenuated pulmonary injury, and reduced level of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β. Interestingly, Zhx2 deletion conferred macrophage tolerance to LPS-induced glycolysis, accompanied by reduced proinflammatory cytokines and lactate. Consistently, treatment of glycolytic inhibitor 2-deoxyglucose almost completely abrogated the protection of mice from LPS-induced sepsis initiated by Zhx2 deletion in macrophages. RNA sequencing and chromatin immunoprecipitation assays confirmed that Zhx2 enhanced transcription of Pfkfb3, the glycolysis rate-limiting enzyme, via binding with Pfkfb3 promoter. Furthermore, Pfkfb3 overexpression not only rescued the reduction of macrophage glycolysis caused by Zhx2 deficiency, displaying as extracellular acidification rates and lactate production but also destroyed the resistance of mice to LPS-induced sepsis initiated by transfer of bone marrow–derived macrophages from MKO mice. These findings highlight the novel role of transcription factor Zhx2 in sepsis via regulating Pfkfb3 expression and reprogramming macrophage metabolism, which would shed new insights into the potential strategy to intervene sepsis.

Natural killer (NK) cells are enriched within the liver. Apart from conventional NK (cNK) cells, recent studies identified a liver-resident NK (LrNK) subset, which constitutes about half of hepatic NK cells and exhibits distinct developmental, phenotypic, and functional features. However, it remains unclear whether and how LrNK cells, as well as cNK cells, participate in the development of hepatocellular carcinoma (HCC) individually. Here, we report that both LrNK and cNK cells are significantly decreased in HCC. The T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was significantly upregulated in both tumor-infiltrating LrNK and cNK cells and suppressed their cytokine secretion and cytotoxic activity. Mechanistically, phosphatidylserine (PtdSer) engagement promoted phosphorylation of Tim-3, which then competed with PI3K p110 to bind p85, inhibiting downstream Akt/mTORC1 signaling and resulting in malfunctioning of both NK-cell subsets. Tim-3 blockade retarded HCC growth in a NK-cell–dependent manner. These studies for the first time report the presence and dysfunction of LrNK cells in HCC and show that Tim-3–mediated PI3K/mTORC1 interference is responsible for the dysfunction of both tumor-infiltrating cNK and LrNK cells, providing a new strategy for immune checkpoint-based targeting. Significance: Tim-3 enhances hepatocellular carcinoma growth by blocking natural killer cell function.

Background: Liver cancer stem cells (CSCs) are critical determinants of cancer relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes (ZHX2) in liver CSCs. Methods: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and cell lines by flow cytometry. Also, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: The expression of ZHX2 was reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of CSCs in supporting tumor initiation, self-renew and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significant poor survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance. Funding Statement: This work was supported by grants from the National Natural Science Fund for Outstanding Youth Fund (81425012), the National Key Research and Development Program (No.2016YFE0127000), the National Science Foundation of China (No. 81830017, 81370527, 81702647), Taishan Scholarship (No.tspd20181201), Shandong Provincial Key Innovation project (No.2018FYJH0503) and Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Informed consent was obtained from all patients before the study was initiated with approval of the Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. All animal procedures were performed in according to protocols approved by the Shandong University Animal Care Committee and conducted with an animal ethical approval.

Background: Liver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs. Methods: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: ZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance. Keywords: ZHX2, Tumor suppressor, Epigenetics, Cancer stem cells, Oncotherapy

As an immune checkpoint, Tim-3 plays roles in the regulation of both adaptive and innate immune cells including macrophages and is greatly involved in chronic liver diseases. However, the precise roles of Tim-3 in nonalcoholic steatohepatitis (NASH) remain unstated. In the current study, we analyzed Tim-3 expression on different subpopulations of liver macrophages and further investigated the potential roles of Tim-3 on hepatic macrophages in methionine and choline-deficient diet (MCD)-induced NASH mice. The results of flow cytometry demonstrated the significantly increased expression of Tim-3 on all detected liver macrophage subsets in MCD mice, including F4/80(+)CD11b(+), F4/80(+)CD68(+), and F4/80(+)CD169(+) macrophages. Remarkably, Tim-3 knockout (KO) significantly accelerated MCD-induced liver steatosis, displaying higher serum ALT, larger hepatic vacuolation, more liver lipid deposition, and more severe liver fibrosis. Moreover, compared with wild-type C57BL/6 mice, Tim-3 KO MCD mice demonstrated an enhanced expression of NOX2, NLRP3, and caspase-1 p20 together with increased generation of IL-1β and IL-18 in livers. In vitro studies demonstrated that Tim-3 negatively regulated the production of reactive oxygen species (ROS) and related downstream pro-inflammatory cytokine secretion of IL-1β and IL-18 in macrophages. Exogenous administration of N-Acetyl-L-cysteine (NAC), a small molecular inhibitor of ROS, remarkably suppressed caspase-1 p20 expression and IL-1β and IL-18 production in livers of Tim-3 KO mice, thus significantly reducing the severity of steatohepatitis induced by MCD. In conclusion, Tim-3 is a promising protector in MCD-induced steatohepatitis by controlling ROS and the associated pro-inflammatory cytokine production in macrophages.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC-associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV-positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV-encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR-155, a well-known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR-155 levels were found in HBV-positive tumors, livers of HBV transgenic mice and HBx-overexpressing hepatoma cell lines. MiR-155 overexpression reduced ZHX2 levels via miR-155 seed sites in the ZHX2 3'UTR, whereas blocking miR-155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC-promoting properties of HBV may include ZHX2 silencing via a miR-155 dependent pathway and suggests a novel therapy for HBV-related HCC.