Tim-4 Inhibits NLRP3 Inflammasome via the LKB1/AMPKα Pathway in Macrophages.

Nonalcoholic fatty liver disease (NAFLD), characterized by excessive inflammation and lipid deposition, is one of the most common metabolic liver diseases. The expression of NLRP3 inflammasome in macrophages is significantly increased in NAFLD, and its activation aggravates NAFLD greatly. Tim-4, as the phosphatidylserine (PS) receptor, is expressed highly in macrophages, and macrophage Tim-4 inhibits inflammation under various conditions of immune activation. However, the precise role of Tim-4 in NLRP3 inflammasome regulation and NAFLD pathogenesis remains completely unknown. Using NAFLD mice models, we confirmed that the expression of Tim-4 was increased in liver tissues by Western blot, real-time PCR, immunohistochemistry, and immunofluorescence, especially higher expression in liver macrophages, and Tim-4 knockout mice displayed more severe liver inflammation and hepatic steatosis than controls in NAFLD mice model. In vitro, we found that Tim-4 could inhibit NLRP3 inflammasome activation, and the inhibition was dependent on PS binding domain in the IgV domain. Mechanistically, Tim-4 induced the degradation of NLRP3 inflammasome components through activating AMPKα-mediated autophagy. Specifically, Tim-4 promoted AMPKα phosphorylation by interacting with LKB1 and AMPKα. In addition, PS binding motif was responsible for Tim-4-mediated AMPKα and LKB1 interaction. In conclusion, NAFLD microenvironments upregulate Tim-4 expression in macrophages, and elevated Tim-4, in turn, suppresses NLRP3 inflammasome activation by activating LKB1/AMPKα-mediated autophagy, thereby ameliorating the release of IL-1ß and IL-18. Collectively, this study unveils the novel function of Tim-4 in suppressing NLRP3 inflammasome, which would shed new lights on intervention of NAFLD or inflammatory liver diseases by targeting Tim-4.

Tim-3 blockade promotes iNKT cell function to inhibit HBV replication.

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.

Progranulin inhibits autophagy to facilitate intracellular colonization of Helicobacter pylori through the PGRN/mTOR/DCN axis in gastric epithelial cells.

Helicobacter pylori (H. pylori) infection is the primary risk factor for the progress of gastric diseases. The persistent stomach colonization of H. pylori is closely associated with the development of gastritis and malignancies. Although the involvement of progranulin (PGRN) in various cancer types has been well-documented, its functional role and underlying mechanisms in gastric cancer (GC) associated with H. pylori infection remain largely unknown. This report demonstrated that PGRN was up-regulated in GC and associated with poor prognosis, as determined through local and public database analysis. Additionally, H. pylori induced the up-regulation of PGRN in gastric epithelial cells both in vitro and in vivo. Functional studies have shown that PGRN promoted the intracellular colonization of H. pylori. Mechanistically, H. pylori infection induced autophagy, while PGRN inhibited autophagy to promote the intracellular colonization of H. pylori. Furthermore, PGRN suppressed H. pylori-induced autophagy by down-regulating decorin (DCN) through the mTOR pathway. In general, PGRN inhibited autophagy to facilitate intracellular colonization of H. pylori via the PGRN/mTOR/DCN axis. This study provides new insights into the molecular mechanisms underlying the progression of gastric diseases, suggesting PGRN as a potential therapeutic target and prognostic predictor for these disorders.

Helicobacter pylori-Induced Progranulin Promotes the Progression of the Gastric Epithelial Cell Cycle by Regulating CDK4.

Helicobacter pylori, a group 1 carcinogen, colonizes the stomach and affects the development of stomach diseases. Progranulin (PGRN) is an autocrine growth factor that regulates multiple cellular processes and plays a tumorigenic role in many tissues. Nevertheless, the mechanism of action of PGRN in gastric cancer caused by H. pylori infection remains unclear. Here, we investigated the role of PGRN in cell cycle progression and the cell proliferation induced by H. pylori infection. We found that the increased PGRN was positively associated with CDK4 expression in gastric cancer tissue. PGRN was upregulated by H. pylori infection, thereby promoting cell proliferation, and that enhanced level of proliferation was reduced by PGRN inhibitor. CDK4, a target gene of PGRN, is a cyclin-dependent kinase that binds to cyclin D to promote cell cycle progression, which was upregulated by H. pylori infection. We also showed that knockdown of CDK4 reduced the higher cell cycle progression caused by upregulated PGRN. Moreover, when the PI3K/Akt signaling pathway (which is promoted by PGRN) was blocked, the upregulation of CDK4 mediated by PGRN was reduced. These results reveal the potential mechanism by which PGRN plays a major role through CDK4 in the pathological mechanism of H. pylori infection.

Design, diversity-oriented synthesis and biological evaluation of novel heterocycle derivatives as non-nucleoside HBV capsid protein inhibitors.

The capsid assembly is a significant phase for the hepatitis B virus (HBV) lifespan and is an essential target for anti-HBV drug discovery and development. Herein, we used scaffold hopping, bioisosterism, and pharmacophore hybrid-based strategies to design and synthesize six series of various heterocycle derivatives (pyrazole, thiazole, pyrazine, pyrimidine, and pyridine) and screened for in vitro anti-HBV non-nucleoside activity. Drug candidate NZ-4 and AT-130 were used as lead compounds. Several compounds exhibited prominent anti-HBV activity compared to lead compound NZ-4 and positive drug Lamivudine, especially compound II-8b, showed the most prominent anti-HBV DNA replication activity (IC50 = 2.2 ± 1.1 µM). Also compounds IV-8e and VII-5b showed the best in vitro anti-HBsAg secretion (IC50 = 3.8 ± 0.7 µM, CC50 > 100 µM) and anti-HBeAg secretion (IC50 = 9.7 ± 2.8 µM, CC50 > 100 µM) respectively. Besides, II-8b can interact HBV capsid protein with good affinity constants (KD = 60.0 µM), which is equivalent to lead compound NZ-4 ((KD = 50.6 µM). The preliminary structure-activity relationships (SARs) of the newly synthesized compounds were summarized, which may help researchers to discover more potent anti-HBV agents.

Tim-3 Hampers Tumor Surveillance of Liver-Resident and Conventional NK Cells by Disrupting PI3K Signaling.

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.

Increased Tim-3 expression alleviates liver injury by regulating macrophage activation in MCD-induced NASH mice.

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-1ß 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-1ß 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-1ß 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.

Tim-3 aggravates podocyte injury in diabetic nephropathy by promoting macrophage activation via the NF-κB/TNF-α pathway.

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.