TIPE1 limits virus replication by disrupting PKM2/ HIF-1α/ glycolysis feedback loop.

OBJECTIVE: Virus infection is a major threat to human health and remains a significant cause of death to date. Macrophages are important innate immune cells that exhibit indispensable roles in controlling virus replication. It was recently reported that metabolic adaption determines the functional state of macrophages. Thus, to further unravel the crucial factors involving in metabolic adaption of macrophages might provide the potential candidates for optimizing their anti-viral capabilities. METHODS: RT-PCR, Western blotting, virus plaque assay and HE were used to evaluate the viral load in virus-infected Tipe1M-KO and Tipe1f/f mice or cultured macrophages. RNA sequencing were performed with Tipe1M-KOor Tipe1f/f BMDMs upon virus infection. Extracellular acidification rate (ECAR) was applied for analyzing glycolysis rate in virus-infected BMDMs. Co-immunoprecipitation (Co-IP) assay and LC-MS/MS were used to determine the potential interacting proteins of TIPE1. RESULTS: TIPE1 level was significantly reduced in BMDMs infected with either RNA viruses or DNA virus. Deficiency of Tipe1 in macrophages increased viral load and aggravated tissue damage. Mechanistically, TIPE1 suppressed the glycolytic capacity of macrophages through interacting with PKM2 and promoting its ubiquitination degradation, which in turn decreased HIF1α transcription and viral replication in macrophages. CONCLUSIONS: TIPE1 functions as a novel regulator for metabolic reprogramming and virus infection in macrophages.

Dynamic Hybrid Module-Driven NK Cell Stimulation and Release for Tumor Immunotherapy.

Natural killer (NK) cells have become a powerful candidate for adoptive tumor immunotherapy, while their therapeutic efficacy in solid tumors remains unsatisfactory. Here, we developed a hybrid module with an injectable hydrogel and hydroxyapatite (HAp) nanobelts for the controlled delivery of NK cells to enhance the therapy of solid tumors. Surface-functionalized HAp nanobelts modified with agonistic antibodies against NKG2D and 4-1BB and cytokines IL-2 and IL-21 support survival and dynamic activation. Thus, the HAp-modified chitosan (CS) thermos-sensitive hydrogel not only improved the retention of NK cells for more than 20 days in vivo but also increased NK cell function by more than one-fold. The unique architecture of this biomaterial complex protects NK cells from the hostile tumor environment and improves antitumor efficacy. The generation of a transient inflammatory niche for NK cells through a biocompatible hydrogel reservoir may be a conversion pathway to prevent cancer recurrence of resectable tumors.

Urolithin A Hijacks ERK1/2-ULK1 Cascade to Improve CD8+ T Cell Fitness for Antitumor Immunity.

According to the latest evidence, the microbial metabolite Urolithin A (UA), known for its role in promoting cellular health, modulates CD8+ T cell-mediated antitumor activity. However, the direct target protein of UA and its underlying mechanism remains unclear. Here, this research identifies ERK1/2 as the specific target crucial for UA-mediated CD8+ T cell activation. Even at low doses, UA markedly enhances the persistence and effector functions of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells both in vitro and in vivo. Mechanistically, UA interacts directly with ERK1/2 kinases, enhancing their activation and subsequently facilitating T cell activation by engaging ULK1. The UA-ERK1/2-ULK1 axis promotes autophagic flux in CD8+ CTLs, enhancing cellular metabolism and maintaining reactive oxygen species (ROS) levels, as evidenced by increased oxygen consumption and extracellular acidification rates. UA-treated CD8+ CTLs also display elevated ATP levels and enhanced spare respiratory capacity. Overall, UA activates ERK1/2, inducing autophagy and metabolic adaptation, showcasing its potential in tumor immunotherapy and interventions for diseases involving ERKs.

Beta-Cell Tipe1 Orchestrates Insulin Secretion and Cell Proliferation by Promoting Gαs/cAMP Signaling via USP5.

Inadequate ß-cell mass and insulin secretion are essential for the development of type 2 diabetes (T2D). TNF-α-induced protein 8-like 1 (Tipe1) plays a crucial role in multiple diseases, however, a specific role in T2D pathogenesis remains largely unexplored. Herein, Tipe1 as a key regulator in T2D, contributing to the maintenance of ß cell homeostasis is identified. The results show that the ß-cell-specific knockout of Tipe1 (termed Ins2-Tipe1BKO) aggravated diabetic phenotypes in db/db mice or in mice with high-fat diet-induced diabetes. Notably, Tipe1 improves ß cell mass and function, a process that depends on Gαs, the α subunit of the G-stimulating protein. Mechanistically, Tipe1 inhibited the K48-linked ubiquitination degradation of Gαs by recruiting the deubiquitinase USP5. Consequently, Gαs or cAMP agonists almost completely restored the dysfunction of ß cells observed in Ins2-Tipe1BKO mice. The findings characterize Tipe1 as a regulator of ß cell function through the Gαs/cAMP pathway, suggesting that Tipe1 may emerge as a novel target for T2D intervention.

Increased Siglec-9/Siglec-9L interactions on NK cells predict poor HCC prognosis and present a targetable checkpoint for immunotherapy.

BACKGROUND & AIMS: Natural killer (NK) cell-based anti-hepatocellular carcinoma (HCC) therapy is an increasingly attractive approach that warrants further study. Siglec-9 interacts with its ligand (Siglec-9L) and restrains NK cell functions, suggesting it is a potential therapeutic target. However, in situ Siglec-9/Siglec-9L interactions in HCC have not been reported, and a relevant interventional strategy is lacking. Herein, we aim to illustrate Siglec-9/Siglec-9L-mediated cell sociology and identify small-molecule inhibitors targeting Siglec-9 that could improve the efficacy of NK cell-based immunotherapy for HCC. METHODS: Multiplexed immunofluorescence staining was performed to analyze the expression pattern of Siglec-7, -9 and their ligands in HCC tissues. Then we conducted docking-based virtual screening combined with bio-layer interferometry assays to identify a potent small-molecule Siglec-9 inhibitor. The therapeutic potential was further evaluated in vitro and in hepatoma-bearing NCG mice. RESULTS: Siglec-9 expression, rather than Siglec-7, was markedly upregulated on tumor-infiltrating NK cells, which correlated significantly with reduced survival of patients with HCC. Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival, further suggesting that Siglec-9/Siglec-9L interactions are a potential therapeutic target in HCC. In addition, we identified a small-molecule Siglec-9 inhibitor MTX-3937 which inhibited phosphorylation of Siglec-9 and downstream SHP1 and SHP2. Accordingly, MTX-3937 led to considerable improvement in NK cell function. Notably, MTX-3937 enhanced cytotoxicity of both human peripheral and tumor-infiltrating NK cells. Furthermore, transfer of MTX-3937-treated NK92 cells greatly suppressed the growth of hepatoma xenografts in NCG mice. CONCLUSIONS: Our study provides the rationale for HCC treatment by targeting Siglec-9 on NK cells and identifies a promising small-molecule inhibitor against Siglec-9 that enhances NK cell-mediated HCC surveillance. IMPACT AND IMPLICATIONS: Herein, we found that Siglec-9 expression is markedly upregulated on tumor-infiltrating natural killer (TINK) cells and correlates with reduced survival in patients with hepatocellular carcinoma (HCC). Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival. More importantly, we identified a small-molecule inhibitor targeting Siglec-9 that augments NK cell functions, revealing a novel immunotherapy strategy for liver cancer that warrants further clinical investigation.

Identification of a small-molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical mouse models.

T cell immunoglobulin and mucin-containing molecule 3 (Tim-3), expressed in dysfunctional and exhausted T cells, has been widely acknowledged as a promising immune checkpoint target for tumor immunotherapy. Here, using a strategy combining virtual and functional screening, we identified a compound named ML-T7 that targets the FG-CC' cleft of Tim-3, a highly conserved binding site of phosphatidylserine (PtdSer) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). ML-T7 enhanced the survival and antitumor activity of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells and reduced their exhaustion in vitro and in vivo. In addition, ML-T7 promoted NK cells' killing activity and DC antigen-presenting capacity, consistent with the reported activity of Tim-3. ML-T7 strengthened DCs' functions through both Tim-3 and Tim-4, which is consistent with the fact that Tim-4 contains a similar FG-CC' loop. Intraperitoneal dosing of ML-T7 showed comparable tumor inhibitory effects to the Tim-3 blocking antibody. ML-T7 reduced syngeneic tumor progression in both wild-type and Tim-3 humanized mice and alleviated the immunosuppressive microenvironment. Furthermore, combined ML-T7 and anti-PD-1 therapy had greater therapeutic efficacy than monotherapy in mice, supporting further development of ML-T7 for tumor immunotherapy. Our study demonstrates a potential small molecule for selectively blocking Tim-3 and warrants further study.

ZHX2 emerges as a negative regulator of mitochondrial oxidative phosphorylation during acute liver injury.

Mitochondria dysfunction contributes to acute liver injuries, and mitochondrial regulators, such as PGC-1α and MCJ, affect liver regeneration. Therefore, identification of mitochondrial modulators may pave the way for developing therapeutic strategies. Here, ZHX2 is identified as a mitochondrial regulator during acute liver injury. ZHX2 both transcriptionally inhibits expression of several mitochondrial electron transport chain genes and decreases PGC-1α stability, leading to reduction of mitochondrial mass and OXPHOS. Loss of Zhx2 promotes liver recovery by increasing mitochondrial OXPHOS in mice with partial hepatectomy or CCl4-induced liver injury, and inhibition of PGC-1α or electron transport chain abolishes these effects. Notably, ZHX2 expression is higher in liver tissues from patients with drug-induced liver injury and is negatively correlated with mitochondrial mass marker TOM20. Delivery of shRNA targeting Zhx2 effectively protects mice from CCl4-induced liver injury. Together, our data clarify ZHX2 as a negative regulator of mitochondrial OXPHOS and a potential target for developing strategies for improving liver recovery after acute injuries.

Design, Synthesis, and Biological Evaluation of Novel Thioureidobenzamide (TBA) Derivatives as HBV Capsid Assembly Modulators.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a promising therapeutic approach for the treatment of HBV infection. In this study, we designed and synthesized five series of benzamide derivatives based on a multisite-binding strategy at the tolerant region and diversity modification in the solvent-exposed region. Among them, thioureidobenzamide compound 17i exhibited significantly increased anti-HBV activity in HepAD38 (EC50 = 0.012 µM) and HBV-infected HLCZ01 cells (EC50 = 0.033 µM). Moreover, 17i displayed a better inhibitory effect on the assembly of HBV capsid protein compared with NVR 3-778 and a inhibitory effect similar to the clinical drug GLS4. In addition, 17i showed moderate metabolic stability in human microsomes, had excellent oral bioavailability in Sprague-Dawley (SD) rats, and inhibited HBV replication in the HBV carrier mice model, which could be considered as a promising candidate drug for further development.

Lipid accumulation-mediated histone hypoacetylation drives persistent NK cell dysfunction in anti-tumor immunity.

Hyperlipidemia impairs anti-tumor immune responses and is closely associated with increased human cancer incidence and mortality. However, the underlying mechanisms are not well understood. In the present study, we show that natural killer (NK) cells isolated from high-fat-diet mice or treated with oleic acid (OA) in vitro exhibit sustainable functional defects even after removal from hyperlipidemic milieu. This is accompanied by reduced chromatin accessibility in the promoter region of NK cell effector molecules. Mechanistically, OA exposure blunts P300-mediated c-Myc acetylation and shortens its protein half-life in NK cells, which in turn reduces P300 accumulation and H3K27 acetylation and leads to persistent NK cell dysfunction. NK cells engineered with hyperacetylated c-Myc mutants surmount the suppressive effect of hyperlipidemia and display superior anti-tumor activity. Our findings reveal the persistent dysfunction of NK cells in dyslipidemia milieu and extend engineered NK cells as a promising strategy for tumor immunotherapy.

Palmitoylation at Residue C221 of Japanese Encephalitis Virus NS2A Protein Contributes to Viral Replication Efficiency and Virulence.

Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.

Zhx2 maintains islet ß-cell mass and function by transcriptionally regulating Pax6.

Emerging evidence shows that pancreatic ß-cell function and quality are key determinants in the progression of type 2 diabetes (T2D). The transcription factor zinc finger homeobox 2 (Zhx2) is involved in proliferation and development of multiple cells. However, the exact role of Zhx2 in ß-cells and T2D remains completely unknown. Here, we report that Zhx2 orchestrates ß-cell mass and function by regulating paired box protein pax-6 (Pax6). We found that ß-cell-specific knockout Zhx2 (Zhx2BKO) mice showed a decrease in ß-cell proliferation and glucose homeostasis. Under prediabetic and diabetic conditions, we discovered glucose intolerance in both Zhx2BKO-HFD mice and Zhx2BKO-db/db mice, with reduced ß-cell mass and insulin secretion. Mechanistically, we demonstrated that Zhx2 targeted the Pax6 promoter region (-1740∼-1563; -862∼-559; -251∼+75), enhanced promoter activity. Overall, Zhx2 maintains ß-cell function by transcriptionally regulating Pax6, which provides a therapeutic target for diabetes intervention.

Lipophagy-mediated cholesterol synthesis inhibition is required for the survival of hepatocellular carcinoma under glutamine deprivation.

Glutamine is critical for tumor progression, and restriction of its availability is emerging as a potential therapeutic strategy. The metabolic plasticity of tumor cells helps them adapting to glutamine restriction. However, the role of cholesterol metabolism in this process is relatively unexplored. Here, we reported that glutamine deprivation inhibited cholesterol synthesis in hepatocellular carcinoma (HCC). Reactivation of cholesterol synthesis enhanced glutamine-deprivation-induced cell death of HCC cells, which is partially duo to augmented NADPH depletion and lipid peroxidation. Mechanistically, glutamine deprivation induced lipophagy to transport cholesterol from lipid droplets (LDs) to endoplasmic reticulum (ER), leading to inhibit SREBF2 maturation and cholesterol synthesis, and maintain redox balance for survival. Glutamine deprivation decreased mTORC1 activity to induce lipophagy. Importantly, administration of U18666A, CQ, or shTSC2 viruses further augmented GPNA-induced inhibition of xenograft tumor growth. Clinical data supported that glutamine utilization positively correlated with cholesterol synthesis, which is associated with poor prognosis of HCC patients. Collectively, our study revealed that cholesterol synthesis inhibition is required for the survival of HCC under glutamine-restricted tumor microenvironment.

Early life gut microbiota sustains liver-resident natural killer cells maturation via the butyrate-IL-18 axis.

Liver-resident natural killer cells, a unique lymphocyte subset in liver, develop locally and play multifaceted immunological roles. However, the mechanisms for the maintenance of liver-resident natural killer cell homeostasis remain unclear. Here we show that early-life antibiotic treatment blunt functional maturation of liver-resident natural killer cells even at adulthood, which is dependent on the durative microbiota dysbiosis. Mechanistically, early-life antibiotic treatment significantly decreases butyrate level in liver, and subsequently led to defective liver-resident natural killer cell maturation in a cell-extrinsic manner. Specifically, loss of butyrate impairs IL-18 production in Kupffer cells and hepatocytes through acting on the receptor GPR109A. Disrupted IL-18/IL-18R signaling in turn suppresses the mitochondrial activity and the functional maturation of liver-resident natural killer cells. Strikingly, dietary supplementation of experimentally or clinically used Clostridium butyricum restores the impaired liver-resident natural killer cell maturation and function induced by early-life antibiotic treatment. Our findings collectively unmask a regulatory network of gut-liver axis, highlighting the importance of the early-life microbiota in the development of tissue-resident immune cells.

TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis.

Mitochondrial function and homeostasis are critical to the proliferation of lung cancer cells. T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM-4) promotes the development and progression of lung cancer. However, the role of TIM-4 in mitochondria homeostasis in tumor cells remains completely unknown. In this study, we found that TIM-4 promoted growth and proliferation of lung cancer cells by the oxidative phosphorylation (OXPHOS) pathway. Consistently, inhibition of OXPHOS reversed TIM-4-induced proliferation of lung cancer cells. Notably, TIM-4 promoted mitochondrial fusion via enhancing L-OPA1 protein expression. Mechanistically, TIM-4 regulated protein of L-OPA1 through the PI3K/AKT pathway, and TIM-4 interacted with ANXA2 to promote the activation of PI3K/AKT signaling. Collectively, TIM-4 promotes oxidative phosphorylation of lung cancer cells to accelerate tumor progress via ANXA2/PI3K/AKT/OPA1 axis, which sheds significant new lights on the potential role of TIM-4 in regulating tumor cell metabolism.

T-cell immunoglobulin- and mucin-domain-containing molecule-4 maintains adipose tissue homeostasis by orchestrating M2 macrophage polarization via nuclear factor kappa B pathway.

Obesity is generally associated with low-grade inflammation. Adipose tissue macrophages (ATMs) orchestrate metabolic inflammation. The classical (M1-like) or alternative (M2-like) activation of ATMs is functionally coupled with the metabolic status of fat tissues. It has been found that T-cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) inhibits inflammation by regulating macrophages. However, the exact role of Tim-4 in macrophage polarization and obesity remains unknown. Here, we identified Tim-4 as a critical switch governing macrophage M1/M2 polarization and energy homeostasis. Tim-4 deletion led to spontaneous obesity in elder mice and promoted obesity severity of db/db mice. Obesity microenvironment enhanced the expression of Tim-4 in white adipose tissue and ATMs. In vitro, we detected an increase in M1-like cells and decrease in M2-like cells in both peritoneal macrophages and bone marrow-derived macrophages from Tim-4 knockout mice. Mechanistically, we demonstrated that Tim-4 promoted M2-like macrophages polarization via suppressing nuclear factor kappa B (NF-κB) signaling pathway. In addition, we found that Tim-4 promoted TLR4 internalization, which might contribute to regulation of NF-κB signaling. Collectively, these results indicated that Tim-4 maintained adipose tissue homeostasis by regulating macrophage polarization via NF-κB pathway, which would provide a new target for obesity intervention.

NAD + salvage governs mitochondrial metabolism, invigorating natural killer cell antitumor immunity.

BACKGROUND AND AIMS: Natural killer (NK) cells are key players in tumor immunosurveillance, and metabolic adaptation manipulates their fate and functional state. The nicotinamide adenine dinucleotide (NAD + ) has emerged as a vital factor to link cellular metabolism and signaling transduction. Here, we identified NAD + metabolism as a central hub to determine the homeostasis and function of NK cells. APPROACH AND RESULTS: NAD + level was elevated in activated NK cells. NAD + supplementation not only enhanced cytokine production and cytotoxicity but also improved the proliferation and viability of NK cells. Intriguingly, the salvage pathway was involved in maintaining NAD + homeostasis in activated NK cells. Genetic ablation or pharmacological blockade of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD + salvage pathway, markedly destroyed the viability and function of NK cells. Mechanistically, NAD + salvage dictated the mitochondrial homeostasis and oxidative phosphorylation activity to support the optimal function of NK cells. However, in human HCC tissues, NAMPT expression and NAD + level were significantly down-regulated in tumor-infiltrating NK cells, which negatively correlated with patient survival. And lactate accumulation in the tumor microenvironment was at least partially responsible for the transcriptional repression of NAMPT in NK cells. Further, deficiency of Nampt in NK cells accelerated the growth of HCC and melanoma. Supplementation of the NAD + precursor nicotinamide mononucleotide (NMN) significantly improved NK antitumor response in both mouse and human cell-derived xenografts. CONCLUSIONS: These findings reveal NAD + salvage as an essential factor for NK-cell homeostasis and function, suggesting a potential strategy for invigorating NK cell-based immunotherapy.

SREBF2-STARD4 axis confers sorafenib resistance in hepatocellular carcinoma by regulating mitochondrial cholesterol homeostasis.

Sorafenib resistance limits its survival benefit for treatment of hepatocellular carcinoma (HCC). Cholesterol metabolism is dysregulated in HCC, and its role in sorafenib resistance of HCC has not been fully elucidated. Aiming to elucidate this, in vitro and in vivo sorafenib resistant models were established. Sterol regulatory element binding transcription factor 2 (SREBF2), the key regulator of cholesterol metabolism, was activated in sorafenib resistant HepG2 and Huh7 cells. Knockdown of SREBF2 resensitized sorafenib resistant cells and xenografts tumors to sorafenib. Further study showed that SREBF2 positively correlated with StAR related lipid transfer domain containing 4 (STARD4) in our sorafenib resistant models and publicly available datasets. STARD4, mediating cholesterol trafficking, not only promoted proliferation and migration of HepG2 and Huh7 cells, but also increased sorafenib resistance in liver cancer. Mechanically, SREBF2 promoted expression of STARD4 by directly binding to its promoter region, leading to increased mitochondrial cholesterol levels and inhibition of mitochondrial cytochrome c release. Importantly, knockdown of SREBF2 or STARD4 decreased mitochondrial cholesterol levels and increased mitochondrial cytochrome c release, respectively. Moreover, overexpression of STARD4 reversed the effect of SREBF2 knockdown on mitochondrial cytochrome c release and sorafenib resistance. In conclusion, SREBF2 promotes STARD4 transcription, which in turn contributes to mitochondrial cholesterol transport and sorafenib resistance in HCC. Therefore, targeting the SREBF2-STARD4 axis would be beneficial to a subset of HCC patients with sorafenib resistance.

ZHX2 in health and disease.

As a transcriptional factor and the negative regulator of alpha fetal protein (AFP), Zinc fingers and homeoboxes 2 (ZHX2) has a well-established role in protection against hepatocellular carcinoma (HCC). However, recent studies have suggested ZHX2 as an oncogene in clear cell renal cell carcinoma (ccRCC) and triple-negative breast cancer (TNBC). Moreover, mounting evidence has illustrated a much broader role of ZHX2 in multiple cellular processes, including cell proliferation, cell differentiation, lipid metabolism, and immunoregulation. This comprehensive review emphasizes the role of ZHX2 in health and diseases which have been more recently uncovered.