Evidence for Hypoxia-Induced Shift in ATP Production from Glycolysis to Mitochondrial Respiration in Pulmonary Artery Smooth Muscle Cells in Pulmonary Arterial Hypertension.

BACKGROUND: The metabolic state of pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) is not well understood. In this study, we examined the balance between glycolysis and mitochondrial respiration in non-PAH-PASMCs and PAH-PASMCs under normoxia and hypoxia. METHODS: We investigated the enzymes involved in glycolysis and mitochondrial respiration, and studied the two major energy-yielding pathways (glycolysis and mitochondrial respiration) by measuring extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) using the Seahorse extracellular flux technology. RESULTS: Under both normoxia and hypoxia, the mRNA and protein levels of pyruvate dehydrogenase kinase 1 and pyruvate dehydrogenase were increased in PAH-PASMCs compared with non-PAH-PASMCs. The mRNA and protein levels of lactate dehydrogenase, as well as the intracellular lactate concentration, were also increased in PAH-PASMCs compared with non-PAH-PASMCs under normoxia. However, these were not significantly increased in PAH-PASMCs compared with non-PAH-PASMCs under hypoxia. Under normoxia, ATP production was significantly lower in PAH-PASMCs (59 ± 5 pmol/min) than in non-PAH-PASMCs (70 ± 10 pmol/min). On the other hand, ATP production was significantly higher in PAH-PASMCs (31 ± 5 pmol/min) than in non-PAH-PASMCs (14 ± 3 pmol/min) under hypoxia. CONCLUSIONS: There is an underlying change in the metabolic strategy to generate ATP production under the challenge of hypoxia.

Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells.

Mannose has anticancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anticancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anticancer activity of mannose.

In order to grow and divide, cells require a variety of sugars. Breaking down sugars provides energy for cells to proliferate and allows them to make more complex molecules, such as DNA. Although this principle also applies to cancer cells, a specific sugar called mannose not only inhibits cancer cell division but also makes them more sensitive to chemotherapy. These anticancer effects of mannose are particularly strong in cells lacking a protein known as MPI, which breaks down mannose. Evidence from honeybees suggests that a combination of mannose and low levels of MPI leads to a build-up of a modified form of mannose, called mannose-6-phosphate, within cells. As a result, pathways required to release energy from glucose become disrupted, proving lethal to these insects. However, it was not clear whether the same processes were responsible for the anticancer effects of mannose. To investigate, Harada et al. removed the gene that encodes the MPI protein in two types of human cancer cells. The experiments showed that mannose treatment was not lethal to these cells but overall slowed the cell cycle ­ a fundamental process for cell growth and division. More detailed biochemical experiments showed that cancer cells with excess mannose-6-phosphate could not produce the molecules required to make DNA. This prevented them from doubling their DNA ­ a necessary step for cell division ­ and responding to stress caused by chemotherapy. Harada et al. also noticed that cancer cells lacking MPI did not all react to mannose treatment in exactly the same way. Therefore, future work will address these diverse reactions, potentially providing an opportunity to use the mannose pathway to search for new cancer treatments.

CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer.

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non-small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti-PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.

Nutrient Condition in the Microenvironment Determines Essential Metabolisms of CD8+ T Cells for Enhanced IFNγ Production by Metformin.

Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMP-activated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as a drug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which are unachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met. Thus, we proposed activating or expanding tumor-infiltrating CD8+ T cells (CD8TILs) in a mouse model by orally administering Met in free drinking water. Here we showed that Met, at around 10 µM and a physiologically relevant concentration, enhanced production of IFNγ,TNFα and expression of CD25 of CD8+ T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involved in enhancing IFNγ production. Under a low-glucose condition, fatty acid oxidation or autophagy-dependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1 (PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced IFNγ production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In addition, IFNγ production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1. Our results revealed a direct effect of Met on IFNγ production of CD8+ T cells that was dependent on differential metabolic pathways and determined by nutrient conditions in the microenvironment.

Metformin-ROS-Nrf2 connection in the host defense mechanism against oxidative stress, apoptosis, cancers, and ageing.

Reactive oxygen species (ROS) acts as a second messenger to trigger biological responses in low concentrations, while it is implicated to be toxic to biomolecules in high concentrations. Mild inhibition of respiratory chain Complex I by metformin at physiologically relevant concentrations stimulates production of low-level mitochondrial ROS. The ROS seems to induce anti-oxidative stress response via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase (GPx), which results in not only elimination of ROS but also activation of cellular responses including resistance to apoptosis, metabolic changes, cell proliferation, senescence prevention, lifespan extension, and immune T cell activation against cancers, regardless of its effect controlling blood glucose level and T2DM. Although metformin's effect against T2DM, cancers, and ageing, are believed mostly attributed to the activation of AMP-activated protein kinase (AMPK), the cellular responses involving metformin-ROS-Nrf2 axis might be another natural asset to improve healthspan and lifespan.

Blocking EP4 down-regulates tumor metabolism and synergizes with anti-PD-1 therapy to activate natural killer cells in a lung adenocarcinoma model.

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis and suppresses the anti-tumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient 3LL lung adenocarcinoma cell line was down-regulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAMs). On the basis of these results, the therapeutic effect of ONO-AE3-208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1 antibody was evaluated. EP4i, but not anti-PD-1 antibody, decreased tumor metabolism including glycolysis, fatty acid oxidation and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2-like to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 antibody treatment. Although CD8 T-cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 antibody treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicated a novel effect of EP4i for the metabolic reprogramming of tumors and revealed unique features of EP4i that can synergize with anti-PD-1 antibody to promote IFNγ production by NK cells, polarize TAMs into the M1 phenotype, and reduce hypoxia through normalization of the tumor vasculature.

Metformin ameliorates chronic colitis in a mouse model by regulating interferon-γ-producing lamina propria CD4+ T cells through AMPK activation.

Metformin, a commonly prescribed drug for type 2 diabetes mellitus, has been shown to activate AMP-activated protein kinase (AMPK). Notably, AMPK activation has recently been observed to be associated with anti-inflammatory responses. Metformin is also reported to elicit anti-inflammatory responses in CD4+ T cells, resulting in improvement in experimental chronic inflammatory diseases, such as systemic lupus erythematosus. To investigate the effect of metformin on inflammatory bowel disease (IBD), we developed a T cell-transfer model of chronic colitis in which SCID mice were injected with CD4+ CD45RBhigh T cells to induce colitis. We examined the effects of metformin via in vitro and in vivo experiments on lamina propria (LP) CD4+ T cells. We observed that metformin suppresses the frequency of interferon (IFN) -γ-producing LP CD4+ T cells in vitro, which were regulated by AMPK activation, a process possibly induced by the inhibition of oxidative phosphorylation. Furthermore, we examined the effects of metformin on an in vivo IBD model. Metformin-treated mice showed AMPK activation in LP CD4+ T cells and ameliorated colitis. Our study demonstrates that metformin-induced AMPK activation in mucosal CD4+ T cells contributes to the improvement of IBD by suppressing IFN-γ production. Moreover, our results indicate that AMPK may be a target molecule for the regulation of mucosal immunity and inflammation. Thus, AMPK-activating drugs such as metformin may be potential therapeutic agents for the treatment of IBD.

Pharmacological effects on anaplerotic pathways alter the metabolic landscape in the tumor microenvironment, causing unpredictable, sustained anti-tumor immunity.

To achieve sustained anti-tumor immunity, tumor-infiltrating effector CD8 T lymphocytes (CD8 TILs) must be able to produce cytokines, including IFNγ, and proliferate robustly within the local tumor tissue upon antigen recognition. IFNγ production by CD8 TILs depends on glycolysis, whereas their proliferation additionally requires oxidative phosphorylation (OxPhos). The level of OxPhos, and hence the oxygen consumption rate, depends on mitochondrial biogenesis and requires the loading of metabolic precursors into the tricarboxylic acid cycle to keep it functioning. This is referred to as anaplerosis. Recent advances in the field of immuno-metabolism have shown the impact of pharmacological agents on anaplerotic pathways, resulting in metabolic down-regulation in tumor cells; in contrast, the agents trigger sustained anti-tumor immunity by up-regulating both glycolysis and OxPhos in CD8 TILs. The opposing effects of pharmacological inhibition (and/or activation) on anaplerosis in tumor cells and CD8 TILs are unpredictable. Careful dissection of the underlying mechanism might confer important knowledge, helping us to step into a new era for cancer immunotherapy.

Mitochondrial reactive oxygen species trigger metformin-dependent antitumor immunity via activation of Nrf2/mTORC1/p62 axis in tumor-infiltrating CD8T lymphocytes.

BACKGROUND: Metformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However, the underlying mechanism of Met-dependent antitumor immunity remains to be elucidated. METHODS: MitoTEMPO, a scavenger of mitochondrial superoxide, abolished the antitumor effect of Met, but not antiprogrammed cell death (PD-1) antibody (Ab) treatment. Consequently, we studied the mechanism of the Met-induced antitumor effect. Expressions of glucose transporter (Glut)-1, mitochondrial reactive oxygen species (mtROS), interferon (IFN)-γ, Ki67, autophagy markers, activation markers for NF-E2-related factor 2 (Nrf2), and mammalian target of rapamaycin complex 1 (mTORC1) in CD8+ tumor-infiltrating T lymphocytes (CD8TILs) were examined by flow cytometry analysis. In addition, conditional knockout mice for Nrf2 and p62 were used to detect these markers, together with the monitoring of in vivo tumor growth. RNA sequencing was performed for CD8TILs and tumor cells. Melanoma cells containing an IFN-γ receptor (IFNγR) cytoplasmic domain deletion mutant was overexpressed and used for characterization of the metabolic profile of those tumor cells using a Seahorse Flux Analyzer. RESULTS: Met administration elevates mtROS and cell surface Glut-1, resulting in the production of IFN-γ in CD8TILs. mtROS activates Nrf2 in a glycolysis-dependent manner, inducing activation of autophagy, glutaminolysis, mTORC1, and p62/SQSTM1. mTORC1-dependent phosphorylation of p62 at serine 351 (p-p62(S351)) is also involved in activation of Nrf2. Conditional deletion of Nrf2 in CD8TILs abrogates mTORC1 activation and antitumor immunity by Met. In synergy with the effect of anti-PD-1 Ab, Met boosts CD8TIL proliferation and IFN-γ secretion, resulting in decreased glycolysis and oxidative phosphorylation in tumor cells. Consequently, Glut-1 is elevated in CD8TILs, together with the expansion of activated dendritic cells. Moreover, tumor cells lacking in IFNγR signaling abolish IFN-γ production and proliferation of CD8TILs. CONCLUSIONS: We found that Met stimulates production of mtROS, which triggers Glut-1 elevation and Nrf2 activation in CD8TILs. Nrf2 activates mTORC1, whereas mTORC1 activates Nrf2 in a p-p62(S351)-dependent manner, thus creating a feedback loop that ensures CD8TILs' proliferation. In combination with anti-PD-1 Ab, Met stimulates robust proliferation of CD8TILs and IFN-γ secretion, resulting in an IFN-γ-dependent reprogramming of the tumor microenvironment.

Multifunctionality of CD8+ T cells and PD-L1 expression as a biomarker of anti-PD-1 antibody efficacy in advanced melanoma.

Anti-programmed cell death protein-1 (PD-1) antibodies have become a standard treatment for advanced melanoma. However, a predictive biomarker for assessing the efficacy of anti-PD-1 antibodies has not been identified. In cancer, CD8+ T cells specific for tumor antigens undergo repeated T-cell receptor stimulation due to the persistence of cancer cells and gradually lose their ability to secrete interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). We aimed to evaluate multi-cytokine production and immune exhaustion of peripheral CD8+ T cells in melanoma patients treated with anti-PD-1 antibodies. Twenty-four melanoma patients treated with nivolumab were included. Effector cytokine production (IL-2, TNF-α, and IFN-γ) and expression of an exhaustion marker (PD-1) in patients' CD8+ cells were analyzed with flow cytometry. The relationships between parameters such as the neutrophil-to-lymphocyte ratio (NLR) and clinical response to nivolumab were examined. Immunohistochemistry for programmed death-ligand 1 (PD-L1) expression in tumor cells and tumor-infiltrating lymphocytes (TILs) and analysis of their association with clinical response were performed. The clinical response rate to nivolumab was 29%. Regarding TILs, NLR, and several other parameters, no significant difference was found between responders and non-responders. The responder group showed an increase in the percentage of PD-1+ CD8+ /TNF-α+ IFN-γ+ or PD-1+ CD8+ /IFN-γ+ IL-2+ TNF-α+ T cells compared to non-responders. Positivity for PD-L1 expression was significantly higher in the responder group than the non-responder group. In advanced melanoma, the percentage of multifunctional CD8+ PD-1+ T cells and PD-L1 expression in the tumors may be a biomarker for a good response to anti-PD-1 antibody monotherapy.

Dysfunction of CD8 + PD-1 + T cells in type 2 diabetes caused by the impairment of metabolism-immune axis.

The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

Metformin Prevents Peritoneal Dissemination via Immune-suppressive Cells in the Tumor Microenvironment.

BACKGROUND/AIM: Metformin, a drug for type 2 diabetes, also exerts anticancer effects. This study addressed the immunological effects of metformin on peritoneal dissemination. MATERIALS AND METHODS: We developed a mouse model of peritoneal dissemination via intraperitoneal injection of RLmale1, an X-ray-induced leukemia cell line, into BALB/c mice. Cell-surface markers, cytokine production, and myeloid-derived suppressor cells (MDSCs) were examined in cells from spleen and peritoneal lavage fluid. RESULTS: Metformin-treated mice exhibited suppressed intraperitoneal tumor growth and extended survival, and these effects were lost in mice with severe combined immunodeficiency. MDSCs induction was inhibited in metformin-treated mice. Although MDSC mobilization into the peritoneal cavity was correlated with suppression of interferon-γ production by tumor-infiltrating lymphocytes, the T-helper 1 ability of these lymphocytes was preserved in metformin-treated mice. CONCLUSION: Our findings demonstrate the action of metformin on both intraperitoneal tumors and immune-suppressive cells and might contribute to the development of immunotherapy against peritoneal dissemination.

Berberine improved experimental chronic colitis by regulating interferon-γ- and IL-17A-producing lamina propria CD4+ T cells through AMPK activation.

The herbal medicine berberine (BBR) has been recently shown to be an AMP-activated protein kinase (AMPK) productive activator with various properties that induce anti-inflammatory responses. We investigated the effects of BBR on the mechanisms of mucosal CD4+T cell activation in vitro and on the inflammatory responses in T cell transfer mouse models of inflammatory bowel disease (IBD). We examined the favorable effects of BBR in vitro, using lamina propria (LP) CD4+ T cells in T cell transfer IBD models in which SCID mice had been injected with CD4+CD45RBhigh T cells. BBR suppressed the frequency of IFN-γ- and Il-17A-producing LP CD4+ T cells. This effect was found to be regulated by AMPK activation possibly induced by oxidative phosphorylation inhibition. We then examined the effects of BBR on the same IBD models in vivo. BBR-fed mice showed AMPK activation in the LPCD4+ T cells and an improvement of colitis. Our study newly showed that the BBR-induced AMPK activation of mucosal CD4+ T cells resulted in an improvement of IBD and underscored the importance of AMPK activity in colonic inflammation.

Metformin induces CD11b+-cell-mediated growth inhibition of an osteosarcoma: implications for metabolic reprogramming of myeloid cells and anti-tumor effects.

CD11b+ myeloid subpopulations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), play crucial roles in the suppression of T-cell-mediated anti-tumor immunity. Regulation of these cell types is a primary goal for achieving efficient cancer immunotherapy. We found that metformin (Met) induces CD11b+-cell-mediated growth inhibition of a K7M2neo osteosarcoma independent of T cells, as growth inhibition of K7M2neo was still observed in wild-type (WT) mice depleted of T cells by antibodies and in SCID; this contrasted with the effect of Met on Meth A fibrosarcoma, which was entirely T-cell-dependent. Moreover, the inhibitory effect seen in SCID was abrogated by anti-CD11b antibody injection. PMN-MDSCs were significantly reduced in both spleens and tumors following Met treatment. In TAMs, production of IL-12 and TNF-α, but not IL-10, became apparent, and elevation of MHC class II with reduction of CD206 was observed, indicating a shift from an M2- to M1-like phenotype via Met administration. Metabolically, Met treatment decreased basal respiration and the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio of CD11b+ cells in tumors, but not in the spleen. In addition, decreased reactive oxygen species (ROS) production and proton leakage in MDSCs and TAMs were consistently observed in tumors. Uptake of both 2-deoxy-2-d-glucose (2-NBDG) and BODIPY® decreased in MDSCs, but only BODIPY® incorporation was decreased in TAMs. Overall, our results suggest that Met redirects the metabolism of CD11b+ cells to lower oxidative phosphorylation (OXPHOS) while elevating glycolysis, thereby pushing the microenvironment to a state that inhibits the growth of certain tumors.

Study Protocol: Phase-Ib Trial of Nivolumab Combined With Metformin for Refractory/Recurrent Solid Tumors.

Although immune checkpoint inhibitors have shown significant survival benefits in the treatment of several cancers, optimal outcomes have been limited to certain subsets of patients. In a previous study, we found that the addition of metformin to nivolumab, an anti-programmed cell death protein 1 (PD-1) antibody, yielded substantial tumor regression in mouse models. Further analysis revealed that the number of tumor-infiltrating CD8 T cells had increased markedly. Based on this result, we have launched an investigator-initiated open-label phase-Ib clinical trial. The objectives of this trial are to investigate the safety, efficacy, and pharmacokinetics of a metformin-nivolumab combination treatment. This study consists of 2 parts. The recommended dose of metformin combined with nivolumab is determined in part 1. The safety and efficacy of the optimal dose of metformin to be delivered in conjunction with nivolumab are examined in part 2. Patient eligibility is based on the following criteria: pathologic diagnosis of refractory/recurrent solid tumor (part 1), and non-small-cell lung cancer or pancreatic cancer refractory to standard primary treatment (part 2); no prior use of immune checkpoint inhibitor; performance status 0 or 1; age ≥ 20 years; and adequate organ function. The primary endpoints are safety in part 1 and safety and pharmacokinetics in part 2. The maximum tolerated dose and recommended dose are determined in part 1 by the 3 + 3 cohort method, and the dose-limiting toxicity evaluation period for each patient is 4 weeks from the start of administration. In part 2, metformin is administered at the optimal dose determined in part 1. Total enrollment is 9 to 18 patients for part 1 and 30 patients for part 2. Enrollment began in 2017, and will be completed by 2019. The University Hospital Medical Information Network registration number for this study is 000028405.

Extracellular and Non-Chaperone Function of Heat Shock Protein-90α Is Required for Skin Wound Healing.

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.

Metformin Promotes the Protection of Mice Infected With Plasmodium yoelii Independently of γδ T Cell Expansion.

Adaptive immune responses are critical for protection against infection with Plasmodium parasites. The metabolic state dramatically changes in T cells during activation and the memory phase. Recent findings suggest that metformin, a medication for treating type-II diabetes, enhances T-cell immune responses by modulating lymphocyte metabolism. In this study, we investigated whether metformin could enhance anti-malaria immunity. Mice were infected with Plasmodium yoelii and administered metformin. Levels of parasitemia were reduced in treated mice compared with those in untreated mice, starting at ~2 weeks post-infection. The number of γδ T cells dramatically increased in the spleens of treated mice compared with that in untreated mice during the later phase of infection, while that of αß T cells did not. The proportions of Vγ1+ and Vγ2+ γδ T cells increased, suggesting that activated cells were selectively expanded. However, these γδ T cells expressed inhibitory receptors and had severe defects in cytokine production, suggesting that they were in a state of exhaustion. Metformin was unable to rescue the cells from exhaustion at this stage. Depletion of γδ T cells with antibody treatment did not affect the reduction of parasitemia in metformin-treated mice, suggesting that the effect of metformin on the reduction of parasitemia was independent of γδ T cells.

Metformin Mediates Protection against Legionella Pneumonia through Activation of AMPK and Mitochondrial Reactive Oxygen Species.

In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow-derived macrophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila-infected bone marrow-derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase-derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macrophages, which may contribute to improved survival in L. pneumophila pneumonia.

[Metabolic Competition in Tumor Microenvironment].

Metabolic pathways tightly regulate T cell response in host defense against infection and cancer. Glycolysis plays a key role in effector T cell differentiation and its function. More recent studies have demonstrated that tumor microenvironment forms hypoxia and metabolic disadvantage of immune cells. These environmental attributions impair the effector T cell survival, proliferation and function. Therefore repurposing of metabolic drugs might develop a novel cancer immunotherapy based on the targeting of T cell immunometabolism. In this review, we abridge basis of cancer cell and T cell metabolism and discuss recent advances elucidating "metabolic competition" exerted on tumor-infiltrating T cells that drive their dysfunction in tumor microenvironment.

Attenuation of CD4+CD25+ Regulatory T Cells in the Tumor Microenvironment by Metformin, a Type 2 Diabetes Drug.

CD4+CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity.