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- Material and Methods
- Supporting Information
The activation of oncogenic signaling pathways induces the reprogramming of glucose metabolism in tumor cells and increases lactic acid secretion into the tumor microenvironment. This is a well-known characteristic of tumor cells, termed the Warburg effect, and is a candidate target for antitumor therapy. Previous reports show that lactic acid secreted by tumor cells is a proinflammatory mediator that activates the IL-23/IL-17 pathway, thereby inducing inflammation, angiogenesis and tissue remodeling. Here, we show that lactic acid, or more specifically the acidification it causes, increases arginase I (ARG1) expression in macrophages to inhibit T-cell proliferation and activation. Accordingly, we hypothesized that counteraction of the immune effects by lactic acid might suppress tumor development. We show that dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinases, targets macrophages to suppress activation of the IL-23/IL-17 pathway and the expression of ARG1 by lactic acid. Furthermore, lactic acid-pretreated macrophages inhibited CD8+ T-cell proliferation, but CD8+ T-cell proliferation was restored when macrophages were pretreated with lactic acid and DCA. DCA treatment decreased ARG1 expression in tumor-infiltrating immune cells and increased the number of IFN-γ-producing CD8+ T cells and NK cells in tumor-bearing mouse spleen. Although DCA treatment alone did not suppress tumor growth, it increased antitumor immunotherapeutic activity of Poly(IC) in both CD8+ T cell- and NK cell-sensitive tumor models. Therefore, DCA acts not only on tumor cells to suppress glycolysis but also on immune cells to improve the immune status modulated by lactic acid and to increase the effectiveness of antitumor immunotherapy.
Many types of immune cells infiltrate tumors. Although these immune cells were classically thought to attack and eliminate tumors, recent studies indicate that they actually induce inflammation within tumors, thereby promoting tumor progression by inducing angiogenesis and tissue remodeling within the tumor microenvironment and tumor invasion and metastasis.[1, 2] Furthermore, immune cells such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), all of which have potent suppressive effects on anticancer immune responses, are also recruited to tumors.[3, 4]
We previously showed that lactic acid secreted by tumor cells enhances the production of IL-23 by monocytes/macrophages stimulated with Toll-like receptor (TLR) ligands. Furthermore, lactic acid acts on monocytes/macrophages to increase antigen-dependent production of IL-17A by effector/memory CD4+ T cells, but suppresses differentiation from naïve to Th17 T cells and inhibits CD4+ T-cell proliferation. IL-23 is an inflammatory cytokine overproduced by tumors, which induces angiogenesis and reduces the infiltration of cytotoxic T lymphocytes into the tumor microenvironment to facilitate tumor growth.[7-9] Therefore, we propose that lactic acid functions as a proinflammatory mediator rather than just a terminal metabolite of anaerobic glycolysis. Recent reports show that lactic acid is an immunosuppressive factor that inhibits the function of dendritic cells (DCs) and cytotoxic T cells.[10, 11] Diclofenac is reported to downregulate lactic acid production and to counteract local immunosuppression by affecting intratumor DCs and Tregs. Lactic acid also acts on endothelial cells and fibroblasts to provide an environment that promotes tumor growth and motility.[13-17] Furthermore, many types of tumor cell produce large amounts of lactic acid; indeed, high concentrations of lactic acid are correlated with distant metastasis and poor prognosis of head and neck, cervical and colorectal cancers. Thus, increasing evidence suggests that lactic acid is a tumor-derived mediator that modulates the tumor microenvironment to promote tumor progression.[13-15]
Cells usually import glucose into the cytoplasm, where it is metabolized to pyruvate. Under aerobic conditions, pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex in the mitochondria and metabolized to CO2, H2O and energy metabolites through the tricarboxylic acid cycle. Tumor cells show increased glucose uptake and lactate production even under normoxic conditions, resulting in a state known as the “Warburg effect.”[19, 20] This reprogramming of glucose metabolism is mediated by activation of canonical oncogenic signaling pathways, including the phosphatidylinositol 3-kinase-AKT pathway, c-Myc, hypoxia-inducible transcriptional factor-1 α, AMP-activated protein kinase and mammalian target of rapamycin.[21, 22] Therefore, glycolytic enzymes that are specifically and commonly activated in cancer cells are expected to be good targets for antitumor chemotherapy. We hypothesized that agents targeting glycolysis in tumor cells may also inhibit the activation of IL-23/IL-17 inflammatory pathway by lactic acid in immune cells to improve the immune status in tumor-bearing patients.
Therefore, this study examined the effects of antiglycolysis agents on immune responses in tumor-bearing mice. We show that dichloroacetate (DCA), which is an inhibitor of PDH kinases (PDKs), is one candidate drug that improves the immune status of tumor-bearing individuals, and enhances the effects of antitumor immunotherapy.
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- Material and Methods
- Supporting Information
This study showed that tumor-secreted lactic acid promotes not only the IL-23/IL-17 inflammatory pathway but also ARG1 expression in macrophages. These effects were suppressed by DCA. Furthermore, DCA treatment suppressed arginase activity in tumor-infiltrating immune cells and enhanced the effects of antitumor immunotherapy with Poly(I:C) in vivo.
ARG1 is an enzyme that metabolizes l-arginine into l-ornithine and urea, and is highly expressed by tumor-associated myeloid cells, including TAMs and MDSCs. Depletion of l-arginine from the tumor microenvironment via ARG1-dependent consumption causes suppression of T-cell activation and proliferation.[3, 35] Accordingly, ARG1 plays an important role in tumor immune escape mechanisms. In this study, we show that lactic acid-pretreated macrophages have increased ARG1 expression and can suppress antigen-specific CD8+ T-cell proliferation. Previous reports have shown that lactic acid inhibits the differentiation of monocytes into DCs and decreases cytokine release from DCs, monocytes and cytotoxic T cells. Here, we identify a new immunosuppressive property of lactic acid: the induction of ARG1 expression in macrophages. We propose that lactic acid acts on myeloid cells to promote tumor development in two independent ways: suppression of T-cell activation and proliferation via an increase in the expression of ARG1, and induction of inflammation through activation of the IL-23/IL-17 pathway.
Interestingly, when macrophages were transiently stimulated by lactic acid, they suppressed CD8+ T-cell proliferation in the absence of lactic acid. This indicates that macrophages are converted into immunosuppressive cells by lactic acid. Therefore, macrophages exposed to high levels of lactic acid within the tumor microenvironment acquire immunosuppressive activity and suppress T-cell proliferation in both the tumor microenvironment and secondary lymphoid tissues.
DCA facilitates PDH activity, leading to a metabolic shift from glycolysis to glucose oxidation. DCA is used clinically to treat patients with lactic acidemia, including PDH complex deficiency and mitochondrial encephalomyopathy.[31, 32, 41, 42] The potential of DCA as an anticancer therapy has been the subject of many clinical and laboratory studies.[43-49] DCA decreases mitochondrial membrane potential, increases the levels of mitochondrial H2O2, and activates potassium channels, leading to inhibition of proliferation and induction of apoptosis in cancer cell lines. However, most reports to date have evaluated the direct effects of DCA on cancer cells. To our knowledge, this is the first report to evaluate the effect of DCA on immune cells in tumor-bearing mice.
In vitro, DCA suppressed the IL-23/IL-17 pathway and lactic acid-induced ARG1 expression in splenocytes and macrophages, but had no effect in the absence of lactic acid. Macrophages pretreated with lactic acid plus DCA increased antigen-specific CD8+ T-cell proliferation compared with macrophages pretreated with lactic acid alone. DCA rescued the immunosuppressive phenotype of macrophages treated with lactic acid. In vivo, DCA treatment decreased ARG1 expression in tumor-infiltrating immune cells and increased the number of IFN-γ-producing CD8+ T cells and NK cells in tumor-bearing mouse splenocytes. Splenocytes derived from DCA-treated mice also increased IFN-γ production induced by Poly(I:C) stimulation. Therefore, DCA treatment may cancel the immunosuppressive effects of lactic acid on tumor-associated myeloid cells and increase antitumor immunoreactivity. In fact, DCA treatment enhanced both the CD8+ T cell- and NK cell-dependent effects of antitumor immunotherapy with Poly(I:C). Interestingly, macrophages pretreated with lactic acid and a high concentration of DCA enhanced CD8+ T-cell proliferation to a much greater extent than untreated macrophages. Thus, DCA may enhance the activity of macrophages that induce CD8+ T-cell proliferation, in addition to counteracting the immunosuppressive effects of lactic acid. This study did not provide any evidence that DCA suppresses the activation of the IL-23/IL-17 pathway in vivo. Therefore, it will be necessary to assess the effect of DCA on the IL-23/IL-17 pathway using other tumor models.
Because tumor cells increase glycolysis and produce a large amount of lactic acid via the Warburg effect, DCA effectively suppressed lactic acid production in tumor cells in vitro, as reported previously.[43, 45, 48] In EG7-bearing mice, DCA also decreased the concentration of lactic acid within tumors in vivo, resulting in loss of the effects of lactic acid on myeloid cells. In contrast, DCA suppressed the effects of lactic acid on macrophages without affecting the level of lactic acid in the surrounding environment in vitro. In B16-bearing mice, DCA did not decrease the concentration of lactic acid within tumors in vivo, but decreased ARG1 expression in tumor-infiltrating immune cells. These results suggest that DCA synergistically suppresses the effects of lactic acid in vivo by two independent mechanisms. First, DCA decreases lactic acid levels in tumor cells, which thereby indirectly suppresses ARG1 expression in tumor-infiltrating myeloid cells. Second, DCA directly acts on myeloid cells to suppress ARG1 expression in these cells.
HCl and lactic acid promoted ARG1 expression and the inhibition of CD8+ cell proliferation by macrophages, suggesting that acidification and not the lactate anion itself is the causative factor. Moreover, DCA suppressed these effects of HCl and lactic acid. As tumor-secreted lactic acid predominantly contributes to acidification within tumors, it is suggested that ARG1 expression in tumor-infiltrating myeloid cells is mainly induced by lactic acid. A previous report indicated that both lactic acid and acidification are involved in the suppression of tumor necrosis factor (TNF) secretion by human monocytes. Acidification also reduces cytokine production such as that of TNF by mouse macrophage via a member of the ovarian cancer G-protein coupled receptor 1 family, T-cell death-associated gene 8. Thereby, not only the lactate anion but also acidification plays an important role in the determination of immune status in tumor-bearing hosts.
We have not yet clarified the molecular mechanisms by which lactic acid induces IL-23 and ARG1 expression in myeloid cells or how DCA suppresses these effects. Different mechanisms probably mediate lactic acid-induced IL-23 and ARG1 expression in myeloid cells as acidification increased the expression of ARG1 but not that of IL-23. DCA also suppressed ARG1 expression induced by lactic acid and the TLR ligand but not that induced by IL-4. A previous report showed that the TLR ligand and IL-4 activate different signaling pathways to induce ARG1 expression. Therefore, ARG1 expression induced by lactic acid and IL-4 could be induced via the activation of different signaling pathways. DCA suppressed the phosphorylation of PDH-E1α at all three serine residues, whereas the degree of phosphorylation induced by lactic acid was different at each residue. Although further studies will be necessary to elucidate how lactic acid regulates phosphorylation, this result suggests that lactic acid increases IL-23p19 and ARG1 expression without the need for direct activation of PDK, which is inhibited by DCA. It is possible that DCA might directly affect other targets involved in the lactic acid signaling pathway or inhibit the IL-23p19 and ARG1 expression independently of the lactic acid signaling pathway. Because anticancer therapies targeting lactic acid production and the lactic acid signaling pathway are now receiving increasing attention, it will be important to elucidate the molecular mechanisms underlying lactic acid signaling pathways.