The HIF-1 transcription complex is essential for translational control of myeloid hematopoietic cell function by maintaining mTOR phosphorylation.

Mammalian myeloid cells are crucial effectors of host innate immune defense. Normal and pathological responses of these cells require adaptation to signaling stress through the hypoxia-inducible factor 1 (HIF-1) transcription complex. Adapted cells activate the mammalian target of rapamycin (mTOR), via S2448 phosphorylation, which induces de novo translation of vital signaling proteins. However, the molecular mechanisms underlying this signaling dogma remain unclear. Here, we demonstrate for the first time that inactivation of HIF-1, by silencing its inducible alpha subunit, significantly decreases mTOR S2448 phosphorylation caused by ligand-dependent activation of human myeloid leukemia cells. This shows that HIF-1 is essential for the activation of mTOR and serves at a crucial juncture of myeloid cell function in both in vitro and in vivo systems.

Gold nanoparticles downregulate interleukin-1ß-induced pro-inflammatory responses.

Interleukin 1 beta (IL-1ß)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1ß-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1ß both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1ß, thus opening potentially novel options for further therapeutic applications.

T lymphocytes induce human cancer cells derived from solid malignant tumors to secrete galectin-9 which facilitates immunosuppression in cooperation with other immune checkpoint proteins.

BACKGROUND: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. METHODS: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. RESULTS: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. CONCLUSION: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes.

Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1ß.

Inflammatory reactions to ssRNA viruses are induced by the endosomal Toll-like receptors (TLRs) 7 and 8. TLR7/8-mediated inflammatory reaction results in activation of the Nalp3 inflammasome via an unknown mechanism. Here we report for the first time that TLR7/8 mediate activation of xanthine oxidase (XOD) in an HIF-1α-dependent manner. XOD produces uric acid and reactive oxygen species, which could activate Nalp3 and therefore induce activation of caspase 1, known to convert inactive pro-IL-1ß into active IL-1ß. Specific inhibition of the XOD activity attenuates TLR7/8-mediated activation of caspase 1 and IL-1ß release. These results were obtained using human THP-1 myeloid macrophages. The findings were verified by conducting in vivo experiments on mice.

Expression of the Immune Checkpoint Protein VISTA Is Differentially Regulated by the TGF-ß1 - Smad3 Signaling Pathway in Rapidly Proliferating Human Cells and T Lymphocytes.

Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-ß)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-ß depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes.

Functional role of galectin-9 in directing human innate immune reactions to Gram-negative bacteria and T cell apoptosis.

Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing ß-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines - interleukin (IL) 6, IL-1ß and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, "opsonisation" of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.

High Mobility Group Box 1 (HMGB1) Induces Toll-Like Receptor 4-Mediated Production of the Immunosuppressive Protein Galectin-9 in Human Cancer Cells.

High mobility group box 1 (HMGB1) is a non-histone protein which is predominantly localised in the cell nucleus. However, stressed, dying, injured or dead cells can release this protein into the extracellular matrix passively. In addition, HMGB1 release was observed in cancer and immune cells where this process can be triggered by various endogenous as well as exogenous stimuli. Importantly, released HMGB1 acts as a so-called "danger signal" and could impact on the ability of cancer cells to escape host immune surveillance. However, the molecular mechanisms underlying the functional role of HMGB1 in determining the capability of human cancer cells to evade immune attack remain unclear. Here we report that the involvement of HMGB1 in anti-cancer immune evasion is determined by Toll-like receptor (TLR) 4, which recognises HMGB1 as a ligand. We found that HGMB1 induces TLR4-mediated production of transforming growth factor beta type 1 (TGF-ß), displaying autocrine/paracrine activities. TGF-ß induces production of the immunosuppressive protein galectin-9 in cancer cells. In TLR4-positive cancer cells, HMGB1 triggers the formation of an autocrine loop which induces galectin-9 expression. In malignant cells lacking TLR4, the same effect could be triggered by HMGB1 indirectly through TLR4-expressing myeloid cells present in the tumour microenvironment (e. g. tumour-associated macrophages).

Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity.

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

Transforming growth factor beta type 1 (TGF-ß) and hypoxia-inducible factor 1 (HIF-1) transcription complex as master regulators of the immunosuppressive protein galectin-9 expression in human cancer and embryonic cells.

Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear. Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-ß1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-ß1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-ß1 signalling pathway as a potential highly efficient target for cancer immunotherapy.

Mitochondrial Defunctionalization Supresses Tim-3-Galectin-9 Secretory Pathway in Human Colorectal Cancer Cells and Thus Can Possibly Affect Tumor Immune Escape.

The Tim-3-galectin-9 secretory pathway is known to protect various types of cancer cells against host immune surveillance. We found that pharmacologically induced mitochondrial dysfunction leads to a reduced galectin-9 expression/exocytosis in human colorectal cancer cells and re-distribution of this protein (the effect described for various cellular proteins) into mitochondria.

Targeting of Basophil and Mast Cell Pro-Allergic Reactivity Using Functionalised Gold Nanoparticles.

Calcineurin inhibitors potentially prevent pro-allergic mediator release from basophils and mast cells but are rarely used systemically due to ubiquitous expressions of target signaling proteins. However, specific targeting of allergic effector cells with these inhibitors could circumvent unwanted side effects. We recently demonstrated the biocompatibility of gold nanoparticles (AuNPs) as a platform for non-toxic delivery of signaling inhibitors due to unique physicochemical properties of these nanomaterials. Since AuNPs can be conjugated with both anti-allergic drugs and antibodies or other proteins that specifically recognize basophils and mast cells, our aims were to assess specific targeting of allergic effector cell function using AuNPs conjugated with the calcineurin inhibitor ascomycin. Purified human basophils and LAD2 human mast cells were used for investigations with AuNPs conjugated either to CD203c antibodies or containing stem cell factor (SCF), respectively, which were amine-coupled to acidic groups of reduced glutathione (GSH). GSH was also used as a spacer for immobilization of ascomycin on the gold surface. AuNPs conjugated with anti-CD203c and ascomycin strikingly blocked IgE-dependent degranulation of both purified basophils and those present in mixed leukocyte preparations, suggesting specific targeting of these cells. In contrast, LAD2 mast cell responses were not inhibited using anti-CD203c-containing nanoconjugates but were when the conjugates contained SCF. Successful targeting of allergic effector cells using gold nanoconjugates indicates that this technology may have therapeutic potential for the treatment of allergies by specifically delivering highly effective signaling inhibitors with reduced side effects.

The Tim-3-Galectin-9 Pathway and Its Regulatory Mechanisms in Human Breast Cancer.

Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.

Protein S-nitrosation in signal transduction: assays for specific qualitative and quantitative analysis.

S-Nitrosation is a type of protein posttranslational modification that has now been found comparable with phosphorylation and acetylation in terms of its contribution to the intracellular signaling networks associated with pathological cell reactions such as host-pathogen interactions, low oxygen availability, cell cycle arrest, and programmed cell death. Therefore, elegant approaches are required to analyze endogenous S-nitrosation of intracellular proteins employed in the regulation of intracellular signal transduction. This chapter describes and discusses recently developed methods that allow both qualitative and quantitative analyses of S-nitrosation of intracellular proteins based on Western blot and enzyme-linked immunosorbent assay.

Biochemical mechanisms implemented by human acute myeloid leukemia cells to suppress host immune surveillance.

Acute myeloid leukaemia (AML) is a blood/bone marrow cancer originating from myeloid cell precusors capable of self-renewing. AML cells implement biochemical mechanisms which allow them not only to survive, but also to successfully escape immune surveillance. ln this work, we discuss crucial molecular mechanisms used by human AML cells in order to evade immune attack.

Highly specific targeting of human acute myeloid leukaemia cells using pharmacologically active nanoconjugates.

In this study we used 5 nm gold nanoparticles as delivery platforms to target cancer cells expressing the immune receptor Tim-3 using single chain antibodies. Gold surfaces were also covered with the cytotoxic drug rapamycin which was immobilised using a glutathione linker. These nanoconjugates allowed highly specific and efficient delivery of cytotoxic rapamycin into human malignant blood cells.

High mobility group box 1 (HMGB1) acts as an "alarmin" to promote acute myeloid leukaemia progression.

High mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1ß) production by healthy leukocytes, leading to IL-1ß-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.

The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

Nitric oxide inhibits HIF-1alpha protein accumulation under hypoxic conditions: implication of 2-oxoglutarate and iron.

Cells exposed to low oxygen conditions respond by initiating defense mechanisms, including the stabilization of hypoxia-inducible factor (HIF) 1alpha, a transcription factor that upregulates genes such as those involved in angiogenesis and glycolysis, which also plays a pivotal role in the regulation of cellular utilization of oxygen and is an essential regulator of angiogenesis in solid tumor and ischemic disorders. Nitric oxide and other inhibitors of mitochondrial respiration prevent the stabilization of HIF-1alpha during hypoxia. In the present study we found that nitric oxide inhibits HIF-1alpha accumulation under low oxygen (1%) conditions. The effect is supported by an increase in 3-nitrotyrosine and is more likely caused by the formation of peroxynitrite in the cells, which leads to the damage of mitochondria and their respiratory chain followed by the increase in 2-oxoglutarate (2-OG) and iron (the components needed to activate HIF-1alpha proline hydroxylases) concentrations in cell cytosol. The inhibiting effect of NO on HIF-1alpha accumulation was not observed in the cells lacking mitochondria. On the other hand the depletion of intracellular glutathione (GSH) was observed upon cell treatment with nitric oxide donors under hypoxic conditions. Treatment of those cells with N-acetyl cysteine (NAC) increased the amount of intracellular GSH and attenuated the NO effect and abolished the damage of mitochondria as well as 2-OG/iron release.

Peroxynitrite as an alternative donor of oxygen in HIF-1alpha proline hydroxylation under low oxygen availability.

In the last years, nitric oxide (NO) mediated signaling became an integral component in understanding physiological and pathophysiological processes of cell proliferation, death or cellular adaptation. Among other activities, NO affects multiple targets that allow regulation of gene expression. Recently, NO was found to attenuate accumulation of hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions because of several mechanisms: redistribution of oxygen toward non-respiratory oxygen-dependent targets (like HIF-1alpha proline hydroxylases--PHDs, which perform hydroxylation of Pro402/564 of HIF-1alpha leading to its proteasomal degradation); in addition, peroxynitrite formed during interactions between NO and mitochondria derived superoxide leads to an increase in cytosolic iron/2-oxoglutarate (2-OG), which required for PHD activation. Here, we propose a hypothesis that peroxynitrite, formed in the cells upon exposure to NO under low oxygen availability, serves as an alternative donor of oxygen for activated PHDs so they can perform HIF-1alpha proline hydroxylation to de-accumulate the protein.