The human G protein-coupled ATP receptor P2Y11 is a target for anti-inflammatory strategies.

BACKGROUND AND PURPOSE: The ATP receptor P2Y11 , which couples to Gq and Gs proteins, senses cell stress and promotes cytoprotective responses. P2Y11 receptors are upregulated during differentiation of M2 macrophages. However, it is unclear whether and how P2Y11 receptors contribute to the anti-inflammatory properties of M2 macrophages. EXPERIMENTAL APPROACH: Transcriptome and secretome profiling of ectopic P2Y11 receptors was used to analyse their signalling and function. Findings were validated in human monocyte-derived M2 macrophages. The suramin analogue NF340 and P2Y11 receptor-knockout cells confirmed that agonist-mediated responses were specific to P2Y11 receptor stimulation. KEY RESULTS: Temporal transcriptome profiling of P2Y11 receptor stimulation showed a strong and tightly controlled response of IL-1 receptors, including activation of the IL-1 receptor target genes, IL6 and IL8. Secretome profiling confirmed the presence of IL-6 and IL-8 proteins and additionally identified soluble tumour necrosis factor receptor 1 and 2 (sTNFR1 and sTNFR2) as targets of P2Y11 receptor activation. Raised levels of intracellular cAMP in M2 macrophages, after inhibition of phosphodiesterases (PDE), especially PDE4, strongly increased P2Y11 receptor-induced release of sTNFR2 through ectodomain shedding mediated by TNF-α converting enzyme (TACE/ADAM17). Both IL-1α and IL-1ß synergistically enhanced P2Y11 receptor- induced IL-6 and IL-8 secretion and release of sTNFR2. During lipopolysaccharide-induced activation of TLR4, which shares the downstream signalling pathway with IL-1 receptors, P2Y11 receptors specifically prevented secretion of TNF-α. CONCLUSIONS AND IMPLICATIONS: Targeting P2Y11 receptors activates IL-1 receptor signalling to promote sTNFR2 release and suppress TLR4 signalling to prevent TNF-α secretion, thus facilitating resolution of inflammation.

Rare, but Severe: Vasculitis and Checkpoint Inhibitors.

In the last years, immunotherapy has become a mainstay of cancer treatment. Owing to its increasing application in clinical practice, novel and rare, but severe, immune-related adverse events such as vasculitis are now being described more frequently. Vasculitis occurs as part of a primary immune disorder but might be induced additionally by substances such as checkpoint inhibitors, which boost the immune system, and thus can also appear as an immune-related adverse event. Several lines of evidence indicate that checkpoint proteins such as programmed death-1 (PD-1) play a major role in the pathophysiology of vasculitis. Such immune checkpoints serve to prevent autoimmunity and to maintain tolerance. We present a case of pronounced vasculitis in a patient with metastatic urothelial carcinoma who received pembrolizumab, and discuss potential pathomechanisms regarding how checkpoint inhibitors can mediate immune-related vascular toxicity. PATIENT SUMMARY: In this report, we looked at potential interactions between checkpoint inhibitors and immune-associated vascular adverse events. Immunotherapy-induced changes of the immune status can favor the occurrence of vascular disease, being fatal in most cases. We conclude that the risk of progressive vascular damage and identification of patients with pre-existing vascular disease should always be borne in mind when treating patients with immunotherapy.

The Human G Protein-Coupled ATP Receptor P2Y11 Is Associated With IL-10 Driven Macrophage Differentiation.

The G protein-coupled P2Y11 receptor is known to sense extracellular ATP during inflammatory and immune responses. The dinucleotide NAD+ has also been proposed to be a P2Y11 receptor ligand but its role is less clear. Here, we have examined for the first time human P2Y11 receptor protein levels and show that the receptor was upregulated during polarization of M2 macrophages. IL-10 reinforced P2Y11 receptor expression during differentiation of M2c macrophages expressing CD163, CD16, and CD274 (PD-L1). Nutlin-3a mediated p53 stabilization further increased P2Y11 receptor, CD16, and PD-L1 expression. AMP-activated kinase (AMPK), which mediates anti-inflammatory effects of IL-10, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, which is under the control of AMPK, were also required for P2Y11 receptor expression. The P2Y11 receptor agonist ATPγS and NAD+ could independently stimulate the production of IL-8 in M2 macrophages, however, only the ATPγS-induced response was mediated by P2Y11 receptor. Both in a recombinant system and in macrophages, P2Y11 receptor-driven IL-8 production predominantly depended on IkB kinase (IKK), and extracellular signal-regulated kinase (ERK). In conclusion, our data indicate that an AMPK-NAMPT-NAD+ signaling axis promotes P2Y11 receptor expression during M2 polarization of human macrophages in response to IL-10. PD-L1 expressing M2c macrophages that secrete the cancer-promoting chemokine IL-8 in response to P2Y11 receptor stimulation may represent an important target in checkpoint blockade immunotherapy.

Mevalonate Metabolism in Cancer Stemness and Trained Immunity.

Mevalonate metabolism provides cancer and immune cells with diverse products to ensure cell functionality. Similar metabolic reprogramming that raises mevalonate metabolism to higher levels appears to drive both, epithelial mesenchymal transition (EMT) of cancer cells, a reverse differentiation program that generates cancer cells with stem cell properties, and immune cell training for increased responsiveness to secondary stimulation. In this review, we address how mevalonate metabolism supports cancer development and stemness on the one hand, and on the other promotes immune responsiveness. In view of this dual nature of mevalonate metabolism, strategies to manipulate this metabolic pathway as part of anti-cancer therapies require careful analysis of risks versus benefits.

Mevalonate Metabolism in Immuno-Oncology.

Immuno-oncology not only refers to the multifaceted relationship between our immune system and a developing cancer but also includes therapeutic approaches that harness the body's immune system to fight cancer. The recognition that metabolic reprogramming governs immunity was a key finding with important implications for immuno-oncology. In this review, we want to explore how activation and differentiation-induced metabolic reprogramming affects the mevalonate pathway for cholesterol biosynthesis in immune and cancer cells. Glycolysis-fueled mevalonate metabolism is a critical pathway in immune effector cells, which may, however, be shared by cancer stem cells, complicating the development of therapeutic strategies. Additional engagement of fatty acidy oxidation, as it occurs in regulatory immune cells as well as in certain tumor types, may influence mevalonate pathway activity. Transcellular mevalonate metabolism may play an as yet unanticipated role in the crosstalk between the various cell types and may add another level of complexity. In humans, a subset of γδ T cells is specifically adapted to perform surveillance of mevalonate pathway dysregulation. While the mevalonate pathway remains an important target in immuno-oncology, in terms of personalized medicine, it may be the type or stage of a malignant disease that determines whether mevalonate metabolism requires training or attenuation.

Mevalonate metabolism governs cancer immune surveillance.

The metabolic reprogramming that drives immunity engages the mevalonate pathway for cholesterol biosynthesis and protein prenylation. The importance of tight regulation of this metabolic route is reflected by the fact that too low activity impairs cellular function and survival, whereas hyperactivity can lead to malignant transformation. Here, we first address how mevalonate metabolism drives immunity and then highlight ways of the immune system to respond to both, limited and uncontrolled flux through the mevalonate pathway. Immune responses elicited by mevalonate pathway dysregulation may be harnessed to increase the clinical efficacy of current cancer therapy regimens.

Intratumoral Th2 predisposition combines with an increased Th1 functional phenotype in clinical response to intravesical BCG in bladder cancer.

Th1-type immunity is considered to be required for efficient response to BCG in bladder cancer, although Th2 predisposition of BCG responders has recently been reported. The aim was to evaluate the relationship of Th1 and Th2 components in 23 patients undergoing BCG treatment. Peripheral blood, serum and urine samples were prospectively collected at baseline, during and after BCG. Th1 (neopterin, tryptophan, kynurenine, kynurenine-to-tryptophan ratio (KTR), IL-12, IFN-γ, soluble TNF-R75 and IL-2Rα) and Th2 (IL-4, IL-10) biomarkers as well as CD4 expression in T helper (Th), effector and regulatory T cells were determined. Local immune cell subsets were measured on formalin-fixed, paraffin-embedded cancer tissue by immunohistochemistry to examine expression of transcription factors that control Th1 (T-bet) and Th2-type (GATA3) immunity. We confirmed a Th2 predisposition with a mean GATA3/T-bet ratio of 5.51. BCG responders showed significantly higher levels of urinary (p = 0.003) and serum neopterin (p = 0.012), kynurenine (p = 0.015), KTR (p = 0.005), IFN-γ (p = 0.005) and IL-12 (p = 0.003) during therapy, whereas levels of IL-10 decreased significantly (p < 0.001) compared to non-responders. GATA3/T-bet ratio correlated positively with serum neopterin (p = 0.008), IFN-γ (p = 0.013) and KTR (p = 0.018) after the first BCG instillation. We observed a significant increase in CD4 expression in the Th cell population (p < 0.05), with only a modest tendency toward higher frequency in responders compared to non-responders (p = 0.303). The combined assessment of GATA3/T-bet ratio, neopterin and KTR may be a useful biomarker in predicting BCG response. Th2-promoting factors such as GATA3 may trigger Th1-type immune responses and thus contribute to the BCG success.

Ecto-ATPase CD39 Inactivates Isoprenoid-Derived Vγ9Vδ2 T Cell Phosphoantigens.

In humans, Vγ9Vδ2 T cells respond to self and pathogen-associated, diphosphate-containing isoprenoids, also known as phosphoantigens (pAgs). However, activation and homeostasis of Vγ9Vδ2 T cells remain incompletely understood. Here, we show that pAgs induced expression of the ecto-ATPase CD39, which, however, not only hydrolyzed ATP but also abrogated the γδ T cell receptor (TCR) agonistic activity of self and microbial pAgs (C5 to C15). Only mevalonate-derived geranylgeranyl diphosphate (GGPP, C20) resisted CD39-mediated hydrolysis and acted as a regulator of CD39 expression and activity. GGPP enhanced macrophage differentiation in response to the tissue stress cytokine interleukin-15. In addition, GGPP-imprinted macrophage-like cells displayed increased capacity to produce IL-1ß as well as the chemokine CCL2 and preferentially activated CD161-expressing CD4(+) T cells in an innate-like manner. Our studies reveal a previously unrecognized immunoregulatory function of CD39 and highlight a particular role of GGPP among pAgs.

T lymphocyte regulation by mevalonate metabolism.

Whereas resting T cells, which have low metabolic requirements, use oxidative phosphorylation (OXPHOS) to maximize their generation of ATP, activated T cells, similar to tumor cells, shift metabolic activity to aerobic glycolysis, which also fuels mevalonate metabolism. Both sterol and nonsterol derivatives of mevalonate affect T cell function. The intracellular availability of sterols, which is dynamically regulated by different classes of transcription factors, represents a metabolic checkpoint that modulates T cell responses. The electron carrier ubiquinone, which is modified with an isoprenoid membrane anchor, plays a pivotal role in OXPHOS, which supports the proliferation of T cells. Isoprenylation also mediates the plasma membrane attachment of the Ras, Rho, and Rab guanosine triphosphatases, which are involved in T cell immunological synapse formation, migration, proliferation, and cytotoxic effector responses. Finally, multiple phosphorylated mevalonate derivatives can act as danger signals for innate-like γδ T cells, thus contributing to the immune surveillance of stress, pathogens, and tumors. We highlight the importance of the mevalonate pathway in the metabolic reprogramming of effector and regulatory T cells.

Mevalonate metabolism in cancer.

Cancer cells are characterized by sustained proliferative signaling, insensitivity to growth suppressors and resistance to apoptosis as well as by replicative immortality, the capacity to induce angiogenesis and to perform invasive growth. Additional hallmarks of cancer cells include the reprogramming of energy metabolism as well as the ability to evade immune surveillance. The current review focuses on the metabolic reprogramming of cancer cells and on the immune system's capacity to detect such changes in cancer cell metabolism. Specifically, we focus on mevalonate metabolism, which is a target for drug and immune based cancer treatment.

Stress-related and homeostatic cytokines regulate Vγ9Vδ2 T-cell surveillance of mevalonate metabolism.

The potentially oncogenic mevalonate pathway provides building blocks for protein prenylation and induces cell proliferation and as such is an important therapeutic target. Among mevalonate metabolites, only isopentenyl pyrophosphate (IPP) has been considered to be an immunologically relevant antigen for primate-specific, innate-like Vγ9Vδ2 T cells with antitumor potential. We show here that Vγ9Vδ2 T cells pretreated with the stress-related, inflammasome-dependent cytokine interleukin 18 (IL-18) were potently activated not only by IPP but also by all downstream isoprenoid pyrophosphates that exhibit combined features of antigens and cell-extrinsic metabolic cues. Vγ9Vδ2 T cells induced this way effectively proliferated even under severe lymphopenic conditions and the antioxidant N-acetylcysteine significantly improved reconstitution of γδ T cells predominantly with a central memory phenotype. The homeostatic cytokine IL-15 induced the differentiation of effector cells in an antigen-independent fashion, which rapidly produced abundant interferon γ (IFNγ) upon antigen re-encounter. IL-15 induced effector γδ T cells displayed increased levels of the cytotoxic lymphocyte-associated proteins CD56, CD96, CD161 and perforin. In response to stimulation with isoprenoid pyrophosphates, these effector cells upregulated surface expression of CD107a and exhibited strong cytotoxicity against tumor cells in vitro. Our data clarify understanding of innate immunosurveillance mechanisms and will facilitate the controlled generation of robust Vγ9Vδ2 T cell subsets for effective cancer immunotherapy.

Essential requirements of zoledronate-induced cytokine and γδ T cell proliferative responses.

The potent nitrogen-containing bisphosphonate zoledronate inhibits farnesyl pyrophosphate synthase, a key enzyme of the mevalonate pathway that is often hyperactive in malignant cells. Zoledronate activates human Vγ9Vδ2 T cells, which are immune sentinels of cell stress and tumors, through upstream accumulation of the cognate Ag isopentenyl pyrophosphate. IL-18 was shown to enhance zoledronate-induced γδ T cell activation. Although monocytes have been considered important accessory cells that provide the Ag isopentenyl pyrophosphate, CD56(bright)CD11c(+) NK cells were postulated to mediate the costimulatory effects of IL-18. We report in this article that downstream depletion of geranylgeranyl pyrophosphate (GGPP), which is required for protein prenylation, caused cell stress in monocytes, followed by caspase-1-mediated maturation and release of IL-18, which, in turn, induced γδ T cell CCL2. Likewise, zoledronate caused a substantial delay in γδ T cell expansion, which could be skipped by GGPP supplementation. Moreover, repletion of GGPP, which prevented acute zoledronate toxicity, and supplementation with IL-18, which strongly upregulated IL-2Rα (CD25) and favored the central memory phenotype, were sufficient to enable zoledronate-induced expansion of highly purified γδ T cells, even when starting cell numbers were as low as 10(4) γδ T cells. Our study reveals essential components of γδ T cell activation and indicates that exogenous IL-18, which can directly costimulate γδ T cells, eliminates the need for any accessory cells. Our findings will facilitate the generation of robust γδ T cells from small blood or tissue samples for cancer immunotherapy and immune-monitoring purposes.

Regulation of mevalonate metabolism in cancer and immune cells.

The mevalonate pathway is a highly conserved metabolic cascade and provides isoprenoid building blocks for the biosynthesis of vital cellular products such as cholesterol or prenyl pyrophosphates that serve as substrates for the posttranslational prenylation of numerous proteins. The pathway, which is frequently hyperactive in cancer cells, is considered an important target in cancer therapy, since prenylated members of the Ras superfamily are crucially involved in the control of proliferation, survival, invasion and metastasis of tumour cells. Upstream accumulation and downstream depletion of mevalonate pathway intermediates as induced for instance by aminobisphosphonates translate into different effects in cancer and immune cells. Thus, mevalonate pathway regulation can affect tumour biology either directly or exhibit indirect antitumour effects through stimulating cancer immune surveillance. The present review summarizes major effects of pharmacologic mevalonate pathway regulation in cancer and immune cells that may collaboratively contribute to the efficacy of cancer therapy.

Novel aspects of mevalonate pathway inhibitors as antitumor agents.

The mevalonate pathway for cholesterol biosynthesis and protein prenylation has been implicated in various aspects of tumor development and progression. Certain classes of drugs, such as statins and bisphosphonates, inhibit mevalonate metabolism and therefore have also been tested as antitumor agents. This concept is strongly supported by the recent finding that mutant p53, which is present in more than half of all human cancers, can significantly upregulate mevalonate metabolism and protein prenylation in carcinoma cells. The first evidence that mevalonate pathway inhibitors may have the potential to reverse the malignant phenotype has already been obtained. Moreover, recently discovered immunomodulatory properties of statins and bisphosphonates may also contribute to their known anticancer effects. Drug-induced inhibition of protein prenylation may induce sequential cellular stress responses, including the unfolded protein response and autophagy, that eventually translate into inflammasome-dependent and caspase-1-mediated activation of innate immunity. This review focuses on these novel capabilities of mevalonate pathway inhibitors to beneficially affect tumor biology and contribute to tumor immune surveillance.

DC-like cell-dependent activation of human natural killer cells by the bisphosphonate zoledronic acid is regulated by γδ T lymphocytes.

Bisphosphonates are mainly used for the inhibition of osteoclast-mediated bone resorption but also have been shown to induce γδ T-cell activation. Using IL-2-primed cultures of CD56(+) peripheral blood mononuclear cells, we show here that zoledronic acid (zoledronate) could induce IFN-γ production not only in γδ T lymphocytes but, surprisingly, also in natural killer (NK) cells in a manner that depended on antigen-presenting cells, which share properties of inflammatory monocytes and dendritic cells (DCs; here referred to as DC-like cells). In the presence of γδ T lymphocytes, DC-like cells were rapidly eliminated, and NK cell IFN-γ production was silenced. Conversely, in the absence of γδ T lymphocytes, DC-like cells were spared, allowing NK cell IFN-γ production to proceed. γδ T cell-independent NK cell activation in response to zoledronate was because of downstream depletion of endogenous prenyl pyrophosphates and subsequent caspase-1 activation in DC-like cells, which then provide mature IL-18 and IL-1ß for the activation of IL-2-primed NK cells. Pharmacologic inhibition of caspase-1 almost abolished IFN-γ production in NK cells and γδ T lymphocytes, indicating that caspase-1-mediated cytokine maturation is the crucial mechanism underlying innate lymphocyte activation in response to zoledronate.

IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells.

Statins are inhibitors of cholesterol biosynthesis and protein prenylation that also have been studied in cancer therapy and chemoprevention. With regard to natural killer (NK) cells, only inhibitory effects of statins such as suppression of granule exocytosis have been reported so far. In this study, we show that statins can cooperate with IL-2 to potently induce the activation of CD56(dim) NK cells in a synergistic, time- and dose-dependent fashion. Supplementation experiments revealed that the statin effect was specific to inhibition of their target hydroxymethylglutaryl coenzyme A reductase and that downstream depletion of geranylgeranyl pyrophosphate was responsible for cooperating with IL-2 in NK cell activation. Mechanistic studies revealed that CD56(+)HLA-DR(+)CD14(+) dendritic cell (DC)-like accessory cells mediated the ability of statin to activate NK cells. In contrast, BDCA-1(+) (CD1c(+)) myeloid DCs, which partially expressed CD56, were somewhat less potent. Conventional blood monocytes, which lack CD56, exhibited the lowest accessory cell capacity. NK cell IFN-γ production was IL-12 independent but required endogenous IL-18, IL-1ß, and caspase-1 activity. Statins directly induced apoptosis in human cancer cell lines and cooperated with NK cell-derived IFN-γ to generate potent cytotoxic antitumor effects in vitro even in the presence of statin-mediated inhibitory effects on granule exocytosis. Our work reveals novel and unexpected immunomodulatory properties of statins, which might be harnessed for the treatment of cancer.

CD56+ human blood dendritic cells effectively promote TH1-type gammadelta T-cell responses.

CD56+ human dendritic cells (DCs) have recently been shown to differentiate from monocytes in response to GM-CSF and type 1 interferon in vitro. We show here that CD56+ cells freshly isolated from human peripheral blood contain a substantial subset of CD14+CD86+HLA-DR+ cells, which have the appearance of intermediate-sized lymphocytes but spontaneously differentiate into enlarged DC-like cells with substantially increased HLA-DR and CD86 expression or into fully mature CD83+ DCs in response to appropriate cytokines. Stimulation of CD56+ cells containing both DCs and abundant gammadelta T cells with zoledronate and interleukin-2 (IL-2) resulted in the rapid expansion of gammadelta T cells as well as in IFN-gamma, TNF-alpha, and IL-1beta but not in IL-4, IL-10, or IL-17 production. IFN-gamma, TNF-alpha, and IL-1beta production were almost completely abolished by depleting CD14+ cells from the CD56+ subset before stimulation. Likewise, depletion of CD14+ cells dramatically impaired gammadelta T-cell expansion. IFN-gamma production could also be blocked by neutralizing the effects of endogenous IL-1beta and TNF-alpha. Conversely, addition of recombinant IL-1beta, TNF-alpha, or both further enhanced IFN-gamma production and strongly up-regulated IL-6 production. Our data indicate that CD56+ DCs from human blood are capable of stimulating CD56+ gammadelta T cells, which may be harnessed for immunotherapy.