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OBJECTIVE: Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN: We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS: We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION: In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.
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Introduction: Myocardial infarction (MI) is a significant contributor to morbidity and mortality worldwide. Many individuals who survive the acute event continue to experience heart failure (HF), with inflammatory and healing processes post-MI playing a pivotal role. Polymorphonuclear neutrophils (PMN) and monocytes infiltrate the infarcted area, where PMN release high amounts of the heme enzyme myeloperoxidase (MPO). MPO has numerous inflammatory properties and MPO plasma levels are correlated with prognosis and severity of MI. While studies have focused on MPO inhibition and controlling PMN infiltration into the infarcted tissue, less is known on MPO's role in monocyte function. Methods and results: Here, we combined human data with mouse and cell studies to examine the role of MPO on monocyte activation and migration. We revealed a correlation between plasma MPO levels and monocyte activation in a patient study. Using a mouse model of MI, we demonstrated that MPO deficiency led to an increase in splenic monocytes and a decrease in cardiac monocytes compared to wildtype mice (WT). In vitro studies further showed that MPO induces monocyte migration, with upregulation of the chemokine receptor CCR2 and upregulation of inflammatory pathways identified as underlying mechanisms. Conclusion: Taken together, we identify MPO as a pro-inflammatory mediator of splenic monocyte recruitment and activation post-MI and provide mechanistic insight for novel therapeutic strategies after ischemic injury.
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Monócitos , Infarto do Miocárdio , Peroxidase , Animais , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Peroxidase/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Humanos , Camundongos , Masculino , Movimento Celular , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Feminino , Neutrófilos/imunologia , Neutrófilos/metabolismo , Camundongos Knockout , Receptores CCR2/metabolismo , Pessoa de Meia-IdadeRESUMO
Macrophages are major players in myocardial infarction (MI) and atherosclerosis, two major cardiovascular diseases (CVD). Atherosclerosis is caused by the buildup of cholesterol-rich lipoproteins in blood vessels, causing inflammation, vascular injury, and plaque formation. Plaque rupture or erosion can cause thrombus formation resulting in inadequate blood flow to the heart muscle and MI. Inflammation, particularly driven by macrophages, plays a central role in both atherosclerosis and MI. Recent integrative approaches of single-cell analysis-based classifications in both murine and human atherosclerosis as well as experimental MI showed overlap in origin, diversity, and function of macrophages in the aorta and the heart. We here discuss differences and communalities between macrophages in the heart and aorta at steady state and in atherosclerosis or upon MI. We focus on markers, mediators, and functional states of macrophage subpopulations. Recent trials testing anti-inflammatory agents show a major benefit in reducing the inflammatory burden of CVD patients, but highlight a necessity for a broader understanding of immune cell ontogeny and heterogeneity in CVD. The novel insights into macrophage biology in CVD represent exciting opportunities for the development of novel treatment strategies against CVD.
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Aterosclerose , Doenças Cardiovasculares , Infarto do Miocárdio , Placa Aterosclerótica , Humanos , Animais , Camundongos , Doenças Cardiovasculares/etiologia , Macrófagos , Aterosclerose/etiologia , Placa Aterosclerótica/etiologia , Infarto do Miocárdio/complicações , InflamaçãoRESUMO
Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.
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Interleucina-18 , Neoplasias Pancreáticas , Camundongos , Animais , Interleucina-2 , Exaustão das Células T , Receptores de Interleucina-18 , Fator de Transcrição STAT5 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Linfócitos T CD8-Positivos/imunologia , Neoplasias Pancreáticas/genética , Serina-Treonina Quinases TOR , Inflamação , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.
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Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Linfócitos T CD8-Positivos , Fibroblastos Associados a Câncer/metabolismo , Interleucina-17/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Proteínas de Homeodomínio , Neoplasias PancreáticasRESUMO
Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.
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Linfócitos T CD8-Positivos/metabolismo , Ácidos Graxos Voláteis/metabolismo , Fatores Imunológicos/metabolismo , Imunoterapia Adotiva/métodos , Microbiota/fisiologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Butiratos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Imunoterapia , Interferon gama , Subunidade alfa de Receptor de Interleucina-2 , Megasphaera , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Receptores Acoplados a Proteínas G/genética , Fator de Necrose Tumoral alfaRESUMO
The development and clinical approval of immunotherapies has revolutionized cancer therapy. Although the role of adaptive immunity in atherogenesis is now well-established and several immunomodulatory strategies have proven beneficial in preclinical studies, anti-atherosclerotic immunotherapies available for clinical application are not available. Considering that adaptive immune responses are critically involved in both carcinogenesis and atherogenesis, immunotherapeutic approaches for the treatment of cancer and atherosclerosis may exert undesirable but also desirable side effects on the other condition, respectively. For example, the high antineoplastic efficacy of immune checkpoint inhibitors, which enhance effector immune responses against tumor cells by blocking co-inhibitory molecules, was recently shown to be constrained by substantial proatherogenic properties. In this review, we outline the specific role of immune responses in the development of cancer and atherosclerosis. Furthermore, we delineate how current cancer immunotherapies affect atherogenesis and discuss whether anti-atherosclerotic immunotherapies may similarly have an impact on carcinogenesis.
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Liver X receptors (LXR) are transcription factors from the nuclear receptor family that are activated by oxysterols and synthetic high-affinity agonists. In this study, we assessed the antitumor effects of synthetic LXR agonist TO901317 in a murine model of syngeneic Lewis Lung carcinoma. Treatment with TO901317 inhibited tumor growth in wild-type, but not in LXR-deficient mice, indicating that the antitumor effects of the agonist depends on functional LXR activity in host cells. Pharmacologic activation of the LXR pathway reduced the intratumoral abundance of regulatory T cells (Treg) and the expression of the Treg-attracting chemokine Ccl17 by MHCIIhigh tumor-associated macrophages (TAM). Moreover, gene expression profiling indicated a broad negative impact of the LXR agonist on other mechanisms used by TAM for the maintenance of an immunosuppressive environment. In studies exploring the macrophage response to GM-CSF or IL4, activated LXR repressed IRF4 expression, resulting in subsequent downregulation of IRF4-dependent genes including Ccl17. Taken together, this work reveals the combined actions of the LXR pathway in the control of TAM responses that contribute to the antitumoral effects of pharmacologic LXR activation. Moreover, these data provide new insights for the development of novel therapeutic options for the treatment of cancer. SIGNIFICANCE: This study reveals unrecognized roles of LXR in the transcriptional control of the tumor microenvironment and suggests use of a synthetic LXR agonist as a novel therapeutic strategy to stimulate antitumor activity.
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Benzoatos/farmacologia , Benzilaminas/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Sulfonamidas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Macrófagos Associados a Tumor/efeitos dos fármacos , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores X do Fígado/agonistas , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Células RAW 264.7 , Linfócitos T Reguladores/patologia , Transcriptoma/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologiaRESUMO
Research over the past years has provided increasing understanding about IL-17-producing CD8+ T cells termed Tc17 or IL-17+ CD8+ T cells, their distribution and role in a range of diverse immune processes. These comprise resistance to pathogens and tissue homeostasis, but also contribution to autoimmunity and cancer, as well as involvement in gut inflammation, lung diseases and graft-versus-host-disease. Tc17 cells are regulated by unique differentiation mechanisms distinguishing them from other IL-17-producing T cells, including Th17, mucosal-associated invariant T cells, and γδ17 T cells, thus ensuring their specific function in immune responses. Here, we review recent advances in understanding Tc17 cell differentiation and function, and highlight experimental evidence from human studies on patients suffering from organ-specific autoimmunity including psoriasis, spondyloarthritis and MS as well as from ulcerative colitis and gastrointestinal tract-associated cancers. We also discuss mouse models analyzing Tc17 characteristics and indicate mechanisms of cross-talk between Tc17 cells and immune or nonimmune cells, enabling their effector function in both protective as well as pathologic immune responses.
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Linfócitos T CD8-Positivos/imunologia , Interleucina-17/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , HumanosRESUMO
Th cells integrate signals from their microenvironment to acquire distinct specialization programs for efficient clearance of diverse pathogens or for immunotolerance. Ionic signals have recently been demonstrated to affect T cell polarization and function. Sodium chloride (NaCl) was proposed to accumulate in peripheral tissues upon dietary intake and to promote autoimmunity via the Th17 cell axis. Here, we demonstrate that high-NaCl conditions induced a stable, pathogen-specific, antiinflammatory Th17 cell fate in human T cells in vitro. The p38/MAPK pathway, involving NFAT5 and SGK1, regulated FoxP3 and IL-17A expression in high-NaCl conditions. The NaCl-induced acquisition of an antiinflammatory Th17 cell fate was confirmed in vivo in an experimental autoimmune encephalomyelitis (EAE) mouse model, which demonstrated strongly reduced disease symptoms upon transfer of T cells polarized in high-NaCl conditions. However, NaCl was coopted to promote murine and human Th17 cell pathogenicity, if T cell stimulation occurred in a proinflammatory and TGF-ß-low cytokine microenvironment. Taken together, our findings reveal a context-dependent, dichotomous role for NaCl in shaping Th17 cell pathogenicity. NaCl might therefore prove beneficial for the treatment of chronic inflammatory diseases in combination with cytokine-blocking drugs.
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Microambiente Celular/efeitos dos fármacos , Citocinas/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Cloreto de Sódio na Dieta/farmacologia , Células Th17/imunologia , Animais , Microambiente Celular/imunologia , Citocinas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Transgênicos , Células Th17/patologiaRESUMO
Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the effect of tumor-infiltrating lymphocyte subpopulations on lung cancer biology by studying in vitro cocultures, in vivo mouse models, and human lung cancer tissue. Lymphocyte conditioned media (CM) induced epithelial-mesenchymal transition (EMT) and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue and correlated with poor survival. Coculturing lung cancer cells with Th9/Th17 cells or exposing them to the respective CM induced EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL-9 and IL-17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Coinjection of Th9/Th17 cells with tumor cells in WT, Rag1-/-, Il9r-/-, and Il17ra-/- mice altered tumor growth and metastasis. Accordingly, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration and metastatic spreading and offering potentially novel therapeutic strategies.
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Movimento Celular/imunologia , Transição Epitelial-Mesenquimal/imunologia , Neoplasias Pulmonares/imunologia , Células Th17/imunologia , Microambiente Tumoral/imunologia , Células A549 , Animais , Humanos , Interleucina-17/imunologia , Interleucina-9/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Células Th17/patologiaRESUMO
IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond.