Role of ZEB1 in macrophages during homeostasis, inflammation and cancer

[eng] ZEB1 is a transcription factor whose expression in cancer cells promotes tumor initiation and progression. In this study, we for the first time characterized Zeb1 and study its function in macrophages under either homeostasis or activation conditions as well as in a murine cancer model. We found that macrophages deficient for Zeb1 showed aberrant characteristics in phenotype and functions, under physiological and pathological conditions. Here we clarified a functional role of Zeb1 on macrophages playing a role in macrophage phagocytosis, migration and inflammation as well as in tumor progression in a non-cell-autonomous manner modulating the tumor microenviroment. In fact the evidence presented indicates that the downregulation of Zeb1 in macrophages is associated with the inhibition of TAM characteristics and inhibition of tumor progression. ZEB1 plays important roles during embryogenesis and deletion of both alleles of Zeb1 in mice results in embryonic lethality. ZEB1 represses key genes involved in the terminal differentiation of multiple tissues, including inter alia epithelial cells, pituitary gland, skeletal and smooth muscle, cartilage, and bone. Although ZEB1 is expressed in lymphoid cells where it represses pivotal hematopoietic transcription factors, there was no evidence for a role of ZEB1 in the regulation of lymphoid or myeloid differentiation. We showed here that downregulation of Zeb1 in bone marrow precursors promoted their differentiation towards macrophages. These data further support a model, best characterized in epithelial tissues and skeletal muscle, where ZEB1 expression needs to decline for early precursors to terminally differentiate. ZEB1 has been extensively characterized in cancer cells where it promotes their stemness, survival and invasiveness. However, its role in the tumor microenvironment remained to be elucidated. Among cancer cells, ZEB1 is not expressed across the entire tumor mass but is rather restricted to a subpopulation of stem-like malignant cells at the invasive front, actually, at the interface where cancer cells and TAMs interact. Although ZEB1 expression among stromal cells has been noted, the identity of the cell types expressing ZEB1 has not been established. This study showed that ZEB1 is also expressed in TAMs and that ZEB1 not only bilaterally regulates the crosstalk between cancer cells and TAMs but that this crosstalk regulates ZEB1 expression itself. Thus, Zeb1 was upregulated in macrophages that have interacted with cancer cells as well as in cancer cells that have interacted with wild-type TAMs. The tumor-promoting role of ZEB1 is therefore supported by a positive feedback of its expression between malignant cells and TAMs. We found that Zeb1 is restricted to the F4/80low macrophage/TAM subpopulation—previously known to display stronger pro-tumor and pro-angiogenic functions—whose share is expanded by ZEB1. Soluble factors produced by the tumor—e.g., CSF1 and CCL2—attract F4/80low CCR2+ monocytes into their microenvironment where they are activated into TAMs. Inhibition of the CCL2–CCR2 axis blocks monocyte recruitment into the tumor stroma and inhibits tumor growth. We found that Zeb1 promotes monocyte migration both in response to chemotactic stimuli (CSF1 and CCL2) and in the context of cancer. Zeb1-deficient TAMs expressed lower levels of Ccr2 and were unable to induce Ccl2 in ID8 cells. At the same time, the maximum effect of ZEB1 as a biomarker of poorer prognosis in ovarian cancer patients depended on high levels of CCL2. These data establish Zeb1 as an important inducer of the pro-tumor and pro-metastatic CCR2-MMP9-CCL2 loop between tumor cells and TAMs. It is important to note that this CCR2-MMP9-CCL2 loop was inhibited by just a partial downregulation of Zeb1 in TAMs. Data here showed that the pro-tumor role of ZEB1 in TAMs also depends on a similarly narrow threshold of expression. Zeb1 (+/-) macrophages still express about half of the Zeb1 mRNA levels of wild-type macrophages, but this downregulation was enough to render Zeb1 (+/-) TAMs unable to promote tumor growth when transplanted into tumor-bearing mice as wild-type macrophages did. As in the case of ZEB1 expression in cancer cells, to the best of our knowledge, this is the first example of a heterozygous gene deletion being sufficient to block the tumor-promoting role of TAMs. Expression of ZEB1 in cancer cells has been associated to increased chemotherapy resistance. In parallel, we found here that expression of ZEB1 in TAMs also increased the cancer cell resistance to chemotherapy. In that line, we showed that Zeb1 in TAMs increased the expression of Il10, Mmp9 and Il1b—that have a suppressor effect on chemotherapy—and of the drug efflux transporter Mdr1. The dual role of ZEB1 promoting tumor progression in cancer cells and in TAMs—albeit through different mechanisms—has translational implications. Targeting ZEB1 in cancer cells is being considered in ongoing clinical trials but data here suggest that improving chemotherapy response would also require the downregulation of ZEB1 in TAMs. The fact that a partial downregulation of Zeb1 in TAMs was sufficient to abolish TAMs’ tumor-promoting function is highly relevant for therapy approaches aiming at blocking ZEB1 expression and/or function. These results establish a new role for ZEB1 promoting tumor progression through its expression in TAMs, thus setting ZEB1 expression as a relevant target in cancer therapy

ZEB1 protects skeletal muscle from damage and is required for its regeneration

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration

Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation.

Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts (Zeb1WT/ApoeKO), male mice with Zeb1 ablation in their myeloid cells (Zeb1∆M/ApoeKO) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1∆M/ApoeKO mice display more pronounced systemic metabolic alterations than Zeb1WT/ApoeKO mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1∆M macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1∆M/ApoeKO mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.S

Marcadores del discurso derivados de los verbos de percepción: un análisis comparativo entre el español y el italiano

Despite the growing interest in discourse markers over the past two decades, few studies are dedicated to the interlinguistic comparison of discourse markers. By means of a corpus-based approach the present study proposes a comparison between the discourse markers derived from verbs of perception in Italian (guarda/guardi, senti/senta) and Spanish (mira/mire, oye/oiga). The results of a comparable corpus study reveals that the discourse markers sharing the original perception modality display a similar formal behaviour (mira/mire ~ guarda/guardi, oye/oiga ~ senti/senta). From the parallel corpus on the other hand it results that the most frequent equivalent of mira/mire is senti/senta, which can be linked to the original semantics of both verbs. These two main observations confirm the relevance of a parallel corpus as a complementary source to a comparable corpus when comparing phenomena in two (or more) languages. This combined corpus approach brings about not only interlinguistic insights but provides at the same time knowledge about the relation and uses of the discourse markers within the languages

Tumor-associated macrophages (TAMs) depend on ZEB1 for their cancer-promoting roles

Accumulation of tumor-associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro-tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial-to-mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor-promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80low pro-tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem-like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor-promoting functions. © 2017 The Author

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation

Acknowledgements: We are grateful to Dr. F. Sanchez-Madrid (Hospital Princesa and CNIC, Madrid, Spain), Dr. D. Cebrian (CNIC, Madrid, Spain), and Dr. A. Valledor (University of Barcelona, Spain) for helpful insights on early versions of the manuscript. We thank Dr. C. Stephan-Otto Attolini (BIST-IRB, Barcelona, Spain) and Dr. J. Rios (IDIBAPS, Hospital Clinic, and Autonomous University of Barcelona, Barcelona, Spain) for their expert guidance on the statistical analyses of the data in the study. We also thank Dr. L. Ribas de Pouplana (BIST-IRB, Barcelona, Spain) for advice on mitochondrial translation experiments. We acknowledge technical assistance by staff in the Flow Cytometry Unit at IDIBAPS, the Molecular Interactions Services Unit at the Biomedical Research Institute of Bellvitge (IDIBELL), and the Transmission Electron Microscopy Unit at the University of Barcelona School of Medicine. We also thank A Téllez (Hospital Clinic, Barcelona, Spain) for his help in collecting samples from septic patients, and Dr. MJ Fernández-Aceñero (Hospital Clinico San Carlos, Madrid, Spain) for help in collecting skin samples from healthy controls, psoriatic patients, and melanoma patients. We are also thankful to Dr. DC Dean (University of Louisville, KY, USA) for his generous gift of an anti-ZEB1 polyclonal antibody. We thank Dr. A. Garcia for the artistic drawing of schematics in the article. IDIBAPS is partly funded by the CERCA Programme of Generalitat de Catalunya. The study was conducted at IDIBAPS’ Centre de Recerca Biomèdica Cellex building, which was partly funded by the Cellex Foundation. The different parts of this study were independently funded by grants to AP from the Leo Foundation (LF-OC-19-000166), the Catalan Agency for Management of University and Research Grants (AGAUR) (2017-SGR-1174 and 2021-SGR-01328), and the Spanish State Research Agency (AEI) of the Ministry of Science and Innovation (MICINN) (PID2020-116338RB-I00) as part of MICINN’s National Scientific and Technical Research and Innovation 2021-2023 Plan, which is co-financed by the European Regional Development Fund (ERDF) of the European Union Commission. AB is a recipient of a PhD scholarship from AGAUR (FI Program, 2021 FI_B 00514).Funder: Government of Catalonia | Agència de Gestió d&apos;Ajuts Universitaris i de Recerca (Agency for Management of University and Research Grants)AbstractAcute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.</jats:p

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation

Acknowledgements: We are grateful to Dr. F. Sanchez-Madrid (Hospital Princesa and CNIC, Madrid, Spain), Dr. D. Cebrian (CNIC, Madrid, Spain), and Dr. A. Valledor (University of Barcelona, Spain) for helpful insights on early versions of the manuscript. We thank Dr. C. Stephan-Otto Attolini (BIST-IRB, Barcelona, Spain) and Dr. J. Rios (IDIBAPS, Hospital Clinic, and Autonomous University of Barcelona, Barcelona, Spain) for their expert guidance on the statistical analyses of the data in the study. We also thank Dr. L. Ribas de Pouplana (BIST-IRB, Barcelona, Spain) for advice on mitochondrial translation experiments. We acknowledge technical assistance by staff in the Flow Cytometry Unit at IDIBAPS, the Molecular Interactions Services Unit at the Biomedical Research Institute of Bellvitge (IDIBELL), and the Transmission Electron Microscopy Unit at the University of Barcelona School of Medicine. We also thank A Téllez (Hospital Clinic, Barcelona, Spain) for his help in collecting samples from septic patients, and Dr. MJ Fernández-Aceñero (Hospital Clinico San Carlos, Madrid, Spain) for help in collecting skin samples from healthy controls, psoriatic patients, and melanoma patients. We are also thankful to Dr. DC Dean (University of Louisville, KY, USA) for his generous gift of an anti-ZEB1 polyclonal antibody. We thank Dr. A. Garcia for the artistic drawing of schematics in the article. IDIBAPS is partly funded by the CERCA Programme of Generalitat de Catalunya. The study was conducted at IDIBAPS’ Centre de Recerca Biomèdica Cellex building, which was partly funded by the Cellex Foundation. The different parts of this study were independently funded by grants to AP from the Leo Foundation (LF-OC-19-000166), the Catalan Agency for Management of University and Research Grants (AGAUR) (2017-SGR-1174 and 2021-SGR-01328), and the Spanish State Research Agency (AEI) of the Ministry of Science and Innovation (MICINN) (PID2020-116338RB-I00) as part of MICINN’s National Scientific and Technical Research and Innovation 2021-2023 Plan, which is co-financed by the European Regional Development Fund (ERDF) of the European Union Commission. AB is a recipient of a PhD scholarship from AGAUR (FI Program, 2021 FI_B 00514).Funder: Government of Catalonia | Agència de Gestió d&apos;Ajuts Universitaris i de Recerca (Agency for Management of University and Research Grants)Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression

Blom7 alpha Is a Novel Heterogeneous Nuclear Ribonucleoprotein K Homology Domain Protein Involved in Pre-mRNA Splicing That Interacts with SNEVPrp19-Pso4

The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEVPrp19-Pso4 complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEVPrp19-Pso4, is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7α. Blom7α is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7α, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7α co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7α to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7α influences splice site selection using two different minigene constructs. Indeed, both 5′- as well as 3′-site selection was altered upon Blom7α overexpression. Thus we suggest that Blom7α is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEVPrp19-Pso4 complex at the splice sites

Antimicrobial de-escalation in the critically ill patient and assessment of clinical cure: the DIANA study

Purpose: The DIANA study aimed to evaluate how often antimicrobial de-escalation (ADE) of empirical treatment is performed in the intensive care unit (ICU) and to estimate the effect of ADE on clinical cure on day 7 following treatment initiation. Methods: Adult ICU patients receiving empirical antimicrobial therapy for bacterial infection were studied in a prospective observational study from October 2016 until May 2018. ADE was defined as (1) discontinuation of an antimicrobial in case of empirical combination therapy or (2) replacement of an antimicrobial with the intention to narrow the antimicrobial spectrum, within the first 3 days of therapy. Inverse probability (IP) weighting was used to account for time-varying confounding when estimating the effect of ADE on clinical cure. Results: Overall, 1495 patients from 152 ICUs in 28 countries were studied. Combination therapy was prescribed in 50%, and carbapenems were prescribed in 26% of patients. Empirical therapy underwent ADE, no change and change other than ADE within the first 3 days in 16%, 63% and 22%, respectively. Unadjusted mortality at day 28 was 15.8% in the ADE cohort and 19.4% in patients with no change [p = 0.27; RR 0.83 (95% CI 0.60-1.14)]. The IP-weighted relative risk estimate for clinical cure comparing ADE with no-ADE patients (no change or change other than ADE) was 1.37 (95% CI 1.14-1.64). Conclusion: ADE was infrequently applied in critically ill-infected patients. The observational effect estimate on clinical cure suggested no deleterious impact of ADE compared to no-ADE. However, residual confounding is likely