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.

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation.

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.

The adaptive antioxidant response during fasting-induced muscle atrophy is oppositely regulated by ZEB1 and ZEB2.

Reactive oxygen species (ROS) serve important homeostatic functions but must be constantly neutralized by an adaptive antioxidant response to prevent supraphysiological levels of ROS from causing oxidative damage to cellular components. Here, we report that the cellular plasticity transcription factors ZEB1 and ZEB2 modulate in opposing directions the adaptive antioxidant response to fasting in skeletal muscle. Using transgenic mice in which Zeb1 or Zeb2 were specifically deleted in skeletal myofibers, we show that in fasted mice, the deletion of Zeb1, but not Zeb2, increased ROS production and that the adaptive antioxidant response to fasting essentially requires ZEB1 and is inhibited by ZEB2. ZEB1 expression increased in fasted muscles and protected them from atrophy; conversely, ZEB2 expression in muscles decreased during fasting and exacerbated muscle atrophy. In fasted muscles, ZEB1 reduces mitochondrial damage and increases mitochondrial respiratory activity; meanwhile, ZEB2 did the opposite. Treatment of fasting mice with Zeb1-deficient myofibers with the antioxidant triterpenoid 1[2-cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoro-ethylamide (CDDO-TFEA) completely reversed their altered phenotype to that observed in fasted control mice. These results set ZEB factors as potential therapeutic targets to modulate the adaptive antioxidant response in physiopathological conditions and diseases caused by redox imbalance.

Zeb1 induces immune checkpoints to form an immunosuppressive envelope around invading cancer cells.

The PDL1-PD1 immune checkpoint inhibits T cell activation, and its blockade is effective in a subset of patients. Studies are investigating how checkpoints are hijacked by cancer cells and why most patients remain resistant to immunotherapy. Epithelial mesenchymal transition (EMT), which drives tumor cell invasion via the Zeb1 transcription factor, is linked to immunotherapy resistance. In addition, M2-polarized tumor-associated macrophages (TAMs), which inhibit T cell migration and activation, may also cause immunotherapy resistance. How EMT in invading cancer cells is linked to therapy resistance and events driving TAM M2 polarization are therefore important questions. We show that Zeb1 links these two resistance pathways because it is required for PDL1 expression on invading lung cancer cells, and it also induces CD47 on these invading cells, which drives M2 polarization of adjacent TAMs. Resulting reprogramming of the microenvironment around invading cells shields them from the hostile inflammatory environment surrounding tumors.

ZEB1 promotes inflammation and progression towards inflammation-driven carcinoma through repression of the DNA repair glycosylase MPG in epithelial cells.

OBJECTIVE: Chronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify new drivers of ulcerative colitis (UC) and inflammatory CRC. DESIGN: The study uses samples from patients with UC, mouse models of colitis and CRC and mice deficient for the epithelial-to-mesenchymal transition factor ZEB1 and the DNA repair glycosylase N-methyl-purine glycosylase (MPG). Samples were analysed by immunostaining, qRT-PCR, chromatin immunoprecipitation assays, microbiota next-generation sequencing and ROS determination. RESULTS: ZEB1 was induced in the colonic epithelium of UC and of mouse models of colitis. Compared with wild-type counterparts, Zeb1-deficient mice were partially protected from experimental colitis and, in a model of inflammatory CRC, they developed fewer tumours and exhibited lower levels of DNA damage (8-oxo-dG) and higher expression of MPG. Knockdown of ZEB1 in CRC cells inhibited 8-oxo-dG induction by oxidative stress (H2O2) and inflammatory cytokines (interleukin (IL)1ß). ZEB1 bound directly to the MPG promoter whose expression inhibited. This molecular mechanism was validated at the genetic level and the crossing of Zeb1-deficient and Mpg-deficient mice reverted the reduced inflammation and tumourigenesis in the former. ZEB1 expression in CRC cells induced ROS and IL1ß production by macrophages that, in turn, lowered MPG in CRC cells thus amplifying a positive loop between both cells to promote DNA damage and inhibit DNA repair. CONCLUSIONS: ZEB1 promotes colitis and inflammatory CRC through the inhibition of MPG in epithelial cells, thus offering new therapeutic strategies to modulate inflammation and inflammatory cancer.

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.

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.

Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine.

Peptide vaccines using specific antigens with poor immunogenicity like GnRH-I are unable to develop an effective adaptive immune response and require the presence of adjuvants, essential to lymphocytic activation. Three chitosan formulations were evaluated for their ability as adjuvant of a poor immunogenic peptide vaccine against GnRH-I. Male Sprague-Dawley rats were immunized subcutaneously with recombinant His-GnRH-tandem-repeat peptide in high, low and phosphorylated high molecular weight chitosan solution at 0.5% (w/v). Freund's complete adjuvant was used as a positive control of immune response. Our results suggest that different chitosan formulations as adjuvant, with high or low viscosity degree allow inducing a high and persistent immune response against a poor immunogenic recombinant peptide. We found that the immune response was mediated by a increasing of IgG isotype 1, which were significantly greater than levels presented by the animals immunized with Freund's complete adjuvant. Nevertheless, chitosan with low molecular weight and highest acetylation degree was able to induce an immune response mediated by IgG isotype 2a. Additionally, high molecular weight phosphorylated chitosan, in which the phosphate groups were linked to N-acetyl-d-glucosamine unit, the immune response was reduced. All the immune responses obtained with chitosan as adjuvant were able to neutralize effectively the GnRH hormone proves by reducing of animal steroidogenesis and spermatogenesis demonstrating its capacity to improve immunogenicity in peptide vaccine.