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Endothelial cells (ECs) rely on glycolysis for energy production to maintain vascular homeostasis and the normalization of hyperglycolysis in tumor vessels has recently gained attention as a therapeutic target. We analyzed the TCGA database and found reduced Foxp1 expression in lung carcinoma. Immunostaining demonstrated reduced expression more restricted at tumor vascular ECs. Therefore, we investigated the function and mechanisms of Foxp1 in EC metabolism for tumor angiogenesis required for tumor growth. EC-Foxp1 deletion mice exhibited a significant increase of tumor and retinal developmental angiogenesis and Hif1α was identified as Foxp1 target gene, and Hk2 as Hif1α target gene. The Foxp1-Hif1α-Hk2 pathway in ECs is important in the regulation of glycolytic metabolism to govern tumor angiogenesis. Finally, we used genetic deletion of EC-Hif1α and RGD-peptide nanoparticles EC target delivery of Hif1α/Hk2-siRNAs to knockdown gene expression which reduced the tumor EC hyperglycolysis state and restricted angiogenesis for tumor growth. This study advances our understanding of EC metabolism for tumor angiogenesis, and meanwhile provides evidence for future therapeutic intervention of hyperglycolysis in tumor ECs for suppression of tumor growth., Competing Interests: Declaration of competing interest There are no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Human FOXP3 + regulatory T (T reg ) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve T reg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate T reg cells into naïve, activated, and effector stages, and resolve the HLA-DR hi , LIMS1 hi , highly suppressive FOXP3 hi , and highly proliferative MKI67 hi effector subsets. Trajectory analysis assembles T reg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3 hi and MKI67 hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3 hi and MKI67 hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of T reg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of T reg complexity in health and disease.

Rationale: Endothelial dysfunction results in sustained and chronic vascular inflammation, which is central to atherosclerotic diseases. However, transcriptional regulation of vascular endothelial inflammation has not been well clarified., Objective: This study aims to explore Foxp (forkhead box P) transcription factor 1 in regulation of endothelial homeostasis, atherogenesis, and its mechanisms., Methods and Results: To assess the importance of Foxp1 in atherosclerosis, Foxp1 expression was analyzed in human coronary artery and mouse artery, and we observed significant downregulation of Foxp1 in atherosclerotic and atherosusceptible endothelium. Endothelial-specific Foxp1 knockout mice (Foxp1 ECKO ) were bred onto Apoe KO mice to generate endothelial Foxp1-deletion hyperlipidemic model Foxp1 ECKO ;Apoe KO , which displayed significant increases in atherosclerotic lesion formation in aortas and aortic roots with enhanced monocyte adhesion, migration, and infiltration into the vascular wall and formation of inflammatory lipid-laden macrophages. In contrast, endothelial-specific Foxp1 overexpression mice Foxp1 ECTg ;Apoe KO exhibited reduced atherosclerotic lesion formation with less monocyte infiltration. Foxp1 was further identified as a gatekeeper of vessel inflammation by direct regulation of endothelial inflammasome components, including Nlrp3 (NLR [nucleotide-binding and leucine-rich repeat immune receptors] family pyrin domain containing 3), caspase-1, and IL (interleukin)-1β. Moreover, endothelial Foxp1 was found to be regulated by Klf2 (Kruppel-like factor 2). Oscillatory shear stress downregulated Foxp1 expression via repressing Klf2 expression in endothelium, and, therefore, promoted endothelial inflammasome activation, leading to atherosclerotic lesion formation. Simvastatin upregulated the reduced expression of Klf2 and Foxp1 in atherosusceptible vascular endothelium and alleviated vascular inflammation contributing to its inhibitory effect in atherosclerosis., Conclusions: These data are the first in vivo experimental validation of an atheroprotective role of endothelial Klf2 and Foxp1, which reveals a Klf2-Foxp1 transcriptional network in endothelial cells as a novel regulator of endothelial inflammasome activation for atherogenesis, therefore, provides opportunities for therapeutic intervention of atherosclerotic diseases and uncovers a novel atheroprotective mechanism for simvastatin.

Background: Pathological cardiac fibrosis and hypertrophy, the common features of left ventricular remodeling, often progress to heart failure. Forkhead box transcription factor P1 (Foxp1) in endothelial cells (ECs) has been shown to play an important role in heart development. However, the effect of EC-Foxp1 on pathological cardiac remodeling has not been well clarified. This study aims to determine the role of EC-Foxp1 in pathological cardiac remodeling and the underlying mechanisms., Methods: Foxp1 EC-specific loss-of-function and gain-of-function mice were generated, and an angiotensin II infusion or a transverse aortic constriction operation mouse model was used to study the cardiac remodeling mechanisms. Foxp1 downstream target gene transforming growth factor-β1 (TGF-β1) was confirmed by chromatin immunoprecipitation and luciferase assays. Finally, the effects of TGF-β1 blockade on EC-Foxp1 deletion-mediated profibrotic and prohypertrophic phenotypic changes were further confirmed by pharmacological inhibition, more specifically by RGD-peptide magnetic nanoparticle target delivery of TGF-β1-siRNA to ECs., Results: Foxp1 expression is significantly downregulated in cardiac ECs during angiotensin II-induced cardiac remodeling. EC-Foxp1 deletion results in severe cardiac remodeling, including more cardiac fibrosis with myofibroblast formation and extracellular matrix protein production, as well as decompensated cardiac hypertrophy and further exacerbation of cardiac dysfunction on angiotensin II infusion or transverse aortic constriction operation. In contrast, EC-Foxp1 gain of function protects against pathological cardiac remodeling and improves cardiac dysfunction. TGF-β1 signals are identified as Foxp1 direct target genes, and EC-Foxp1 deletion upregulates TGF-β1 signals to promote myofibroblast formation through fibroblast proliferation and transformation, resulting in severe cardiac fibrosis. Moreover, EC-Foxp1 deletion enhances TGF-β1-promoted endothelin-1 expression, which significantly increases cardiomyocyte size and reactivates cardiac fetal genes, leading to pathological cardiac hypertrophy. Correspondingly, these EC-Foxp1 deletion-mediated profibrotic and prohypertrophic phenotypic changes and cardiac dysfunction are normalized by the blockade of TGF-β1 signals through pharmacological inhibition and RGD-peptide magnetic nanoparticle target delivery of TGF-β1-siRNA to ECs., Conclusions: EC-Foxp1 regulates the TGF-β1-endothelin-1 pathway to control pathological cardiac fibrosis and hypertrophy, resulting in cardiac dysfunction. Therefore, targeting the EC-Foxp1-TGF-β1-endothelin-1 pathway might provide a future novel therapy for heart failure.

Regulatory T (Treg) cells play central roles in maintaining immune homeostasis and self-tolerance. However, the molecular mechanisms underlying Treg cell homeostasis and suppressive function are still not fully understood. Here, we report that the deletion of another P subfamily members of the forkhead box (Foxp) subfamily member Foxp1 in Treg cells led to increased numbers of activated Treg (aTreg) cells at the expense of quiescent Treg cells, and also resulted in impaired Treg suppressive function. Mice with Foxp1-deficient Treg cells developed spontaneous inflammatory disease with age; they also had more severe inflammatory disease in colitis and experimental autoimmune encephalomyelitis (EAE) models. Mechanistically, we found that Foxp1 bound to the conserved noncoding sequence 2 (CNS2) element of the Foxp3 locus and helped maintain Treg suppressive function by stabilizing the Foxp3 expression. Furthermore, we found that Foxp1 and Foxp3 coordinated the regulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression levels. Taken together, our study demonstrates that Foxp1 plays critical roles in both maintaining Treg cell quiescence during homeostasis and regulating Treg suppressive function., Competing Interests: The authors have declared that no competing interests exist.

FOXP3 + Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis and tolerance. Disruption of Foxp3 expression results in the generation of instable Treg cells and acquisition of effector T-cell-like function. Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells. Mice depleted of Usp21 specifically in Treg cells display immune disorders characterized by spontaneous T-cell activation and excessive T-helper type 1 (Th1) skewing of Treg cells into Th1-like Treg cells. USP21 stabilizes FOXP3 protein by mediating its deubiquitination and maintains the expression of Treg signature genes. Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.

Previously we have shown that transcription factor Foxp1 plays an essential role in maintaining naive T cell quiescence; in the absence of Foxp1, mature naive CD8(+) T cells proliferate in direct response to homeostatic cytokine IL-7. In this study, we report that the deletion of Foxp1 in naive CD8(+) T cells leads to enhanced activation of the PI3K/Akt/mammalian target of rapamycin signaling pathway and its downstream cell growth and metabolism targets in response to IL-7. We found that Foxp1 directly regulates PI3K interacting protein 1, a negative regulator of PI3K. Additionally, we found that deletion of Foxp1 in naive CD8(+) T cells results in increased expression levels of E2fs, the critical components for cell cycle progression and proliferation, in a manner that is not associated with increased phosphorylation of retinoblastoma protein. Taken together, our studies suggest that Foxp1 enforces naive CD8(+) T cell quiescence by simultaneously repressing key pathways in both cellular metabolism and cell cycle progression., (Copyright © 2016 by The American Association of Immunologists, Inc.)

CD4(+) follicular helper T cells (T(FH) cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4(+) T cells deficient in the transcription factor Foxp1 'preferentially' differentiated into T(FH) cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of T(FH) cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4(+) T cells partially resistant to blockade of the ICOS ligand (ICOSL) during T(FH) cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of T(FH) cell differentiation.

The molecular mechanisms that underlie T cell quiescence are poorly understood. Here we report that mature naive CD8(+) T cells lacking the transcription factor Foxp1 gained effector phenotype and function and proliferated directly in response to interleukin 7 (IL-7) in vitro. Foxp1 repressed expression of the IL-7 receptor α-chain (IL-7Rα) by antagonizing Foxo1 and negatively regulated signaling by the kinases MEK and Erk. Acute deletion of Foxp1 induced naive T cells to gain an effector phenotype and proliferate in lympho-replete mice. Foxp1-deficient naive CD8(+) T cells proliferated even in lymphopenic mice deficient in major histocompatibility complex class I. Our results demonstrate that Foxp1 exerts essential cell-intrinsic regulation of naive T cell quiescence, providing direct evidence that lymphocyte quiescence is achieved through actively maintained mechanisms that include transcriptional regulation.