Downregulated Copper Homeostasis-Related Gene FOXO1 as a Novel Indicator for the Prognosis and Immune Response of Breast Cancer.

Copper (Cu) is one of the essential microelements for all living systems. Studies have illustrated the biological significance of Cu homeostasis in human cancers, including breast cancer (BRCA). Nevertheless, the detailed roles of Cu homeostasis in BRCA need to be further explored. Here, we identified a downregulated Cu homeostasis-related gene FOXO1 and investigated the potential functions of FOXO1 in BRCA through several bioinformation databases. The BRCA patients with high level of FOXO1 displayed favorable prognostic values. Subsequently, enrichment analysis of FOXO1 coexpressed genes revealed that the top three enriched KEGG pathways were spliceosome, oxidative phosphorylation, and ribosome. Immunoinfiltration analysis indicated that aberrantly expressed FOXO1 showed positive correlations with the subcellular infiltration of macrophages and neutrophils in BRCA. Moreover, FOXO1 expression was positively associated with multiple immune checkpoints, such as sialic acid-binding immunoglobulin-like lectin 15 (SIGLEC15), indoleamine 2,3-dioxygenase 1 (IDO1), programmed cell death 1 ligand 1 (PD-L1/CD274), hepatitis A virus cellular receptor 2 (HAVCR2), programmed cell death 1 (PDCD1), cytotoxic T lymphocyte antigen 4 (CTLA4), and programmed cell death 1 ligand 2 (PDCD1LG2). Overall, these findings would deepen our understanding of FOXO1 in BRCA prognosis and immunotherapy response, representing a promising therapeutic strategy for BRCA patients.

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development.

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

Regulation of autoreactive CD4 T cells by FoxO1 signaling in CNS autoimmunity.

Myelin-specific CD4 T effector cells (Teffs), Th1 and Th17 cells, are encephalitogenic in experimental autoimmune encephalomyelitis (EAE), a well-defined murine model of multiple sclerosis (MS) and implicated in MS pathogenesis. Forkhead box O 1 (FoxO1) is a conserved effector molecule in PI3K/Akt signaling and critical in the differentiation of CD4 T cells into T helper subsets. However, it is unclear whether FoxO1 may be a target for redirecting CD4 T cell differentiation and benefit CNS autoimmunity. Using a selective FoxO1 inhibitor AS1842856, we show that inhibition of FoxO1 suppressed the differentiation and expansion of Th1 cells. The transdifferentiation of Th17 cells into encephalitogenic Th1-like cells was suppressed by FoxO1 inhibition upon reactivation of myelin-specific CD4 T cells from EAE mice. The transcriptional balance skewed from the Th1 transcription factor T-bet toward the Treg transcription factor Foxp3. Myelin-specific CD4 T cells treated with the FoxO1 inhibitor were less encephalitogenic in adoptive transfer EAE studies. Inhibition of FoxO1 in T cells from MS patients significantly suppressed the expansion of Th1 cells. Furthermore, FoxO1 inhibition with AS1842856 promoted the development of functional iTreg cells. The immune checkpoint programmed cell death protein-1 (PD-1)-induced Foxp3 expression in CD4 T cells was impaired by FoxO1 inhibition. These data illustrate an important role of FoxO1 signaling in CNS autoimmunity via regulating autoreactive Teff and Treg balance.

Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4+ T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer.

HIV latency is a challenge to the success of antiretroviral therapy (ART). Hence patients may benefit from interventions that efficiently reactivate the latent virus to be eliminated by ARTs. Here we show that plasma extracellular vesicles (pEVs) can enhance HIV infection of activated CD4+ T cells and reactivate the virus in latently infected J-Lat 10.6 cells. Evaluation of the extravesicular miRNA cargo by a PCR array revealed that pEVs from HIV patients express miR-139-5p. Furthermore, we found that increased levels of miR-139-5p in J-Lat 10.6 cells incubated with pEVs corresponded with reduced expression of the transcription factor, FOXO1. pEV treatment also corresponded with increased miR-139-5p expression in stimulated PD1+ Jurkat cells, but with concomitant upregulation of FOXO1, Fos, Jun, PD-1 and PD-L1. However, J-Lat 10.6 cells incubated with miR-139-5p inhibitor-transfected pEVs from HIV ART-naïve and on-ART patients expressed reduced levels of miR-139-5p than cells treated with pEVs from healthy controls (HC). Collectively, our results indicate that pEV miR-139-5p belongs to a network of miRNAs that can promote cell activation, including latent HIV-infected cells by regulating the expression of FOXO1 and the PD1/PD-L1 promoters, Fos and Jun.

Serum- and Glucocorticoid-Inducible Kinase 1 Promotes Alternative Macrophage Polarization and Restrains Inflammation through FoxO1 and STAT3 Signaling.

Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-α, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre-mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage-priming conditions, respectively. Combined with decreased FoxO1 phosphorylation and the subsequent suppressed cytoplasmic translocation observed, SGK1 deficiency robustly enhances FoxO1 activity and drives macrophage to preferential M1 phenotypes. Furthermore, FoxO1 inhibition abrogates M1 phenotypes, and STAT3 overexpression results in a significant increase of M2 phenotypes, indicating that both FoxO1 and STAT3 are involved in SGK1-mediated macrophage polarization. Additionally, SGK1 differentially regulates the expression of M1 and M2 molecular markers, including CD68 and F4/F80 and CD163 and CD206, respectively, and protects against Porphyromonas gingivalis-induced alveolar bone loss in a mouse model. Taken together, these results have demonstrated that SGK1 is critical for macrophage polarization and periodontal bone loss, and for the first time, to our knowledge, we elucidated a bifurcated signaling circuit by which SGK1 promotes alternative, while suppressing inflammatory, macrophage polarization.

FOXO1 constrains activation and regulates senescence in CD8 T cells.

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span.

Pathogenic CARD11 mutations affect B cell development and differentiation through a noncanonical pathway.

Pathogenic CARD11 mutations cause aberrant nuclear factor κB (NF-κB) activation, which is presumably responsible for multiple immunological disorders. However, whether there is an NF-κB-independent regulatory mechanism contributing to CARD11 mutations related to pathogenesis remains undefined. Using three distinct genetic mouse models, the Card11 knockout (KO) mouse model mimicking primary immunodeficiency, the CARD11 E134G point mutation mouse model representing BENTA (B cell expansion with NF-κB and T cell anergy) disease, and the mouse model bearing oncogenic K215M mutation, we show that CARD11 has a noncanonical function as a negative regulator of the AKT-FOXO1 signal axis, independent of NF-κB activation. Although BENTA disease-related E134G mutant elevates NF-κB activation, we find that E134G mutant mice phenotypically copy Card11 KO mice, in which NF-κB activation is disrupted. Mechanistically, the E134G mutant causes exacerbated AKT activation and reduced FOXO1 protein in B cells similar to that in Card11 KO cells. Moreover, the oncogenic CARD11 mutant K215M reinforces the importance of the noncanonical function of CARD11. In contrast to the E134G mutant, K215M shows a stronger inhibitory effect on AKT activation and more stabilized FOXO1. Likewise, E134G and K215M mutants have converse impacts on B cell development and differentiation. Our results demonstrate that, besides NF-κB, CARD11 also governs the AKT/FOXO1 signaling pathway in B cells. The critical role of CARD11 is further revealed by the effects of pathogenic CARD11 mutants on this noncanonical regulatory function on the AKT-FOXO1 signaling axis.

Disruption of Immune Homeostasis in Human Dendritic Cells via Regulation of Autophagy and Apoptosis by Porphyromonas gingivalis.

As fundamental processes of immune homeostasis, autophagy, and apoptosis must be maintained to mitigate risk of chronic inflammation and autoimmune diseases. Periodontitis is a chronic inflammatory disease characterized by oral microbial dysbiosis, and dysregulation of dendritic cell (DC) and T cell responses. The aim of this study was to elucidate the underlying mechanisms by which the oral microbe Porphyromonas gingivalis (P. gingivalis) manipulates dendritic cell signaling to perturb both autophagy and apoptosis. Using a combination of Western blotting, flow cytometry, qRT-PCR and immunofluorescence analysis, we show a pivotal role for the minor (Mfa1) fimbriae of P. gingivalis in nuclear/cytoplasmic shuttling of Akt and FOXO1 in human monocyte-derived DCs. Mfa1-induced Akt nuclear localization and activation ultimately induced mTOR. Activation of the Akt/mTOR axis downregulated intracellular LC3II, also known as Atg8, required for autophagosome formation and maturation. Use of allosteric panAkt inhibitor MK2206 and mTOR inhibitor rapamycin confirmed the role of Akt/mTOR signaling in autophagy inhibition by P. gingivalis in DCs. Interestingly, this pathway was also linked to induction of the anti-apoptotic protein Bcl2, decreased caspase-3 cleavage and decreased expression of pro-apoptotic proteins Bax and Bim, thus promoting longevity of host DCs. Addition of ABT-199 peptide to disrupt the interaction of antiapoptotic Bcl2 and its proapoptotic partners BAK/BAX restored apoptotic death to P. gingivalis-infected DC cells. In summary, we have identified the underlying mechanism by which P. gingivalis promotes its own survival and that of its host DCs.

TGFß signaling in germinal center B cells promotes the transition from light zone to dark zone.

B cells in germinal centers (GCs) cycle between light zone (LZ) and dark zone (DZ). The cues in the GC microenvironment that regulate the transition from LZ to DZ have not been well characterized. In Peyer's patches (PPs), transforming growth factor-ß (TGFß) promotes IgA induction in activated B cells that can then differentiate into GC B cells. We show here that TGFß signaling occurs in B cells in GCs and is distinct from signaling that occurs in activated B cells in PPs. Whereas in activated B cells TGFß signaling is required for IgA induction, in the GC it was instead required for the transition from LZ to DZ. In the absence of TGFß signaling, there was an accumulation of LZ GC B cells and reduced antibody affinity maturation likely due to reduced activation of Foxo1. This work identifies TGFß as a microenvironmental cue that is critical for GC homeostasis and function.

Discs large homolog 1 regulates B-cell proliferation and antibody production.

Antibody production results from B-cell activation and proliferation upon antigen binding. Discs large homolog 1 (Dlg1), a scaffold protein from the membrane-associated guanylate kinase family, has been shown to regulate the antigen receptor signaling and cell polarity in lymphocytes; however, the physiological function of Dlg1 in humoral responses is not completely clear. Here, we addressed this question using a conditional knockout (KO) mouse model with Dlg1 deficiency in different B-cell subsets by crossing dlg1fl/fl mice with either mb1cre/+ or aicdacre/+ mice, respectively. In both mouse models, we observed that Dlg1 deficiency in B cells (Dlg1-KO B cells) led to obvious hyper-antibody responses upon immunization, the effect of which was more obvious in antigen-recall responses. Mechanistically, we found that Dlg1-KO B cells exhibited hyper-proliferation compared with wild-type B cells upon antigen stimulation, suggesting that the hyper-antibody responses are likely induced by the hyper-proliferation of Dlg1-KO B cells. Indeed, further studies demonstrated that Dlg1 deficiency in B cells led to the down-regulation of a tumor suppressor, FoxO1. Thus, all these results reveal an unexpected function of Dlg1 in restraining hyper-antibody responses through the inhibition of FoxO1 and thus antigen-binding-induced proliferation in B cells.

Hematopoietic-Specific Deletion of Foxo1 Promotes NK Cell Specification and Proliferation.

We previously reported that deletion of Foxo1, via Ncr1-iCre mice from the expression of NKp46 onward, led to enhanced natural killer (NK) cell maturation and effector function. In this model, however, the role of Foxo1 in regulating NK cell specification and early development remains exclusive. Herein, we utilized a murine model of hematopoietic-specific deletion of Foxo1 before lymphoid specification, by crossing mice carrying floxed Foxo1 alleles (Foxo1fl/fl) with Vav1-iCre mice, to revisit the role of Foxo1 on NK cell specification and early development. The data showed that hematopoietic-specific deletion of Foxo1 resulted in increased proportion and numbers of common lymphoid progenitors (CLP) (Lin-CD127+c-Kit+Sca-1+), pre-pro NK b cells (Lin-Sca-1+c-Kit-CD135-CD127+), as well as committed Lin-CD122+ cells and CD3-CD19-NKp46+ NK cells in bone marrow. Hematopoietic-specific deletion of Foxo1 also promoted NK cells proliferation in a cell-intrinsic manner, indicated by increased Ki-67 expression and more expansion of NK cell after ex vivo stimulation with IL-15. The reason for Foxo1 suppressing NK cell proliferation might be its direct transcription of the cell-cycle inhibitory genes, such as p21cip1, p27kip1, p130, Gadd45a, and Ccng2 (cyclin G2) in NK cells, supported by the evidence of decreased mRNA expression of p21cip1, p27kip1, p130, Gadd45a, and Ccng2 in Foxo1-deficient NK cells and direct binding of Foxo1 on their promoter region. Furthermore, hematopoietic-specific deletion of Foxo1 resulted in increased ratio of mature NK subsets, such as CD11b+CD27- and CD43+KLRG1+ NK cells, but decreased ratio of immature NK subsets, such as CD27+CD11b- and CD27+CD11b+ NK cells, consistent with the findings in the murine model of Ncr1-iCre mediated Foxo1 deletion. Conclusively, Foxo1 not only acts as a negative checkpoint on NK cell maturation, but also represses NK cell specification and proliferation. The relative higher expression of Foxo1 in CLP and early NK precursors may also contribute to the later NK cell proliferation and responsiveness, which warranties another separate study in the future.

Fisetin supplementation prevents high fat diet-induced diabetic nephropathy by repressing insulin resistance and RIP3-regulated inflammation.

Obesity-related renal disease is related to caloric excess promoting deleterious cellular responses. However, a full understanding of the molecular mechanisms involved in progressive kidney disease, as well as a therapeutic strategy, is still absent. Fisetin (FIS), as a natural flavonoid, possesses various bioactivities in a number of disease models. However, its role in obesity-associated kidney injury is still unclear and requires elucidation. In our study, an obesity animal model was established using C57BL/6 mice fed with a normal chow diet (NCD) or high fat diet (HFD) for 16 weeks with or without FIS administration (20, 40 or 80 mg kg-1). Our results indicated that chronic HFD feeding led to a significant body weight gain in mice compared to the normal control group, accompanied by a marked insulin resistance and glucose intolerance, whereas FIS treatment exerted prominently protective effects. In addition, FIS significantly attenuated HFD-induced histological alterations in renal tissue samples. Moreover, FIS treatment down-regulated expression of kidney injury molecule-1 (KIM-1), and up-regulated nephrin and podocin expression levels in the kidneys of HFD-fed mice, improving their renal dysfunction. After HFD feeding, mice treated with FIS exhibited a decrease in phosphorylated IRS1Ser307, and an increase in phosphorylated glycogen synthase kinase 1 (IRS1Tyr608), AKT, forkhead box protein O1 (FOXO1) and glycogen synthase kinase (GSK)-3ß. Furthermore, FIS administration markedly restrained the inflammatory response in the kidneys of HFD-challenged mice, as evidenced by the reduced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-1ß and IL-18, which was attributed to the blockage of nuclear factor κB (NF-κB) signaling. Importantly, FIS-treated obese mice exerted a remarkable decrease in RIP3 expressions in the kidneys compared to obese mice in the absence of FIS, along with an evident reduction in the NOD-like receptor protein 3 (NLRP3), an apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) and Caspase-1. The protective effects of FIS against HFD-induced renal injury were verified in vitro using palmitate (PAL)-treated HK2 cells, an immortalized proximal tubule epithelial cell line from the adult human kidney. In summary, our results supported the notion that FIS functions as a promising agent to improve insulin resistance and inflammatory response against metabolic stress-induced renal injury.

The E3 ubiquitin ligase Itch is required for B-cell development.

The E3 ubiquitin ligase Itch interacts with Foxo1 and targets it for ubiquitination and degradation during follicular helper T-cell differentiation, whereas the transcription factor Foxo1 plays a critical role in B-cell development. Thus, we proposed that Itch mediates B-cell differentiation. Unexpectedly, we found that Itch deficiency downregulated Foxo1 expression in B cells. Itch cKO (conditional knock out in B cells) mice had fewer pro-B cells in the bone marrow, more small resting IgM-IgD-B cells in the periphery, and lower B-cell numbers in the lymph nodes through decreased Foxo1-mediated IL-7Rα, RAG, and CD62L expression, respectively. Importantly, Itch deficiency reduced Foxo1 mRNA expression by up-regulating JunB-mediated miR-182. Finally, Foxo1 negatively regulated JunB expression by up-regulating Itch. Thus, we have identified a novel regulatory axis between Itch and Foxo1 in B cells, suggesting that Itch is essential for B-cell development.

MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes.

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor- and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)-mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells.

Akt2 Regulates the Differentiation and Function of NKT17 Cells via FoxO-1-ICOS Axis.

As a critical linker between mTORC1 and mTORC2, Akt is important for the cell metabolism. The role of Akt in the function and development of B and T cells is well characterized, however, the role of Akt for development and function of iNKT cells is unknown. iNKT cells bridge the adaptive and innate immunity, and in this study, we found that the differentiation of NKT17 cells and IL17 production of NKT17 cells were disrupted in Akt2 KO mice. ICOS has been demonstrated to be critical for the differentiation of NKT17 cells and we found that ICOS mRNA and protein expression was reduced in Akt2 KO iNKT cells. As a consequence, phosphorylation of FoxO-1 was downregulated in Akt2 KO thymocytes but the sequestration of FoxO-1 in the nucleus of Akt2 KO iNKT cells was increased. The negative feedback loop between ICOS and FoxO-1 has been demonstrated in CD4+T follicular helper cells. Therefore our study has revealed a new intracellular mechanism in which Akt2 regulates ICOS expression via FoxO-1 and this signaling axis regulates the differentiation and function of NKT17 cells. This study provides a new linker between cell metabolism and function of iNKT cells.

Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Expression of FOXO1.

Immunity following an acutely resolved infection or the long-term equipoise of chronic viral infections often depends on the maintenance of antigen-specific CD8+ T cells, yet the ongoing transcriptional requirements of these cells remain unclear. We show that active and continuous programming by FOXO1 is required for the functional maintenance of a memory population. Upon Foxo1 deletion following resolution of an infection, memory cells rapidly lost their characteristic gene expression, gradually declined in number, and were impaired in self-renewal. This was extended to chronic infections, as a loss of FOXO1 during a persistent viral infection led to a rapid decline of the TCF7 (a.k.a. TCF1)-expressing memory-like subset of CD8+ T cells. We further establish FOXO1 regulation as a characteristic of human memory CD8+ T cells. Overall, we show that the molecular and functional longevity of a memory T cell population is actively maintained by the transcription factor FOXO1.

MicroRNA-425 facilitates pathogenic Th17 cell differentiation by targeting forkhead box O1 (Foxo1) and is associated with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic autoimmune disease, and its pathogenesis remains mostly unknown. MicroRNAs (miRs) has drawn much attention as a crucial regulator of autoimmune diseases. In this study, we demonstrated, for the first time, that miR-425 was significantly up-regulated in peripheral blood mononuclear cells (PBMC) and mucosa of patients with IBD. In note, T helper (Th) 17 cells were found to be the major source of miR-425 expression. Using gain-of-function approaches, we demonstrated that miR-425 could facilitate the differentiation of CD4+ T cells into Th17 lineage. In addition, forkhead box O1 (Foxo1) was identified as a novel target gene of miR-425, which was able to inhibit Th17 cell differentiation, and it was observed to be markedly decreased in PBMC and mucosa of patients with IBD. Notably, in vivo inhibition of miR-425 significantly alleviated the disease severity of TNBS-induced colitis in mice, with down-regulated levels of IL-17A. Our data reveal a novel mechanism in which the elevated miR-425 in IBD mediates pathogenic Th17 cell generation through down-regulation of Foxo1. In vivo blockade of miR-425 may serve as a novel therapeutic approach in the treatment of IBD.

Imaging flow cytometry: A method for examining dynamic native FOXO1 localization in human lymphocytes.

While flow cytometry can reliably assess surface and intracellular marker expression within small cell populations, it does not provide any information on protein localization. Several key transcription factors (TF) downstream of lymphocyte surface receptors are regulated by nuclear versus cytoplasmic localization, and one such TF is Forkhead box O1 (FOXO1). FOXO1 integrates antigen-binding, co-receptor activation and metabolic signals in lymphocytes, leading to proliferation and differentiation. Importantly, the nuclear or cytoplasmic localization of FOXO1 is key for gene expression leading to different lymphocyte phenotypes. In effector lymphocytes (Teff), for example, lymphocyte receptor (TCR) signaling leads to an Akt-dependent phosphorylation of FOXO1. Phosphorylated FOXO1 is excluded from the nucleus, promoting proliferation and effector functions. In contrast, nuclear retention of FOXO1 is essential for early and late development of T and B cells and for the thymic development and stability of regulatory T cells. Given the critical role of FOXO1 localization as an indicator and determinant of function, quantification of FOXO1 cellular localization in human lymphocytes can help determine immune cell activation and activity in experimental and clinical scenarios. The standard method used to determine subcellular protein localization is the analysis of nuclear and cytoplasmic protein extracts by Western blotting (WB). However, available techniques, such as WB, are limited by a requirement for a large number of cells and inability to determine FOXO1 localization in individual cells or sub-populations. In contrast, a standardized method using an imaging flow cytometer (IFC) such as the Amnis ImagestreamX Mark II, would provide both qualitative, per-cell localization information, as well as quantitative data on gated sub-populations. To this end, we report the development and optimization of an IFC protocol to examine native FOXO1 localization in human lymphocytes. A human CD4+ lymphocyte line, HuT102, as well as primary human T cells, were assessed for dynamic FOXO1 localization after treatment with a lymphocyte receptor signaling mimic (PMA/Ionomycin). IFC nuclear translocation analysis permitted us to precisely quantify the alterations over time in nuclear and cytoplasmic localization of native FOXO1 on a per cell basis, including within specific, user-defined sub-populations of cells. For human lymphocytes, using IFC to assess and quantify dynamic FOXO1 localization allows the user to simultaneously study multiple lymphocyte subpopulations as well as to delineate differing effects of dynamic FOXO1 localization that may be lost when other available methods are used.

Atorvastatin prevents endothelial dysfunction in high glucose condition through Skp2-mediated degradation of FOXO1 and ICAM-1.

OBJECTIVE: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin has been reported to exert vasculo-protective action in diabetes. We investigated the vasculo-protective mechanism of atorvastatin by evaluating its effect on two major pathogenic molecules, FOXO1 and ICAM1, mediated by S-phase kinase-associated protein 2 (Skp2) in diabetic endothelial dysfunction. APPROACH AND RESULTS: [1] FOXO1: Hyperglycemic condition increased FOXO1 protein level in endothelial cells, which was reversed by atorvastatin. This atorvastatin effect was obliterated by treatment of protease inhibitor, suggesting that atorvastatin induces degradation of FOXO1. Immunoprecipitation showed that atorvastatin facilitated the binding of Skp2 to FOXO1, leading to ubiquitination and degradation of FOXO1. [2] ICAM-1: Increased ICAM1 in high glucose condition was reduced by atorvastatin. But this effect of atorvastatin was obliterated when Skp2 was inhibited, suggesting that atorvastatin enhances binding of Skp2 to ICAM1 leading to degradation. Actually, ubiquitination and degradation of ICAM-1 were reduced when Skp2 was inhibited. In vitro monocyte adhesion assay revealed that atorvastatin reduced monocyte adhesion on endothelial cells in high glucose condition, which was reversed by Skp2 knock-down. CONCLUSION: Atorvastatin strengthens Skp2 binding to FOXO1 or ICAM1, leading to ubiquitination and degradation. Skp2-dependent ubiquitination of major pathogenic molecules is the key mechanism for statin's protective effect on endothelial function in diabetes.

Continuous activity of Foxo1 is required to prevent anergy and maintain the memory state of CD8+ T cells.

Upon infection with an intracellular pathogen, cytotoxic CD8+ T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.